Phthalocyanine-peptide conjugates: Receptor-targeting bifunctional agents for imaging and photodynamic therapy

Article abstract of DOI:10.1021/jm301311c

The synthesis of a series of new zinc phthalocyanine-peptide conjugates targeting the gastrin-releasing peptide (GRP) and integrin receptors is reported. Two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated zinc phthalocyanine with acetylenic bombesin or arginine-glycine-aspartic acid (RGD) derivatives, either in solution or on solid phase, are presented. The water-soluble conjugates were screened for their photodynamic efficacy against several cancer cell lines expressing different levels of GRP and integrin receptors, and their intracellular localization was evaluated via confocal fluorescence microscopy. Variations in photocytotoxicity between the conjugates correlate to differences in hydrophobicity as well as receptor-mediated cell uptake. In the case of the phthalocyanine-bombesin conjugate, competition experiments confirm the involvement of the GRP receptor in both the phototherapeutic activity as well as intracellular localization. These findings warrant further in vivo studies to evaluate the potential of this conjugate as photosensitizer for photodynamic therapy (PDT) of cancers overexpressing the GRP receptor. Published 2013 by the American Chemical Society.

Full text of DOI:10.1021/jm301311c

Article
pubs.acs.org/jmc
Phthalocyanine−Peptide Conjugates: Receptor-Targeting
Bifunctional Agents for Imaging and Photodynamic Therapy
Elena Ranyuk,† Nicole Cauchon,† Klaus Klarskov,‡ Brigitte Guerin,*,† and Johan E. van Lier*,†
́
‡
†Department of Nuclear Medicine and Radiobiology and Department of Pharmacology Faculty of Medicine and Health Sciences,
́ ́
Universite de Sherbrooke, Sherbrooke, Quebec, Canada
S
* Supporting Information
ABSTRACT: The synthesis of a series of new zinc
phthalocyanine−peptide conjugates targeting the gastrin-
releasing peptide (GRP) and integrin receptors is reported.
Two alternative synthetic methods based on Sonogashira
cross-coupling of an iodinated zinc phthalocyanine with
acetylenic bombesin or arginine−glycine−aspartic acid
(RGD) derivatives, either in solution or on solid phase, are
presented. The water-soluble conjugates were screened for their photodynamic efficacy against several cancer cell lines expressing
different levels of GRP and integrin receptors, and their intracellular localization was evaluated via confocal fluorescence
microscopy. Variations in photocytotoxicity between the conjugates correlate to differences in hydrophobicity as well as receptor-
mediated cell uptake. In the case of the phthalocyanine−bombesin conjugate, competition experiments confirm the involvement
of the GRP receptor in both the phototherapeutic activity as well as intracellular localization. These findings warrant further in
vivo studies to evaluate the potential of this conjugate as photosensitizer for photodynamic therapy (PDT) of cancers
overexpressing the GRP receptor.
can identify malignant areas of a tumor may further contribute
to improve Pc-PDT effectiveness.8 Sulfonated Pc (PcS) are
anionic water-soluble phthalocyanines that are effective PS to
kill tumor cells in vitro9 and cause tumor regression in vivo.10
Because of the presence of negatively charged sulfonate groups,
PcS show higher water-solubility and reduced aggregation as
compared to nonsubstituted Pc. Combined these properties
make PcS attractive PS for PDT as well as molecular probes for
fluorescence imaging.
The successful outcome of PDT depends to a large extent on
the tissue and intracellular localization of the PS. Nonspecific
localization often leads to suboptimal treatment outcome and
toxicity to healthy tissues. Therefore, the development of PS
with improved specificity, selectivity, and efficacy is highly
desirable for the successful PDT of tumors while preserving
healthy adjacent tissues. To achieve efficient and reliable
delivery of chemotherapeutics and diagnostics to cancer cells, a
number of delivery agents have been investigated in recent
years.11 Among them, tumor cell-targeting peptides have
emerged as the most valuable nonimmunogenic tools. In
contrast to larger molecules such as monoclonal antibodies
(mAbs), short synthetic peptides have excellent tumor
penetration properties, which in combination with their
selective binding and rapid internalization, make them ideal
carriers of therapeutics to both primary and metastatic tumor
sites. Unlike viral delivery vectors and mAbs, peptides are
nearly invisible to the immune system and are expected to
INTRODUCTION
■
Multifunctional agents for simultaneous imaging and targeted
therapy of cancer, known as theranostics, have become of
increasing interest over the past decade. Various noninvasive
imaging techniques can be used in tandem with therapeutic
modalities for image-guided therapy, and particularly the
combination of photodynamic therapy (PDT) and fluorescence
imaging has gained growing attention over the last decades.1,2
PDT combines a photosensitizer (PS) and light of an
appropriate wavelength to impart cytotoxicity via the
generation of reactive molecular species.3 The light-induced
electronic excitation of a PS can result not only in a cytotoxic
effect but also in the emission of fluorescence due to relaxation
of the excited-singlet-state PS back to the ground state.4 The
phenomenon that the same entity (PS) can act both as
therapeutic and imaging agent due to ability of the PS to
fluoresce is a unique advantage of PDT. The PS’s fluorescence
can be used in diagnostics, therapy guidance, monitoring,
treatment assessment, and mechanistic studies.1
Phthalocyanines (Pc) are of particular interest as PS for PDT
due to their suitable physical and chemical properties. They can
be synthesized in a straightforward manner and modified to
alter hydrophilicity, absorption, and emission wavelengths for
different applications.5,6 Pc exhibit high photo- and chemical
stability, which is desirable for chemical modifications as well as
in vitro and in vivo applications. They show long-wavelength
absorption with high extinction coefficients (ε > 105 M−1
cm−1), red fluorescence emission approaching NIR wavelengths
for deeper tissue imaging, and high singlet oxygen quantum
yields.7 Recent evidence that Raman imaging of Pc distribution
Received: September 11, 2012
Published: January 28, 2013
Published 2013 by the American Chemical
Society
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Scheme 1. Solid-Phase Synthesis of ZnPc(t-Bu)3−YVG Conjugate 3
cause minimal or no side effects.12 Although natural peptides
have a short biological half-life, structural modifications of the
amino acid sequence can be easily done in order to improve
their in vivo stability.13
shown that the presence of a carbon−carbon triple bond in the
PcS side chain does not affect in vitro stability.10 Very mild
conditions and tolerance to different functional groups are
among the advantages of this procedure. Hence Sonogashira
coupling was a logical target for the development of solid-phase
organic synthesis (SPOS) offering several advantages including
facile purification by simple filtration.32 Herein we report two
alternative strategies to synthesize Pc−peptide conjugates based
on Sonogashira cross-coupling. First, acetylenic peptides of
different lengths and structures (YVG, RGD, BBN[6−14])
linked to a solid support were screened in a solid-phase
Sonogashira cross-coupling with different types of iodinated
phthalocyanines (metalated or metal-free Pc, neutral or anionic
Pc). Second, the Sonogashira cross-coupling of an iodinated Pc
with an unprotected acetylenic peptide in solution was
investigated. The water-soluble conjugates were then screened
for their PDT efficacy against several cancer cell lines
expressing different levels of GRPR and integrin receptors,
and their intracellular localization was evaluated via confocal
fluorescence microscopy. The involvement of receptor-
mediated processes in both the PDT action and localization
of the PS was verified by receptor blocking experiments.
Bombesin (BBN) is a 14-amino acid peptide (Tyr-Gln-Arg-
Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) showing
high affinity for the gastrin-releasing peptide receptors (GRPR).
A variety of tumors overexpress receptors to BBN and gastrin,
including those of breast, prostate, gastric, colon, pancreatic,
and small cell lung cancer.14 In the case of prostate cancer, the
receptor subtype GRPR is expressed early in tumor develop-
ment and correlated with tumor aggressiveness.15 BBN
analogues have been labeled with both fluorescent moieties,
and radioisotopes and in vitro and in vivo studies confirm their
potential for fluorescence imaging, conventional nuclear
medicine, or PET imaging as well as radiation-mediated
therapy.16 These analogues generally show good stability,
high receptor binding affinity, and rapid tumor uptake.17,18
Hence we selected [D-Tyr6,βAla11,Thi13,Nle14]bombesin[6−
14]NH2, a potent modified GRPR agonist peptide19 previously
developed by Robert T. Jensen and co-workers as a site-specific
delivery agent for Pc. We also included the integrin-binding
motif arginine−glycine−aspartic acid (RGD) as another
RESULTS AND DISCUSSION
interesting peptide carrier for Pc. Integrins are a large class of
■
heterodimeric cell surface receptors that are overexpressed on
tumor vasculature. RGD is a recognition motif of numerous
ligands (fibronectin, collagen, prothrombin, adenovirus penton
base protein, vitronectin, as well as many other glycoproteins)
for different integrins including α3β1, α5β1, α8β1, αvβ1, αvβ3,
αvβ5, αvβ6, and αIIbβ3.20−22 Of these RGD binding integrins,
αvβ3 and αvβ5 have received increasing interest as therapeutic
and diagnostic targets23,24 because of their role in tumor growth
and angiogenesis.25 As a nonspecific peptide that does not
target any peptide receptor involved in cancer, we used the
tyrosine−valine−glycine (YVG) motif.
As a carrier for Pc, a BBN or RGD peptide moiety can be
expected not only to improve cell targeting but also to increase
water solubility and reduce aggregation, consequently leading
to improved photosensitizing activity and augmented fluo-
rescence. Only a few examples of Pc functionalization with
peptides have been reported,26,27 generally involving peptide
bond formation, an approach that is also commonly used for
the preparation of covalently linked Pc−protein conjugates.28
More recently the conjugation of Pc azide linked to solid
support and alkyne functionalized peptide was achieved using
Click chemistry.29 We recently demonstrated a new strategy for
the introduction of Pc at the N/C-terminal positions or on a
phenylalanine side chain of protected model peptides utilizing
palladium-catalyzed cross-coupling reactions.30 Sonogashira
cross-coupling is one of the most important and widely used
sp2−sp carbon−carbon bond forming reaction in organic
synthesis, frequently employed in the synthesis of natural
products and biologically active molecules.31 We previously
Solid-Phase Synthesis. Although Sonogashira coupling
using aryl-halide or triflate attached to solid support has been
extensively applied to SPOS, the alternative method using
resin-supported alkyne is less common.24 The first approach we
envisaged for the synthesis of Pc−peptide conjugates involved
Sonogashira coupling of a resin-bound peptide functionalized
with the terminal triple bond and an iodinated phthalocyanine.
Use of Sonogashira coupling for modification of peptides, while
still attached to solid support, seems to be not well-exploited.
The “on resin” Sonogashira coupling reaction employed for the
macrocyclization of a resin-bound peptide to form a 65-
membered ring appears to be the only example found in the
literature.33 In our studies, a hydrophilic, polyethylene glycol-
based TGR resin was chosen as a solid support. The application
of this type of resin in solid-phase variant of Sonogashira has
been previously reported and shown to be advantageous over
Rink amide resin (cross-linked polystyrene resin with Rink
linker) in some cases.34 Accordingly, the solid-supported
acetylenic peptides 2a−2c were synthesized via the Fmoc
(fluorenylmethyloxycarbonyl) strategy to be employed in
Sonogashira coupling with iodinated Pc.
Typical literature procedures for Sonogashira couplings
utilize palladium catalyst with a metal cocatalyst and a base.
The most widely employed cocatalysts are copper salts which
also mediate Hay/Glaser reaction.35 Thus homocoupling of the
alkyne is a major problem when the supply of acetylene is
limited. Indeed, when our first experiments were carried out
using 1a and a resin-supported peptide 2a as a model system
under the conditions reported for the cyclization of the resin-
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a
Table 1. Analytical Data for the Pc−Peptide Conjugates Obtained via the Solid-Phase Sonogashira Cross-Coupling
[M + H]+
b
d
d
g
entry Pc
peptide ratio Pc:peptide product
solvent
DMF
calcd
found
UV−vis λmax, nm (DMF)
conversion , %
yield %
35
1
1a
2a
1:2
3
1189.8
1211.8
1198.7
2137.9
2159.9
1190.0
1212.6
1198.5
2138.1
2160.8
348, 612 (sh), 678
80
e
e
e
e
e
e
e
e
e
e
c
c
2
3
1a
1a
2b
2c
1:2
1:2
4
5
DMF
THF
351, 613 (sh), 679
349, 612 (sh), 680
60 (75 )
21 (30 )
50
75
10
30
f
1b
2a
1:2
6
THF
1126.4
1148.4
1135.3
2074.5
2096.5
1126.2
1148.8
1135.1
2074.7
2097.0
340, 607 (sh), 641 (sh), 665, 698
5
6
1b
1b
2b
2c
1:1.7
1:1.7
7
8
THF
THF
344, 610 (sh), 639 (sh), 669, 696
344, 610 (sh), 638 (sh), 670, 694
70
30
30
10
c
7
1c
2a
1:3
9
DMF
1261.6
1283.6
1270.6
2209.7
1261.7
1283.7
1270.9
2209.9
354, 613 (sh), 680
50
54
11
c
8
9
1c
1c
2b
2c
1:3
1:3
10
11
DMF
352, 614 (sh), 681
355, 614 (sh), 681
16
2
c
DMF
20
a
b
Conditions: TGR resin (1.5−3 equiv), Pc (1 equiv), Pd(OAc)2 (50 mol %, (o-tolyl)3P (150 mol ), DIPEA (15 equiv), rt, 2 d. It should be noted
c
that the Pc 1a−1c were obtained via statistical condensation and consequently are composed of mixtures of positional isomers. The reaction was run
d
e
f
for 1 d at 80 °C. Average mass values obtained by MALDI-TOF spectroscopy. Mass value is given for [M + H]+ adduct. THF was used as a
g
solvent. The conversion of Pc to Pc−peptide conjugate was estimated from the amount of Pc left in the solution and calculated based on the molar
amount of starting Pc.
bound peptide via solid-phase Sonogashira coupling [Pd-
(PPh3)2Cl2 (10 mol %), PPh3 (20 mol %), CuI (20 mol %)],29
the desired product 3 was formed in moderate ∼25% yield
along with the product of Glazer coupling as a major
compound (Scheme 1).
the reaction is indeed sensitive to water. For the best result, the
reaction should be performed under anhydrous conditions and
inert atmosphere, with special attention being paid to careful
degassing all the solvents and reagents. The quality of solvent
used in the reaction has been observed to play an important
role as well; in all the reactions described herein, anhydrous
amine-free solvent was employed. It should also be noted that
in all the reactions the recuperated phthalocyanine was the
reduction product and not the starting iodinated phthalocya-
nine.
To examine the general applicability of our approach, we
performed a series of experiments employing different peptides
and phthalocyanines (Table 1). The coupling of neutral 1a and
1b with solid-supported peptides was carried out at room
temperature for two days, while all the reactions of 1c with
peptides were performed at 80 °C for one day. Generally,
peptide was taken in excess to achieve higher conversion of Pc
to an appropriate conjugate.
tert-Butyl-substituted ZnPc 1a was successfully coupled with
the on-bead model YVG−peptide 2a and the RGD−peptide 2b
to give the corresponding conjugates 3 and 4 in moderate 30−
35% yields (Table 1, entries 1, 2). The coupling in DMF of the
solid-supported BBN 2c and 1a gave only trace amount of the
desired product, whereas the reaction in THF resulted in 50%
conversion of 1a to the Pc−BBN conjugate 5, which was
isolated in 10% yield (Table 1, entry 3). Similarly, the cross-
coupling of metal-free 1b with the resin-bound tripeptides 2a
and 2b gave the corresponding conjugates 6 and 7 in moderate
This result was somewhat surprising as less favorable
interference between alkynes linked to the solid support was
expected. Thus it is well-known that under the conditions of
solid-phase Sonogashira coupling, the oxidative dimerization of
alkyne is almost avoided when the acetylenic reactant is
covalently bound to the polymer.36 To confirm our result, a test
experiment was performed: the reaction was repeated using the
same conditions but omitting the addition of Pc. After one day
at room temperature, only a few percent of starting peptide
remained whereas the diyne appeared as a major compound.
Considering these results, copper-free Sonogashira coupling
conditions37 were then tried. Reacting 1.5 equiv of 1a with the
on-bead 2a in the presence of Pd(OAc)2, a bulky, electron-rich
tri(o-tolyl)phosphine [(o-tolyl)3P] and N,N-diisopropylethyl-
amine [DIPEA] in N,N-dimethylformamide (DMF) resulted in
the formation of the desired product in 60% crude yield,
meaning that only 40% of Pc was converted into the conjugate
3. We subsequently inverted the Pc:peptide ratio and
performed the reaction of 1a with 2 equiv of solid-supported
2a, resulting in 80% conversion of Pc into conjugate 3. We also
performed the same reaction in 5% v/v aqueous/DMF solution
to test the sensitivity of the reaction to water. This resulted in a
decrease of Pc conversion to desired conjugate, confirming that
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Table 2. Synthesis of Water-Soluble Pc−Peptide Conjugates 9, 11, and Organo-soluble Conjugate 3 via the Sonogashira Cross-
a
Coupling in Solution
b
T
[°C]
time
[h]
conversion
[%]
yield
[%]
entry Pc peptide product
catalyst
base
solvent
1
2
3
1c
1c
1c
12a
12a
12a
9
9
9
5 mol % Pd(OAc)2/25 mol % TPPTS
50 mM NaOAc buffer,
rt
24
traces
pH 5.5
10 mol % Pd(OAc)2, 30 mol % TXPTS, 10
mol % CuI
Et3N
1:1 H2O: CH3CN
DMF
80
70
1
85
50 mol % Pd(OAc)2, 150 mol % (o-tolyl)3P
EtN(i-
Pr)2
1
90
70
4
5
6
1c
1c
1c
12b
12b
12b
11
11
11
50 mol % Pd(OAc)2, 150 mol % TXPTS, 5
mol % CuI
Et3N
1:1 H2O: CH3CN
1:1 H2O: DMF
DMF
80
80
70
24
12
1
traces
65
50 mol % Pd(OAc)2, 150 mol % TXPTS, 5
mol % CuI
EtN(i-
Pr)2
50 mol % Pd(OAc)2, 150 mol % (o-tolyl)3P
EtN(i-
Pr)2
75
50
60
b
c
7
1a
12a
3
50 mol % Pd(OAc)2, 150 mol % (o-tolyl)3P
EtN(i-
Pr)2
DMF
70
1
quant
a
b
Reactions were run under conditions given in the table. Conversion of Pc to the cross-coupled product was estimated from HPLC profile of the
c
reaction mixture, unless otherwise stated. Conversion was estimated by TLC (SiO2, CH2Cl2:MeOH 10:1).
30% yields (Table 1, entries 4, 5), while the Pc−BBN conjugate
8 was isolated in 10% yield (Table 1, entry 9). The reactions of
the metal-free 1b with the peptides 2a and 2b were carried out
in tetrahydrofuran (THF) because of the low solubility of 1b in
DMF. However, when the anionic 1c was subjected to copper-
free Sonogashira coupling with the solid-supported 2a in DMF
at room temperature for two days, only trace amounts of the
desired conjugate 9 were formed, together with the
homocoupling product. But, when the reaction was heated at
80 °C for one day, the desired product 9 was isolated in 11%
yield (Table 1, entry 10). Similarly, the reaction of 1c with the
on-resin RGD−peptide 2b at elevated temperature gave the
target conjugate 10 in 16% (Table 1, entry 11). However, when
trying to couple the on-bead bombesin 2c with 1c under
optimized conditions, the desired conjugate 11 was isolated in
only 2% yield (Table 1, entry 12). We speculate that the rather
low isolated yields of the coupling products 9−11 considering
the quite high estimated conversion of Pc to the conjugates
might by caused by instability of the TentaGel support at
elevated temperatures. Indeed, Hutchins and Chapman, when
describing Fisher indole synthesis on a solid support, reported
that at higher temperatures the yields began to decline and
impurities related to the PEG grafted solid support became
problematic.38
conjugates from starting peptides and/or peptide homodimers
can be performed rather easily via two-step reversed-phase
high-performance liquid chromatography (RP-HPLC). At first,
peptides are eluted with a 0−100% linear gradient of CH3CN
in water (containing 0.05% TFA) in 20 min while Pc−peptide
conjugates remain on the column. Afterward, tert-butyl
substituted Pc−peptide conjugates are eluted with THF
whereas sulfonated derivatives are eluted with a linear gradient
of 0−100% MeOH in phosphate buffer (10 mM, pH 5) in 40
min.39
Generally, the yields of the Pc−peptides conjugated with
shorter peptides such as YVG and RGD are higher (30−35%
for tert-butyl substituted Pcs, 11−16% for sulfonated Pc) than
those with longer peptide BBN (6−14) (10% for tert-butyl
substituted PcS, 2% for sulfonated Pc). The coupling of
sulfonated Pc 1c with on-bead peptides is not favored at room
temperature and requires elevated temperatures. However, the
instability of the solid support at elevated temperatures gives
rise to rather low isolated yields of the desired products. The
yields can be likely improved by substitution of TGR resin for
another solid support with higher thermal stability.
Synthesis in Solution. An alternative strategy that we
explored for the synthesis of Pc−peptide conjugates was based
on Sonogashira coupling of unprotected acetylenic peptide and
iodinated Pc in solution. It has been previously demonstrated
that Sonogashira cross-coupling on proteins allows the
regioselective linking of iodo-aryl and alkyne moieties because
these groups are orthogonal in their reactivity to almost all
other functional groups in proteins.40 Bong and Ghadiri41
reported the efficient chemoselective Pd(0)-catalyzed Sonoga-
shira coupling of synthetic peptides of 17- and 33-residue
Although the yields of the Pc−peptide conjugates obtained
via solid-phase synthesis are relatively moderate, the method
offers an advantage of rather ease of product purification. After
the reaction is completed, the reduced product, catalyst, and
base are removed by simple filtration followed by a cleavage of
reaction products from the resin with the TFA:H2O:thioanisole
(92:2:6, v/v/v) cocktail. The purification of the Pc−peptide
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length bearing iodo-phenyl moieties to a trialkyne in water
employing catalyst derived from Pd(OAc)2 and commercially
available trisodium tri(3-sulfonatophenyl)phosphane (TPPTS).
The authors showed that peptides containing free amines,
carboxylates, guanidines, hydroxyls, and thiolesters were
efficiently coupled to a trialkyne, whereas the presence of free
thiols, thioethers, and bipyridyl moieties was not tolerated. In
our preliminary experiments, model peptide 12a was allowed to
react with water-soluble trisulfonated Pc 1c in 1:1.1 ratio under
the reported conditions (Table 2, entry 1). In contrast to our
expectations, only traces of the desired conjugate 9 were
detected using MALDI-TOF spectroscopy even after prolong-
ing the reaction for one day, while the bulk of the starting
peptide remained unreacted. More recently, Cho and co-
workers reported markedly higher activity of the catalyst
derived from more bulky trisodium tri(2,4-dimethyl-5-
sulfonatophenyl)phosphane (TXPTS) compared to catalyst
formed from TPPTS in the Sonogashira coupling of nucleo-
sides.42 They found that the reaction was the most efficient
when stirring alkyne and halonucleoside in 2:1 ratio in the
presence of 10 mol % Pd(OAc)2, 10 mol % CuI, 30 mol %
TXPTS, and 1 equiv of triethylamine in 1:1 H2O:CH3CN at 80
°C for 1 h. The same authors noted that at least one equivalent
of base was required for the reaction albeit higher
concentrations of base did not affect the catalyst performance.
We employed the reported conditions (Table 2, entry 2) for
the coupling of 12a with 1c in 1.5:1 ratio. The reaction was
complete within 1 h, and the formation of the cross-coupled
product 9 was confirmed by MALDI-TOF spectroscopy and
analysis of RP-HPLC profile of the reaction mixture at 223 and
675 nm. When we employed the copper-free Sonogashira
coupling conditions for the same reaction, a similar reaction
course was observed. Compound 1c was allowed to react with
the peptide 12a in DMF at 70 °C in the presence of Pd(OAc)2,
(o-tolyl)3P, and DIPEA (Table 2, entry 3). Monitoring the
reaction showed the clean formation of the cross-coupled
product 3 in 90% yield within 1 h (Supporting Information
Figure S41). However, when acetylenic bombesin 12b was
subjected to a reaction with water-soluble 1c in the presence of
10 mol % Pd(OAc)2, 10 mol % CuI, 30 mol % TXPTS, and 5
equiv of triethylamine in 1:1 H2O:CH3CN, almost no 1c
consumption was observed even after 24 h of stirring.
Increasing the catalyst loading (50 mol % Pd(OAc)2 and 150
mol % TXPTS) gave only traces of the desired conjugate 11
along with starting Pc, while a peak of the starting peptide
completely disappeared in the HPLC trace of the reaction
mixture (Table 2, entry 4). This failure can probably be
attributed to some heterogeneity of the reaction; BBN 12b is
not sufficiently soluble in the reaction mixture. Using DMF
instead of acetonitrile drastically changed the course of the
reaction; the desired product was formed in ∼65% in the
reaction mixture when 12b was allowed to react with 1c in 1:1
H2O:DMF in the presence of 50 mol % Pd(OAc)2, 5 mol %
CuI, 150 mol % TXPTS, and 5 equiv of triethylamine, at 80 °C
overnight (Table 2, entry 5). The copper-free Sonogashira
conditions afforded the desired conjugate 11 in ∼75% crude
yield along with the reduced product after the reaction was
stirred at 70 °C for 1 h (Table 2, entry 6, Supporting
Information Figure S48). Similarly, the organo-soluble
conjugate 3 was easily prepared from 1a and 12a in good
yield using the optimized copper-free conditions (Table 2,
entry 7). The purification of water-soluble conjugates 9 and 11
was achieved via two-step RP-HPLC: at first the unreacted
peptide and/or peptide homodimer was eluted with a 0−100%
linear gradient of CH3CN in water (containing 0.05% TFA) in
20 min while Pc−peptide conjugates remained on the column.
Then a linear gradient of 0−100% MeOH in phosphate buffer
(10 mM, pH 5.0) in 40 min was employed in order to purify
the target compound from the reduction product and catalyst.
Following this procedure, the desired conjugates 9 and 11 were
isolated in ∼70 and ∼50% yields, respectively. The organo-
soluble conjugate 3 was purified by flash chromatography on
silica using 1−10% gradient of MeOH in CH2Cl2. In summary,
we have demonstrated that both Sonogashira coupling on solid
phase and in solution are feasible routes to Pc−peptide
conjugates.
Cell Lines. Cell lines used in these studies express different
levels of the GRPR and integrin receptors, including the
estrogen-independent human breast cancer cell line MDA-MB-
231, human lung adenocarcinoma A549, androgen-independent
human prostate cancer PC-3, and murine mammary carcinoma
EMT-6. The expression of the integrin αv by MDA-MB-231,
PC-3, and A549 cells is well documented using various standard
methods43−45 but has not been reported for EMT-6 cells. With
regard to the expression of subtype integrins αvβ1, αvβ3, αvβ6,
and αvβ5 by MDA-MB-231 cells, reports are controversial. Thus
in some publications, MDA-MB-231 expresses high integrin
αvβ3 receptor levels and undetectable amounts of GRPR.46
Other studies support the expression of αvβ5 and αvβ1 but
detect no significant levels of αvβ3 and αvβ6.44 GRPR levels in
MDA-MB-231 are detected in moderate levels, comparable to
those found in PC3 cells.47 A549 cells express very low levels of
GRPR48 but high levels of integrin αv, in particular αvβ1, αvβ5
receptors that exert a high binding affinity for the RGD motif.43
The PC-3 prostate cancer cell line has been used extensively as
in vitro and in vivo models to evaluate the receptor-mediated
uptake of fluorescent or radiolabeled BBN conjugates.49 This
cell line expresses high levels of GRPR that are 1000× higher
than those of the integrin αvβ3 receptor density (e.g., 2.70 × 106
vs 2.76 × 103 receptors/cell).50 Literature values for surface
expression of α2, α3, α6, β1, and β4 integrin subunits, and mRNA
expression of α6, β1, and β4 by PC-3 cells, were established by
flow cytometry and Northern blot analysis, respectively.51
Finally, EMT-6 murine mammary carcinoma cells were selected
as a control because they express very little integrin αvβ3.52−54
To verify the content of α3, αvβ3, αvβ5, and GRPR by the
selected cell lines, we investigated their expression by Western
blot (Supporting Information Figure S45). Their concen-
trations were calculated relative to the β-actin concentration
using Image J software (Table 3). Our results show that all the
four cell lines express high levels of integrin α3. The integrin/β-
Table 3. Receptor Concentration Relative to β-Actin as
Determined by Western Blot
cell lines
a
receptors
PC3
EMT-6
A549
MDA-MB-231
GRPR glycosylated
2.88
1.78
1.57
0.79
0.15
0.60
0.92
1.04
0.61
0.10
0.10
0.09
1.42
1.53
2.82
0.91
0.13
1.60
0.29
1.35
1.63
1.05
0.92
1.08
GRPR endogenous
α3
αv
β3
β5
a
GRPR or integrin concentrations relative to β-actin as determinate by
Image J software analysis.
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Figure 1. Structures of the reference phthalocyanines.
Table 4. Cell Uptake and Phototoxicity of ZnPc Derivatives
a
b
c
d
cell lines
PC3
ZnPc
LD50 (J cm−2
)
cell uptake (nmol Pc/mg protein)
cells killed per photon absorbed (%)
ZnPcS3C5−COOH (13)
ZnPc−BBN (11)
ZnPc−RGD (10)
ZnPc−YVG (9)
ZnPcS2 (14)
9.31 0.60
1.81 0.20
16.16 0.74
9.83 0.76
0.52 0.03
2.20 0.28
9.19 0.64
0.38 0.06
0.78 0.12
0.26 0.04
1.93 0.29
4.76 0.71
2.82 0.36
0.35 0.05
8.47
98.25
4.58
0.00
66.44
59.08
8.25
ZnPcS3C6 (16)
ZnPcS3 (15)
A549
ZnPcS3C5−COOH (13)
ZnPc−BBN (11)
ZnPc−RGD (10)
ZnPc−YVG (9)
ZnPcS2 (14)
15.49 1.84
3.70 0.46
26.45 2.44
7.76 0.38
0.52 0.03
2.30 0.24
8.71 0.77
0.66 0.10
0.61 0.09
0.47 0.07
0.70 0.11
1.84 0.27
2.53 0.38
0.69 0.10
3.3
1.9
0.0
0.0
91.3
37.7
1.6
ZnPcS3C6 (16)
ZnPcS3 (15)
MDA-MB-231
ZnPcS3C5−COOH (13)
ZnPc−BBN (11)
ZnPc−RGD (10)
ZnPc−YVG (9)
ZnPcS2 (14)
7.95 0.37
2.78 0.17
10.21 1.00
9.59 0.94
0.24 0.08
0.91 0.06
5.52 0.42
0.33 0.05
0.53 0.08
0.41 0.06
0.01 0.00
0.53 0.08
0.20 0.03
1.00 0.15
21.22
36.86
22.17
0.00
50.81
50.46
28.87
ZnPcS3C6 (16)
ZnPcS3 (15)
EMT-6
ZnPcS3C5−COOH (13)
ZnPc−BBN (11)
ZnPc−RGD (10)
ZnPc−YVG (9)
ZnPcS2 (14)
8.43 0.69
4.48 0.51
16.13 1.97
5.33 0.60
0.69 0.08
2.24 0.27
8.62 0.47
0.73 0.11
0.91 0.14
0.59 0.09
1.11 0.17
3.72 0.56
1.02 0.15
0.00
7.40
0.00
0.00
93.41
25.44
37.17
ZnPcS3C6 (16)
ZnPcS3 (15)
1.58 0.24
a
b
The relative concentrations of GRPR and integrin receptors are summarized in Table 4. Tumor cells were incubated with the various ZnPc
derivatives (2 μM) for 24 h prior to PDT. LD50 values represent the fluence required for 50% cell survival, calculated from the survival curves
c
presented in Supporting Information (Figure S52). Tumor cells were incubated with the various ZnPc derivatives (2 μM) for 24 h prior to
d
extraction. The % of cells killed/photon absorbed were calculated from cell survival after exposure to 3 J·cm−2 and by taking into account cell
uptake and absorbance of the different conjugates.55
actin ratios derived from the Western blots indicate that the
relative level of integrin expression respects the following order:
MDA-MB-431 ≈ A549 > PC-3 > EMT-6 (Table 3). The
expression of αv was detected in all cell lines, although in very
low levels in EMT-6. Integrin αvβ3 levels were detected only in
MDA-MB-231 cells. None of the other three cell lines tested
expressed substantial levels of this receptor, including the A549
line, which confirms reported data.43 In contrast, αvβ5
expression was shown to be substantially higher in A549 cells
as compared to levels found in MDA-MB-231 cells, while low
levels were detected in PC-3 cells and none in EMT-6 cells.
GRPR endogenous was expressed by all four cell lines (PC-3 ≥
A549 > MDA-MB-231 ≫ EMT-6), but the GRPR glycosylated
form was absent in MDA-MB-231 cells while present in the
other three cell lines (PC-3 ≫ A549 > EMT-6).
Photocytotoxicity and Cellular Uptake. The cellular
uptake results for the water-soluble ZnPc−peptide conjugates
9−11, as well as the reference phthalocyanines, e.g., the
carboxylated analogue ZnPcS3C5COOH (13), disulfonated
ZnPcS2 (14), trisulfonated ZnPcS3 (15), and ZnPcS3C6 (16)
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■
▲
Figure 2. Comparison of LD50 ( ) and cell uptake of Pc−peptide conjugates after 24 h incubation ( ) with different cell lines vs retention times on
a C18 reversed-phase HPLC column. Retention times and n-octanol/water partition coefficients of substituted PcS show a linear correlation10 and
can be used as a measure of the relative hydrophobicity of the PcS and their peptide conjugates. Retention times of the conjugates and reference Pc
are: 27.4 min, ZnPc−RGD (10); 29.0 min, ZnPcS3C5−COOH (13); 29.8 min, ZnPc−YVG (9); 31.6 min, ZnPc−BBN (11) and 34.2 min,
ZnPcS3C6 (16).
(Figure 1) by four different tumor cell lines after 24 h
incubation with 2 μM PS, together with the light dose required
for 50% cell survival (LD50), are summarized in Table 4.
The average LD50s, together with the standard deviation,
were estimated with Prism graph software from the survival
curves presented as Supporting Information (Figure S52). The
phototoxicity is also expressed as number of cells killed per
photon absorbed (Table 4) by calculating cell survival after
exposure to 3 J·cm−2 and by taking into account cell uptake and
absorbance of the different conjugates. To illustrate a possible
correlation between the hydrophobicity of the Pc−peptide
conjugates and phototoxicity, the LD50 and cell uptake values
were plotted against the HPLC retention times on a reversed
phase column (Figure 2).
We have previously shown that the photocytotoxicity of
sulfonated ZnPc depends on cellular uptake and intracellular
localization, which in turn correlates to the overall hydro-
phobicity of the derivatives.9,10 Using the retention time (tR) on
a reversed-phase HPLC column as a measure of relative
hydrophobicity, the three ZnPc−peptide conjugates 9−11,
when compared to the nonconjugated ZnPcS3C5−COOH (13)
and ZnPcS3C6 (16) analogues, are shown likewise to obey this
structure−activity relationship (SAR) in all four cell lines tested
(Figure 2). Thus the most hydrophilic RGD conjugate 10 (tR =
27.4 min) is the least phototoxic (e.g., highest LD50) in each
cell line, coinciding with the lowest level of cell uptake. Only in
the MDA-231 cell line, this correlation is less evident, which is
largely due to the relative low uptake of all PS tested in this cell
line. The photocytotoxicity decreases with decrease in hydro-
phobicity as observed for the other cell lines. The highest
cellular uptake paired with low LD50 is observed with the
nonconjugated ZnPcS3C6 (16) parent compound (Figure 2).
However, when expressing photocytotoxicity at a fixed light
dose as the percentage of cells killed per photon absorbed, a
new SAR emerges, indicative of the involvement of a receptor
binding parameter in the overall PDT response (Table 4).
Thus, the amphiphilic, nonconjugated ZnPcS2 (14) and
ZnPcS3C6 (16) as well as the hydrophilic ZnPcS3 (15) show
high percentages of cell kill in the receptor-poor EMT-6 cells
while all ZnPc−peptide conjugates (9, 10 and 11) as well as
ZnPcS3C5−COOH (13) exhibit strongly reduced activity. In
contrast, in both GRPR-rich PC3 and MDA-231 cell lines, the
ZnPc−BBN conjugate 11 shows a high percentage cell kill.
Likewise, the ZnPc−RGD conjugate 10 shows a high
percentage of cell kill in the integrin-rich MDA-231 cell line,
while this conjugate is inactive in EMT-6 cells that lack this
receptor. The RGD-specific integrin receptor (αvβ3) is highly
expressed in the MDA-231 cell line, while PC3 and A549 cells
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Figure 3. Inhibition of PDT response by BBN and RGD. Left graph: PDT response with ZnPc−BBN (11) as PS and BBN as inhibitor. Right graph:
PDT response with ZnPc−RGD (10) as PS and RGD as inhibitor. Cell lines: in EMT-6(blue diamonds), A549 (red triangles), PC3 (black squares),
and MDA-MB-231 (green circles).
Figure 4. Effect of preincubation with 1 nM BBN on cell survival of EMT-6, A549, PC3, and MDA-MB-231 cells after PDT (12 J·cm−2) using
ZnPc−BBN (11) and several nonconjugated reference Pc as photosensitizers: ZnPcS3C5COOH (13), ZnPcS2 (14), ZnPcS3 (15), and ZnPcS3C6
(16).
express only αvβ5 receptors that exhibit lower affinity for the
RGD peptide (Table 3).43 This correlates with the high cell kill
observed with the RGD conjugate 10 in MDA-231 cells (Table
4). Furthermore, the highest percentage cell kill (98%) was
observed with the BBN conjugate 11 in the case of the PC-3
prostate cancer cell line, while the RGD conjugate 10 showed
low activity in this cell line. Conjugation of the ZnPc with YVG
(9) reduced the PDT efficacy substantially, as indicated by the
negligible number of cells killed per photon absorbed for all
four cell lines studied (Table 4). These data also confirm the
nonspecificity of compound 9 for either the GRPR or integrin
receptors.
Inhibiting PDT Efficacy with BBN or RGD. The
involvement of a receptor-mediated photodynamic response
using the RGD conjugate 10 and the BBN conjugate 11 on the
different cell lines was evaluated by preincubating the cells with
either free RGD or BBN prior to the photoinactivation assay.
For this assay, a light dose of 12 J·cm−2 was selected to ensure
adequate cell inactivation with all four cell lines. Figure 3 shows
the effect of BBN or RGD on the photodynamic activity of
ZnPc−BBN (11) (left panel) and ZnPc−RGD (10) (right
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Figure 5. Confocal fluorescence microscopy images of selected MDA-MB-231 (left panels) and PC3 (right panels) cells after 24 h incubation with
ZnPc−RGD (10) (top panels, red fluorescence) and ZnPc−BBN (11) (lower panels, red fluorescence), and various tracker dyes (blue and green
fluorescence), including TMADPH for plasmic and endocytic membranes (A), Lyso-Tracker Green for lysosomes (B), Myto-Tracker Green for
mitochondria (C), NBDC6 Ceramide for golgi (D), and DAPI for the nucleus (E). Colocalization of ZnPc and tracker dye is presented as white
fluorescence.
panel), respectively. The PDT inhibition shows a reversed
sigmoid curve indicative of a receptor-blocking, cell type-
dependent relationship. The concentration of BBN required to
block 50% of the maximum PDT response (IC50) for each cell
line increases in the following order: EMT-6 (4.0 0.1 pM) ≪
A549 ∼ PC3 (1.2 0.2 nM) < MDA-MB-231 (53 5 nM).
The order differs slightly from that observed for the GRP
receptor content, where MDA-MB-231 express lower GRPR
levels as compared to PC3 or A549 cells (Table 3). This
confirms that BBN can interact with other receptors expressed
by these cell lines and/or that nonspecific cell uptake is
involved. In the case of RGD, no effect was observed on the
EMT-6 cell line that only expresses negligible levels of αvβ3 and
αvβ5 receptors (Table 3). In contrast, we observed 50%
decrease in PDT response for the RGD receptor positive cell
lines A549 and MDA-MB231 (Table 3) at similar RGD
concentration (1.56 0.33 and 1.88 0.29 nM, respectively).
In the case of the ZnPc−RGD (10), using the receptor-poor
EMT-6 cells, no RGD inhibition of PDT response could be
observed, even at the maximum concentration of 0.5 μM used
in these experiments (Figure 3, right graph). With the MDA-
MB-231 and A549 cell lines that both express the RGD
receptor, PDT response could be inhibited up to 34% and 22%,
respectively, with IC50 RGD concentrations of 1.6 0.7 nM
(the IC50 is defined as the concentration inhibiting 50% of PDT
response). In the case of the bombesin analogue ZnPc−BBN
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Figure 6. PC3 and MDA-MB-231 cellular uptake visualized by confocal fluorescence microscopy. Effect of preincubation: (A) with 1 nM BBN on
ZnPc−BBN (11) uptake, (B) with 1 nM RGD on ZnPc−RGD (10) uptake.
(11), PDT efficacy could be inhibited in all four cell lines by the
addition of free BBN, e.g., up to 52% with the GRPR-rich PC3
cells (IC50 = 1.4 0.1 nM), 48% with the MDA-MB-231 cells
(IC50 = 53 4.9 nM), 40% with the A549 cells (IC50 = 1.0
0.3 nM), and 35% with the EMT-6 cells (IC50 ∼ 4 0.1 pM)
(Figure 3, left graph). The lower IC50 value observed with the
EMT-6 cells likely reflects the presence of low concentrations
of the GRPR, in accordance with the receptor levels detected
by our Western blot analysis (Table 3). EMT-6 cell growth
inhibition by vaccine that specifically targets GRPR also
suggests the presents of low levels of this receptor.56
The role of the BBN receptor in the PDT response obtained
with ZnPc−BBN (11) was verified on all four cell lines by
comparing the latter’s activity with that of nonconjugated
analogues (Figure 1), e.g., ZnPcS2 (14), ZnPcS3 (15),
ZnPcS3C6 (16), and ZnPcS3C5COOH (13), with and without
addition of 1 nM BBN (Figure 4). Prior incubation with free
BBN only affected cell survival after PDT in the case of
conjugate 11, confirming the involvement of a substantial
receptor-mediated component in the PDT response induced by
this new ZnPc−BBN conjugate. Our data also indicate that all
four cell lines express the GRPR to some extent and that the
level of BBN inhibition correlates to the level of BBN receptor
expression by each cell line (Table 3). These data also exclude a
possible involvement of free BBN on nonspecific cell uptake of
the nonconjugated ZnPc.
Intracellular Distribution. The intracellular localization of
ZnPc−RGD (10) and ZnPc−BBN (11) was studied in the two
receptor-positive PC3 and MDA-MB-231 cell lines (Figure 5).
The extent of colocalization with various tracker dyes was
estimated by superimposing fluorescence images with those of
the following tracker dyes: DAPI for the nucleus, Myto-Tracker
Green for mitochondria, Lyso-Tracker Green for lysosomes,
NBDC6 Ceramide for Golgi, and TMADPH for membranes.
The presence of the ZnPc−peptide conjugates 10 and 11 is
evident from their red fluorescence, while tracker dyes are
detected by their intense green or blue fluorescence.
Colocalization is presented as white coloration as determined
with FluoView software. Both the ZnPc−RGD (10) and
ZnPc−BBN (11) conjugates localize at various intracellular
organelles and membrane structures in a similar pattern as
previously demonstrated for the nonconjugated sulfonated
ZnPcS3C6 (16).10 This latter study showed that 16 mainly
localized in the cytoplasm, e.g., mitochondria, Golgi, and
lysosomes, while almost excluded from the cell nucleus.
Peptide conjugation of ZnPcS3C6 (16) increases plasmic and
perinuclear membrane localization, reflecting cell uptake by
integrin and GRP receptor-mediated processes, while local-
ization at the mitochondria, Golgi, and lysosomes reveals that
part of the cellular uptake involves passive diffusion and
nonspecific endocytosis.
The receptor mediated cellular uptake of the RGD and BBN
conjugates 10 and 11 is also evident from blocking experiments
(Figure 6). These images show that preincubation of the
receptor-rich PC3 and MDA-MB-231 cells (Table 3) with
RGD or BBN reduces the red fluorescence from the ZnPc−
peptide conjugates. This reduction in intracellular localization is
particularly strong in the case of BBN with the ZnPc−BBN
(11) conjugate. The images also reveal that the cell uptake of
the RGD−peptide conjugate (10) is substantially lower as
compared to that of the BBN analogue 11, which likely reflects
the higher hydrophilicity of the former (Figure 2).
CONCLUSION
■
The synthesis of a series of Pc−peptide conjugates was achieved
via the solid-phase Sonogashira cross-coupling in moderate
isolated yields for organo-soluble (∼10−35%) and low to
moderate yields for water-soluble analogues (∼2−15%).
Although the water-soluble conjugates were obtained in
relatively low yields using solid-phase strategy, they were
successfully prepared in rather high yields using an alternative
approach, which also works well for organo-soluble analogues.
In this approach, an iodinated water-soluble ZnPc was allowed
to react with acetylenic model tripeptide and unprotected
bombesin in solution to give corresponding conjugates in 70
and 50% isolated yields, compared to 11 and 2%, respectively,
when using SPOS. The ZnPc−RGD conjugate 10 showed
receptor binding character in photodynamic activity in vitro
blocking studies, whereas its PDT efficacy was rather low,
which could possibly be improved by conjugating the ZnPc to
multimeric RGD peptide. Thus it has been shown that receptor
binding characteristics of dimeric and multimeric RGD peptides
are more advantageous than those of the monomeric RGD
peptide.57,58 In the case of the water-soluble ZnPc−BBN
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in 20% piperidine in dimethylformamide (DMF). At the final step, 6-
heptynoic acid was introduced at the N-terminal position after peptide
assembly. The on-bead acetylenic peptides 2a−2c thus obtained were
used directly for the solid-phase Sonogashira cross-coupling with the
iodinated phthalocyanines 1a−1c. Purities of the resin-bound peptides
were assessed by analytical reverse-phase HPLC after cleavage of ∼10
mg of corresponding resin with the same cleavage cocktail, and their
identities were confirmed using ESI mass spectrometry.
In parallel, the resin-bound 2a and 2c were treated with a cocktail of
TFA:H2O:thioanisole (92:2:6, v/v/v) at room temperature for 3 h to
afford unprotected acetylenic peptides 12a and 12b, respectively. The
crude peptides 12a and 12b were precipitated with cold diethyl ether
(Et2O) and then purified via reverse-phase HPLC on a semi-
preparative C18 column using a linear gradient of 20−100%
acetonitrile in water with 0.05% TFA in 30 min. The isolated peptides
12a and 12b were then freeze-dried and stored at −20 °C. Their
purities were found to be >95% (analytical reverse-phase HPLC), and
their structures were confirmed by ESI mass spectrometry as well as by
1H and 13C NMR for tripeptide 12a.
Synthesis of Iodinated Phthalocyanines. Monoiodo tri-tert-Bu-
ZnPc 1a1 and monoiodo trisulfonated ZnPc 1c2 were prepared
following previously described procedures. The trisulfonated metal-
free analogue 1b was prepared from 1a using a recently reported
demetalation procedure3 for ZnPc. Zinc phthalocyanine 1a (50 mg,
0.06 mmol) was stirred in pyridine (5 mL), and pyridine−HCl (1.5 g)
under argon at 110 °C for 17 h. After evaporation of the volatiles, the
dark-green residue was redissolved in dichloromethane and extracted
three times with water to remove pyridinium chloride salt. Then
organic fraction was dried over anhydrous magnesium sulfate, filtered,
and concentrated in vacuo. The resulting dark-blue residue was further
purified by passing it down a short silica gel column (elution with 10%
dioxane in hexane). The appropriate fraction was pooled and freed of
solvent under reduced pressure to furnish the pure 1b as dark-blue
crystals (40 mg, 81%). HPLC profiles of the starting Pc and the Pc−
peptide conjugates comprised of several peaks representing structural
isomers because the statistical condensation route was used to obtain
monoiodinated unsymmetrical zinc phthalocyanines 1a and 1c.
General Procedure for Sonogashira Cross-Coupling of Solid-
Supported Acetylenic Peptides and Iodinated Pc. A reaction vial was
charged with a solid-supported acetylenic peptide (25.0 mg, assumed
∼0.24 mmol g−1), Pd(OAc)2 (0.34 mg, 50 mol %), (o-tolyl)3P (1.18
mg, 150 mol %), and an iodinated phthalocyanine (0.003 mmol).
Then the reaction vial was evacuated and purged with argon three
times. Subsequently, 300 μL of degassed DMF was added under argon,
followed by addition of DIPEA (8 μL, 15 equiv) degassed beforehand
by gentle bubbling Ar through it for 30 min. After that, the reaction
vial was tightly sealed and the resulting suspension was shaken at room
temperature for 2 d in the case of conjugates 3−8 or gently stirred at
80 °C for 1 d in the case of conjugates 9−11. After cooling, the resin
was separated by filtration and washed with DMF (3 × 5 mL) and/or
THF (3 × 5 mL), CH2Cl2 (3 × 5 mL), and MeOH (3 × 5 mL) until
the filtrate runs through colorless. The resin was then dried and
treated with TFA−thioanisol−H2O (92:6:2 v/v/v) for 4 h. Following
filtration, the crude product was precipitated with Et2O. The resulting
solid was purified via two-step semipreparative RP-HPLC: at first the
unreacted peptide and/or peptide homodimer was eluted with a 0−
100% linear gradient of CH3CN in water (containing 0.05% TFA) in
20 min while Pc−peptide conjugates were not eluted under these
conditions. Afterward the tert-butyl substituted Pc−peptide conjugate
3−8 were eluted with THF while sulfonated Pc−peptide conjugates
9−11 were eluted with a linear gradient of 0−100% MeOH in
phosphate buffer (10 mM, pH 5.0) in 40 min.
conjugate 11, our data show its potential to function as both a
fluorescence imaging probe and receptor-targeting photo-
dynamic agent. These results warrant further in vivo studies
to evaluate the potential of the Pc−bombesin conjugate for
PDT of prostate, breast, and lung cancer that overexpress these
receptors. Also, selective localization in these tumors would
render the conjugates good candidates for PET imaging via
substitution of the central Zn ion for positron emitting 64Cu or
67Cu isotopes.59
EXPERIMENTAL SECTION
■
Synthesis. General Procedures. Unless otherwise stated, all
reactions were carried out under argon atmosphere. All chemicals
and solvents were used as supplied without further purification. 1H and
13C NMR spectra were recorded on a Bruker Spectrospin 300
instrument in DMSO-d6 with the chemical shifts reported as δ in ppm
and coupling constants expressed in Hz. The following abbreviations
were used to describe spin multiplicity: s = singlet, d = doublet, t =
triplet, q = quintet, dd = doublet of doublets, m = multiplet. LC−
electrospray ionization (ESI+) mass spectra of the peptides were
obtained with a Waters Micromass ZQ single-quadrupole mass
spectrometer. MALDI-TOF mass spectra of the phthalocyanines
(Pc), and the Pc−peptide conjugates were acquired in linear mode
using a Micromass Tof Spec2E spectrometer, with α-cyano-4-
hydroxycinnamic acid as the matrix (positive mode). Visible
absorption spectra were recorded with Beckman DU 530 spectropho-
tometer. Analytical reverse-phase HPLC of the peptides 2a−2c, 12a,
and 12b, the water-soluble Pc 1c and 13−16, and Pc−peptide
conjugates 9−11 was performed on an Agilent 1200 system equipped
with an Agilent Eclipse XDB-C18 reverse phase column (4.6 mm ×
250 mm, 5 μ) and an Agilent 1200 series diode array UV/vis detector
at a flow rate of 1 mL/min. Linear gradient of 0−100% acetonitrile in
water (0.05% TFA) over 30 min was used for the peptides, whereas a
linear gradient of 0−100% MeOH in phosphate buffer (10 mM, pH
5.0) over 40 min was employed for sulfonated Pc−peptide conjugates.
Analytical reverse-phase HPLC of tert-butyl substituted Pc 1a−1b and
Pc−peptide conjugates 3−8 was carried out on a Hewlett-Packard
1050 system equipped with a Phenomenex Luna C18 reverse phase
column (10 mm × 250 mm, 5 μ) at a flow rate of 2 mL/min using
isocratic elution with 65% aqueous THF as a mobile phase.
Purification of peptides 12a and 12b was performed on the
Hewlett-Packard 1050 system equipped with the Phenomenex Luna
C18 semipreparative reverse phase column (10 mm × 250 mm, 5 μ) at
a flow rate of 2 mL/min using linear gradient of 20−100% acetonitrile
in water with 0.05% TFA in 30 min. Conjugates 3−11 were purified
via two-step semipreparative reverse phase HPLC on the Hewlett-
Packard 1050 system equipped with the Phenomenex Luna C18
reverse phase column (10 mm × 250 mm, 5 μ) at a flow rate of 2 mL/
min. Linear gradient of 0−100% acetonitrile in water with 0.05% TFA
in 20 min was first employed in order to eliminate peptides (Pc−
peptide conjugates are not eluted in these conditions) followed by
isocratic elution with THF for tert-butyl substituted Pc−peptide
conjugates 3−8 or a linear gradient of 0−100% MeOH in phosphate
buffer (10 mM, pH 5.0) in 40 min for sulfonated conjugates 9−11.
Silica gel column chromatography was carried out using 40_63 μm
silica gel. All tested compounds were purified until at least 95% purity
as judged by reverse phase HPLC. The identity of all the new
compounds was confirmed by MALDI-TOF or ESI mass-spectrom-
etry, UV−vis spectroscopy, as well as by 1H and 13C NMR for
tripeptide 2a.
Peptides Functionalized with Terminal Triple Bond. All peptides
were synthesized manually using the Fmoc (fluorenylmethyloxycar-
bonyl) strategy and NovaSyn TGR resin. A 2.5-fold excess of Fmoc-
protected amino acids over resin substitution rate was utilized for
coupling. The amino acids were activated with an equimolar amount of
HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) and 2-fold excess of N,N-diisopropylethylamine
(DIPEA). After each coupling step, Fmoc deprotection was performed
General Procedure for Sonogashira Cross-Coupling of Unpro-
tected Acetylenic Peptides and Iodinated Pcs in Solution. Pd(OAc)2
(0.68 mg, 50 mol %), (o-tolyl)3P (2.36 mg, 150 mol %), an iodinated
phthalocyanine (0.006 mmol), and an acetylenic unprotected peptide
(0.009 mmol, 1.5-fold excess) were assembled in a round-bottomed
flask sealed with a septum. The reaction flask was evacuated and
purged with argon three times. Subsequently, 500 μL of degassed
DMF was added under argon, followed by addition of DIPEA (16 μL,
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15 equiv) degassed beforehand by gentle bubbling Ar through it for 30
min. The reaction mixture was stirred at 70 °C until RP-HPLC
showed complete conversion (0.5−1 h). The solvent was evaporated
and the crude product was purified via two-step semipreparative RP-
HPLC as described above.
software (Wayne Rasband, NIH, USA). The protein intensity was
determinate by intensity multiply by protein band area.
Cellular Uptake. Tumor cells (1.5 × 105 cells per well) in 1 mL of
their specific growth medium were incubated in 24-well plates
(Falcon) overnight at 37 °C under 5% CO2. The medium was
removed, and the monolayer was rinsed twice with 1% PBS and then
overlaid with 500 μL of the dye solution (2 μM) in a combination
medium prepared from equal amounts of the four specific growth
media supplemented with 4% FBS. After 24 h incubation, the dye
solution was removed and the cells were washed twice with 1% PBS.
The cells were lysed with 500 μL of 0.1 N NaOH. An aliquot (50 μL)
was removed to determine cellular protein according to the Bradford
method (Bio-Rad protein assay).61 DMF (450 μL) and 1% PBS (100
μL) were added to the remaining cell lysate, the mixture was
centrifuged for 30 min at 4 °C, 3500 rpm, and dye concentration in
the clear supernatant was assayed by fluorescence spectroscopy (F-
2000, Hitachi) (λex 608 nm, 5 nm band-pass, λem 680 nm, 10 nm band-
pass). Standard curves were obtained using cell lysates treated as above
with known amounts of the appropriate Pc solution. The cellular ZnPc
concentration is expressed as nmole of Pc per mg of cellular protein.
Cell Photoinactivation. Cell cultures were trypsinated to give a 1.5
× 105 cells/mL suspension of which 100 μL per well were plated in 96-
well plates and incubated overnight at 37 °C under 5% CO2. Attached
cells were rinsed twice with PBS and incubated for 24 h with 50 μL of
dye solution (2 μM) in a combination medium prepared from equal
amounts of the four specific growth media supplemented with 4%
FBS. One column did not receive cells to serve as a blank, while a
second control column was filled with dye-free medium only. The cells
were then rinsed twice with PBS, reefed with 100 μL of specific culture
medium, and exposed for varying time intervals to red light (10
mW·cm−2 at 660−700 nm). For illumination, we used a homemade
device consisting of two 500 W tungsten−halogen lamps fitted with an
aqueous rhodamine B (OD580 = 1.25) (Sigma, Canada) filter
connected to a cold water bath. The emitted light intensity was
calibrated by a photometer (model LI-185B, LI-COR Biosciences inc.
Nebraska USA). Plates were incubated overnight at 37 °C under 5%
CO2. Cell survival was measured by a colorimetric method, using the
tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide) (Sigma). Eight replicates were run, and experiments
were repeated at least three times. The survival curves were plotted as
a function of light dose and LD50 values were calculated.
Reference Phthalocyanines. ZnPc 13 was prepared from ZnPc 1c
under the copper-free Sonogashira cross-coupling conditions. 1c (40
mg, 0.04 mmol) was reacted with 5-fold excess of 5-hexynoic acid (22
mg, 0.2 mmol) in the presence of 50 mol % Pd(OAc)2 (4.4 mg, 0.02
mmol)/100 mol % (o-tolyl)3P (12.2 mg, 0.04 mmol) and 15 equiv of
DIPEA (0.6 mmol, 104 μL) in anhydrous DMF (3 mL) at 70 °C
overnight. Then solvent was evaporated under reduced pressure, the
residue was redissolved in minimal amount of water, and Pc was
precipitated by adding an excess of ethanol. The blue precipitate was
filtered off, washed on the filter with ethanol, and dried to give the
desired product 13 as dark-blue powder (29 mg, 73%). The structures
of 1b and 13 were confirmed by MALDI-TOF mass-spectrometry and
UV−vis spectroscopy. The purity of 13 was estimated by analytical
HPLC on a C18 reverse phase column using a linear gradient of 0−
100% MeOH in phosphate buffer (5 mM, pH 5.0) in 40 min and
found to be greater than 95%. MALDI-TOF MS (m/z): [M+•] calcd
for C38H22N8O11S3Zn, 928.23, found 928.47; UV−vis λmax (DMF):
680, 613 (sh), 354; HPLC (676 nm): three close peaks, tR 27.8−30.2
min. Three other reference compounds, e.g., ZnPcS2 (14), ZnPcS3
(15),4 and ZnPcS3C6 (16)2 were prepared as previously described.
Biology. Cell Cultures. All cell lines were purchased from American
Type Culture Collection (ATCC, Manassas, VA, USA) and
maintained in T75 flasks (Falcon, Mississauga, Canada) in specific
media at 37 °C, under 5% CO2 and 95% air. All culture media were
supplemented with 1% L-glutamine, 1% penicillin−streptomycin
(GIBCO, Burlington, Canada). The cell lines used in these studies
include the murine mammary EMT-6 (ATCC CRL-2755), human
breast adenocarcinoma MDA-MB-231 (ATCC HTB-26), human lung
adenocarcinoma A549 (ATCC CCL-185), and human prostate cancer
PC3 (ATCC CRL-1435). EMT-6 cells were maintained in Waymouth
MB 752/1 medium fortified with 15% fetal bovine serum (FBS),
MDA-MB-231 cells in EMEM with 10% FBS, A549 in RPMI 1640
with 10% fetal calf serum, and PC3 in HAM F12K with 10% fetal calf
serum. The cells were transferred periodically following treatment with
trypsin (0.05%).
Western Blot Analysis.60 The relative concentrations of integrin
and GRP receptors in the cell lines were established by Western blot
analysis. Briefly, cells were washed with PBS (3 times) and total
protein was extracted (10 mM Tris base-HCl, 150 mM NaCl, 1% NP-
40, 1% Triton X-100, 5 mM EDTA, 0.1% SDS, 1% sodium
deoxicholate, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin,
1 μg/mL pepstatin, 1 μg/mL leupeptin, 1% sodium orthonovadate,
and 50 mM sodium fluoride). Cellular debris was cleared by
centrifugation, and supernatants (lysates) were aliquoted and stored
at −20 °C. The concentration of lysates was determined by the
Bradford method (Bio-Rad protein assay).61 Briefly, the protein
extracts were applied on a 4−20% mini-protean TGX Stain Free
Precast gels at 160 V during 1 h and transferred to a PVDF membrane
(Millipore, Bedford, MA, USA) using the Mini Trans-Blot Cell (Bio-
Rad) settled at 100 V for 1 h. In all cases, the membrane was blocked
during 2 h at room temperature with 5% reconstituted skim milk
powder in TBST solution (10 mM Tris-HCl pH 7.5 containing 150
mM NaCl and 0.05% Tween 20). Membranes were incubated
overnight at 4 °C under continuous agitation with primary antibodies
β-actin, sc-47778, integrin α3, sc-374242 and GRPR(F6), SC377316
(Santa Cruz Biotechnology, CA, USA), integrin αvβ5, MAB 1961
(Millipore, MA, USA), and integrin αvβ3, CD61 (Biosciences, ON,
Canada) and washed 3 times with TBS-T. Then they were incubated
with secondary antibodies goat antimouse IgG-HRP, sc-2005 (Santa
Cruz Biotechnology, CA, USA) for 2 h at room temperature.
Detection of immunoreactive bands was performed using the
enhanced chemiluminescence (Plus-ECL) detection kit (PerkinElmer
Inc., MA, USA).62 The protein levels that corresponded to the
immunoreactive bands were quantified using the Image J analysis
PDT-Inhibition with BBN and RGD. PDT-inhibition assays were
performed on all four tumor cell lines detailed in the cell culture
section. Cell cultures were trypsinated to give a 1.5 × 105 cells/mL
suspension, of which 100 μL per well were incubated in 96-well plates
overnight at 37 °C under 5% CO2. Attached cells were rinsed twice
with PBS and incubated for 24 h with 50 μL of 2 μM ZnPc−RGD
(10) or ZnPc−BBN (11) in a mixture of the four culture media (25%
each), supplemented with 4% FBS. One row did not receive cells to
serve as a blank, while a second control row was filled with dye-free
medium only. Two hours before PS addition, the other rows received
50 μL/well of BBN or RGD in increasing concentrations from 10−12 to
10−5 M. The plates were incubated for 24 h at 37 °C, where after the
incubation medium was removed, cells were washed three times with
PBS, reefed with 100 μL of specific culture medium, and exposed for
20 min to red light (10 mW·cm−2, 12 J·cm−2, at 660−700 nm). Plates
were incubated overnight at 37 °C under 5% CO2. Cell survival was
measured by the MTT test. Eight replicates were run, and experiments
were repeated at least three times. Finally, data were analyzed with
GraphPad Prism 5 software to determine the IC50 value, e.g., the BBN
or RGD concentration required to reduce the PDT response by 50%.
A similar PDT inhibition protocol was used to determine the effect of
BBN on 2 μM nonconjugated ZnPc (13−16). To allow a significant
PDT response, inhibitor concentration (BBN or RGD) and light dose
were fixed at 1 nM and 12 J·cm−2, respectively.
Intracellular Distribution. The PC3 and MDA-MB-231 cell
suspensions (104 cells/mL) were plated onto 12 mm diameter glass
coverslips (Fisher Scientific) placed in 24-well plates and incubated for
18−24 h at 37 °C, under 5% CO2, to allow for cell adhesion. The
medium was removed, and the cells were rinsed twice with PBS and
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then overlaid with 0.1 mL of the ZnPc−peptide solution (10 μM) in
their specific medium culture supplemented with 1% FBS. After 18 h
at 37 °C, the solution was removed and cells were washed twice with
PBS and twice with HBSS/HEPES buffer. The cells were incubated at
37 °C under 5% CO2 with different tracker dyes (Invitrogen Inc.,
Ontario, Canada) including Myto-Tracker green (150 nM, 45 min),
Lyso-Tracker Green (200 nM, 1 h), TMA-DPH or 1-[4-
(methylamino)phenyl]-6-phenylhexa-1,3,5-triene (5 μM, 1 h), and
NBDC6 Ceramide (5 μM, 1 h). Only the latter dye needed to be
rinsed three times, after which the cells were incubated for another 30
min. After the last incubation, all samples were washed twice with PBS.
For TMA-DPH staining, with and without ZnPc−peptide conjugates
(10 and 11), slides were mounted for microscopic observations using
Vectashield mounting media (Vector Laboratories, Peterborough,
U.K.). The Vectashield including DAPI mounting media (Vector
Laboratories, Peterborough, U.K.) was used for all other staining.
For blocking studies, PC3 and MDA-MB-231 suspensions (104
cells/mL) were plated onto 12 mm diameter glass coverslips (Fisher
Scientific) placed in 24-well plates and incubated for 18−24 h at 37
°C, under 5% CO2, to allow for cell adhesion. The medium was
removed, and the cells were rinsed twice with PBS and then overlaid
with 0.1 mL of new specific culture medium, with or without 1 nM
final concentration of specific inhibitor (BBN or RGD). After 2 h, the
medium was removed, and the cells were rinsed twice with PBS and
then replaced by 0.1 mL of ZnPc−RGD (10) or ZnPc−BBN (11) (10
μM), or their specific medium culture alone, supplemented with 1%
FBS in the corresponding well (e.g., BBN, RGD, or blank). After 18 h
at 37 °C, the solution was removed and the cells were washed twice
with PBS and twice more with HBSS/HEPES buffer. The cells were
mounted for microscopic observations with Vectashield mounting
medium.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +1 819 564 5409. E-mail: Johan.E.vanLier@
USherbrooke.ca (J.E.v.L.); Brigitte.Guerin2@USherbrooke.ca
(B.G.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
J.E.v.L. and B.G. are members of the Fonds de la Recherche en
́
Sante
́
du Queb
́
ec-supported CRC Etienne-Le Bel of the Centre
Hospitalier Universitaire de Sherbrooke, Sherbrooke, QC,
Canada. This work was supported by an NSERC Strategic
́
Projects Grant (Canada) and the Jeanne and J.-Louis Levesque
Chair in Radiobiology.
ABBREVIATIONS USED
■
PDT, photodynamic therapy; BBN, bombesin; GRP, gastrin-
releasing peptide; GRPR, gastrin-releasing peptide receptor;
RGD, arginine−glycine−aspartic acid, integrin binding motif;
YVG, tyrosine−valine−glycine, nonspecific peptide; αvβ3, RGD
binding integrin; αvβ5, RGD binding integrin; mAbs,
monoclonal antibodies; PS, photosensitizer; Pc, phthalocya-
nines; PcS, sulfonated phthalocyanines; Fmoc, fluorenylmethy-
loxycarbonyl; LD50, fluence required for 50% cell survival; IC50,
BBN or RGD concentration required for 50% inhibition of
PDT response; SPOS, solid-phase organic synthesis
Confocal Microscopic Analysis. Cells were examined with a
scanning confocal microscope (FV1000, Olympus, Tokyo, Japan)
coupled to an inverted microscope with a 63× oil immersion objective
(Olympus). Specimens were laser-excited at 405 nm (DAPI and TMA-
DPH), 488 nm (Myto-Tracker green, Lyso-Tracker Green and
NBDC6 Ceramide), or 633 nm (ZnPc), and the fluorescence was
collected with FL1 (blue, 440−470 nm), FL2 (green, 500−530 nm),
and FL3 (red, >640 nm), respectively. Serial horizontal optical sections
of 512 × 512 pixels with two times line averaging were taken at 0.4 μm
intervals through the entire thickness of the cell (optical resolution:
lateral −0.2 μm, axial −0.8 μm). Images were acquired during the
same day, typically from five cells of similar size, from each
experimental condition using identical settings of the instrument.
For merged fluorescence images the dots fluorograms were obtained
by plotting pixel values of each component toward horizontal and
vertical axis, respectively. Quadrant markers were placed forming
background (C), red-only (D), green-only (A), and colocalization
areas (B). Colocalization index were calculated as (B)/(B + D), and %
of colocalization as (B)/(B + D) × 100.63 Quantitative analysis was
performed on five size-matched cells for each experimental condition,
and experiments were performed three times. For illustration
purposes, images were contrast enhanced, pseudocolored accordingly
to their original fluorochromes, merged (FluoView software,
Olympus), and then cropped and assembled (Adobe Photoshop
software, Mountain View, CA).
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S
* Supporting Information
General synthetic procedures and analytical data for 1b, 13,
1
2a−2c, 3−11, 12a, and 12b, including copies of the H NMR,
13C NMR, UV−vis, MALDI-TOF, ESI-MS spectra, and HPLC
traces, cell survival curves, and Western blots. This material is
available free of charge via the Internet at http://pubs.acs.org.
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