Synthesis of new zinc(II) phthalocyanine conjugates with block copolymers for cancer therapy

Article abstract of DOI:10.24820/ark.5550190.p009.764

Synthetic routes towards new conjugates of hydrophilic zinc(II) phthalocyanine (ZnPc) with poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic P123) and poly(L-lactide) (PLLA), are described. The main semiproduct ZnPc was obtained by heating 4-nitrophthalimide with urea and zinc chloride, followed by the reduction step. Steglich esterification was used to synthesize two ZnPc-conjugated block copolymers, further utilized in fabrication of polymeric micelles (PMs) - functionalized with the zinc(II) phthalocyanine-type moiety. Biological evaluation of the PMs indicated an acceptable biocompatibility level in accord with requirements in the field of nanotheranostics and nanomedicine.

Full text of DOI:10.24820/ark.5550190.p009.764

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Organic Chemistry
Arkivoc 2017, part ii, 433-445
Synthesis of new zinc(II) phthalocyanine conjugates with block copolymers
for cancer therapy
Łukasz Lamch,a Marta Tsirigotis-Maniecka,a Julita Kulbacka,b Kazimiera A. Wilka*
a Department of Organic and Pharmaceutical Technology, Faculty of Chemistry,
Wroclaw University of Science and Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland
b Department of Medical Biochemistry, Medical University of Wrocław, Chałubińskiego 10,
50-368 Wrocław, Poland
E-mail: kazimiera.wilk@pwr.edu.pl
Dedicated to Prof. Jacek Młochowski on the occasion of his 80th anniversary
Received 06-27-2016
Accepted 10-23-2016
Published on line 12-14-2016
Abstract
Synthetic routes towards new conjugates of hydrophilic zinc(II) phthalocyanine (ZnPc) with poly(ethylene
oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic P123) and poly(L-lactide) (PLLA), are
described. The main semiproduct ZnPc was obtained by heating 4-nitrophthalimide with urea and zinc
chloride, followed by the reduction step. Steglich esterification was used to synthesize two ZnPc-conjugated
block copolymers, further utilized in fabrication of polymeric micelles (PMs) - functionalized with the zinc(II)
phthalocyanine-type moiety. Biological evaluation of the PMs indicated an acceptable biocompatibility level in
accord with requirements in the field of nanotheranostics and nanomedicine.
R
HN
O
+ Pluronic P123
3 steps
=
N
NH
P123
NH
R
Steglich
Steglich
N
N
N
N
O
amidation
esterification
Zn
N
N
R
N
+ PLLA
R
NH
PLLA
HO CH2CH2O
H
90
2 steps
NH
O
O
C
C
CH2
O
CH2CH2O CH2
90
OH
R=
Keywords: ZnPc-conjugated block copolymers; cyclotetramerization; Steglich esterification; fluorescent
polymeric micelles; diagnostic marker; in vitro biological evaluation
DOI: https://doi.org/10.24820/ark.5550190.p009.764
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Introduction
One of the most interesting groups of porphyrin-like compounds, considering physicochemical properties, are
the zinc(II) phthalocyanines - light-sensitive molecules, known for their exceptional stability and light
absorption properties in the red/near IR region (ε may exceed 105 M-1 cm-1 for the Q band, often located at
650-700 nm).1 These compounds comprise not only a variety of components of biosensors in imaging bio-
applications, but also are one of the most promising second generation photosensitizers for photodynamic
therapy of cancers.2-4 Moreover, phthalocyanines exhibit a high fluorescence quantum yield and their physical
and photochemical properties can be easily modified by exchange of peripheral groups.5
The principal limitation of most of the zinc(II) phthalocyanine-type derivatives, used as photosensitizers or
fluorescent markers, is their low solubility, even in organic solvents, their instability, and tendency to
aggregate in water solutions, which severely limits their potential application in the physiological
environment.6 To overcome these drawbacks, hydrophilic groups such as sulfonate,7,8 phosphonate,9
saccharide,10 carboxylate10-12 or tertiary amine13-15 groups can be introduced into the molecules.
Unfortunately, those zinc(II) phthalocyanine derivatives may aggregate in aqueous systems, resulting in loss of
absorbance, fluorescence and photoactivity.16 That is why the present study has been focused on the
synthesis and characterization of poly(ethylene oxide)-modified zinc(II) phthalocyanine, a nonionic and water-
soluble compound, with its application as a fluorescent marker for cancer therapy.
Generally, the synthesis of substituted phthalocyanines is a multistep process, including preparation of the
appropriate phthalimide or phthalonitrile derivative, their further high-temperature condensation
(cyclotetramerization) to obtain the phthalocyanine ring and eventually peripheral group exchange, if needed.
Amino-substituted phthalocyanines cannot be synthesized by self-condensation of substrates containing
amino groups, due to interference by the amino groups in the cyclotetramerization process.17 To solve this
problem, two synthetic routes are usually employed. One approach is that tetranitrophthalocyanines are
prepared in the first step, followed by reduction to amino-derivatives. The other is that the amino group in the
phthalonitrile or phthalimide derivative is protected by acyl or tosyl groups before the substrate is
cyclotetramerized, the product then hydrolyzed to the tetraaminophthalocyanine. The first method needs
relatively fewer reaction steps, so is usually preferred.18
A convenient starting material for zinc phthalocyanine possessing peripheral amine groups is zinc
tetranitrophthalocyanine, obtained upon heating 4-nitrophthalimide with an appropriate catalyst, metal salt
(e.g. zinc chloride or acetate) and urea. The latter derivative can be easily reduced to zinc tetraamino-
phthalocyanine utilizing sodium sulfide18 or hydrazine.17 An additional advantage of this procedure is that
4-nitrophthalimide is conveniently prepared by nitration of phthalimide.19
Poly(ethylene oxide), a nonionic compound commonly used to enhance water solubility or dispersability of
many substances, can be attached to zinc tetraaminophthalocyanine by two approaches: zinc
tetraaminophthalocyanine transformation to zinc tetra(N-carbonylacrylic)aminophthalocyanine utilizing
reaction with maleic anhydride,18 followed by coupling with poly(ethylene glycol) or synthesis of carboxyl
group terminated poly(ethylene glycol) that can react directly with zinc tetraaminophthalocyanine. Both
synthetic routes can be conducted utilizing Steglich esterification or its amide modification – a general method
of preparation of labile esters or amides under very mild conditions.20 By this approach it is also possible to
avoid product degradation in the ZnPc-conjugated block copolymers.
To overcome the poor solubility, target specificity and difficulties in tracking of anticancer compounds in
tissue systems, many carriers, such as oil-core polyelectrolyte nanocarriers, solid lipid nanoparticles, polymer
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conjugates, polymeric nanoparticles and micelles, have been developed.21-25 Polymeric micelles are
nanoscopic structures (hydrodynamic diameter is often below 100 nm) generated by the self-assembly of
amphiphilic block copolymers in water in a concentration exceeding the critical micelle concentration. Such
structures have attracted much attention as nanocarriers of anticancer drugs due to the possibility of
modifying their hydrophilic corona with ligands or fluorescence markers.5
In order to evaluate application properties of ZnPc as a fluorescent agent, we have synthesized new
conjugates with two block copolymers, which were further utilized to prepare functionalized polymeric
micelles and characterized by physical and biological methods. Polymeric nanostructures, in the form of the
above-mentioned micelles, have attracted our attention as delivery vehicles in cancer therapy and diagnostics,
due to their ability to selectively accumulate within the tumor tissue with vestigial or no uptake by non-target
cells without the loss of their activity. Thus, the ZnPc-functionalized polymeric micelles may constitute a
promising addition to the inventory of nanotheranostics and to monitor the efficiency of chemotherapy.
Results and Discussion
Synthetic studies
In order to prepare a water soluble zinc(II) phthalocyanine (ZnPc), the most appropriate starting materials
were compounds which it was possible to synthesize in convenient reactions: carboxyl group terminated
poly(ethylene glycol) 1 and 4-nitrophthalimide 2, as well as commercially available urea and zinc chloride
hexahydrate. The carboxyl group terminated poly(ethylene glycol) 1 was synthesized in a multistep reaction as
outlined in Scheme 1, starting from commercially available dihydroxy-terminated poly(ethylene oxide). The
1
resulting compound 1 analyses performed by H NMR and FT-IR spectroscopies were in a good agreement
with the structurally similar compounds obtained from slightly different methods by Niculescu-Duvaz et al.26
4-Nitrophthalimide 2 was synthesized by standard, low temperature nitration, starting from the readily-
available phthalimide (Scheme 2). After recrystallization, the purity and identity of the compound 2 was
1
confirmed by a very sharp melting point (194.5 – 195 °C) and H NMR spectroscopy, fully consistent with
literature data.27 The yield obtained of compound 2 was only slightly lower than that reported by Young et al.
(61% in comparison to 70%).27
CH3
CH3CH2 C O- K+
CH3
-
K+
HO CH2CH2O H
+ O CH2CH2O CH2CH2O-
K
90
89
O
O
O
O
O
BrCH2 C OCH2CH3
NaOH
CH3CH2O C CH2 O CH2CH2O CH2 C OCH2CH3
HO C CH2 O CH2CH2O CH2 C OH
HCl
90
90
1
Scheme 1. Synthesis of carboxyl terminated poly(ethylene glycol) 1.
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Lamch, Ł. et al
O2N
NO2
N
Zn
N
.
O
O
CO(NH2)2, ZnCl2 2H2O
N
N
N
N
O2N
HNO3/H2SO4
(NH4)2MoO4 (cat.)
N
NH
N
NH
O
O
O2N
NO2
2
3
R
H2N
HN
NH2
NH
R
N
Zn
N
N
Zn
N
O
O
HO C CH2 O CH2CH2O CH2
C OH
N
N
N
N
N
Na2S.9H2O
90
N
N
N
N
DCC, NHS, DMAP (cat.)
N
N
N
R
H2N
HN
NH2
NH
R
O
O
C CH2 O CH2CH2O CH2 C OH
R=
90
5
4
Scheme 2. Synthesis of zinc(II) phthalocyanine hydrophilic derivative (ZnPc) 5.
The synthesis of zinc tetraaminophthalocyanine 4 was accomplished via a two-step reaction with 4-
nitrophthalimide 2, urea and zinc chloride hexahydrate as starting materials in presence of ammonium
molybdate as the catalyst. The obtained zinc tetranitrophthalocyanine 3, was converted into zinc
tetraaminophthalocyanine 4 by reduction with sodium sulfide. Strong absorptions at 3330 and 3206 cm-1 in
the FT-IR spectrum of 4 were attributed to stretching vibrations of peripheral amine groups, while a strong
absorption at 1604 cm-1 was contributed to their bending vibrations. Generally, peaks observed in the FT-IR
spectra of compound 4 were nearly the same as for very similar nickel tetraaminophthalocyanine, described
by Pavaskar et al.18 The chemical identity of the obtained zinc tetraaminophthalocyanine 4 was additionally
confirmed by 1H NMR analysis, which revealed signals from aromatic protons at 8.05-9.65 ppm confirming the
presence of aromatic moieties characteristic for phthalocyanine structures. The desired compound was
obtained in very good yield (over 90%), exceeding the yields of similar nickel tetraaminophthalocyanine (about
61%), synthesized by Pavaskar et al..18
For further synthesis of poly(ethylene oxide) modified zinc(II) phthalocyanine 5, employment of a tetra (N-
carbonylacrylic) aminophthalocyanine derivative was initially considered, because esterification of the
carboxylic acid group with standard poly(ethylene glycol) is an approved and easy method of its PEG-ylation.
According to Pavaskar et al.18 a tetra (N-carbonylacrylic) aminophthalocyanine derivative has been synthesized
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by zinc tetraaminophthalocyanine reaction with maleic anhydride, and coupled with poly(ethylene oxide) by
Steglich esterification. Unfortunately the obtained product was scarcely soluble in water, which made it
impossible to apply it as a hydrophilic fluorescent marker, although introducing a carboxyl group into zinc
tetraaminophthalocyanine is easier than into poly(ethylene oxide). For this reason, the product of carboxylic
acid terminated poly(ethylene glycol) and zinc tetraaminophthalocyanine coupling, which was more difficult to
obtain, was synthesized and used further in conjugation with block copolymers.
1
Signals observed in the H NMR spectra at 8.05-9.65 ppm from CH protons of the obtained poly(ethylene
oxide) modified zinc(II) phthalocyanine 5 and both ZnPc-conjugated block copolymers revealed that these
compounds contain aromatic rings, and was confirmed by characteristic bands observed in the FT-IR spectrum
1
at about 730-744 cm-1, attributed to C-C and C-H bonds present also in aromatic moieties. Moreover in H
NMR spectra recorded for poly(ethylene oxide) modified zinc(II) phthalocyanine, performed in D2O, complete
structural resolution of the whole molecule was possible, due to presence of both PEG (3.67 ppm and 4.15
ppm for end groups) and aromatic (8.05-9.65 ppm) protons with proper integration, while the fluorescence
emission (λex 354 nm) spectrum of its aqueous solution contained a characteristic band with the maximum at
about λ 430 nm (Figure S1). These results clearly show that the obtained compound is molecularly dissolved in
aqueous solution with no sign of aggregation. According to our investigations, the obtained ZnPc-conjugated
block copolymers are good candidates for preparation of polymeric micelles with functionalized corona.
R
6
HN
CH3
CHCH2O
8
NH
HO CH2CH2O
CH2CH2O H
CH3
CHCH2O
R
20
70
20
N
Zn
N
HO CH2CH2O
CH2CH2O ZnPc
20
70
20
DCC, DMAP (cat.)
N
N
N
N
N
R
N
O
DCC, DMAP (cat.)
O
CH3
O C CH O ZnPc
R
HN
70
CH3
CH3
O C CH OH
9
70
NH
CH3
7
O
O
C CH2 O CH2CH2O CH2 C OH
R=
90
5
Scheme 3. Synthetic routes to Pluronic and PLLA block copolymers conjugated with zinc(II) phthalocyanine
derivatives (ZnPc) 8 and 9.
In order to prepare poly(ethylene oxide) modified zinc(II) phthalocyanine (ZnPc) 5, as well as to prepare
both zinc(II) phthalocyanine modified block copolymers 8 and 9, reactions in Steglich conditions were
necessary, due to the risk of poly(ethylene glycol) derivatives or block copolymers degradation. For
poly(ethylene oxide) modified zinc(II) phthalocyanine (5) reaction with N-hydroxysuccinimide was performed
(Scheme 2), while for both block copolymers – the Steglich esterification with N,N-dicyclohexylcarbodiimide
(Scheme 3) was used. In order to avoid residues of unreacted poly(ethylene oxide) (difficult to remove)
modified zinc(II) phthalocyanine 5, excess of Pluronic P123 ((poly(ethylene oxide)-b-poly(propylene oxide)-b-
poly(ethylene oxide)) 6 or poly(L-lactide) (PLLA) 7 was used in the reaction mixture. Purification of 5, as well as
purification of zinc phthalocyanine modified Pluronic P123 8, involved dialysis to remove excess reagents -
carboxylic terminated poly(ethylene glycol) 1 or Pluronic P123 6, respectively. For zinc phthalocyanine
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modified PLLA 9 excess of polyester was removed by precipitation from THF, followed by filtration. Generally,
both zinc(II) phthalocyanine modified block copolymers 8 and 9 were obtained conveniently in multistep
reactions with high yields (at least 60%) (Scheme 3).
Preparation and characterization of polymeric micelles
Polymeric micelle systems were prepared using two different approaches: the thin film method for Pluronic
micelles (System 1) and modified interfacial polymer deposition – solvent evaporation technique for poly-
(L-lactide)-derived polymeric micelles (System 2), due to different properties of these block copolymers.
Pluronic P123 and its functionalized derivatives 8 are waxy solids or pastes with an ability to self-assemble
o
o
during temperature decrease from about 60-50 C to below 35 C, so the most appropriate and efficient
method for micelle preparation of these is the thin film method. Poly(L-lactide) (PEG-b-PLLA) block copolymers
are semi-crystalline solids and the process of micelle formation is driven by the surface tension on the liquid-
liquid interface, so methods utilizing dropwise addition of cosolvent and/or emulsification are required for
their preparation.
The average size of the obtained polymeric micelles (expressed as hydrodynamic diameter, DH) ranged
from around 15 nm (Pluronic micelles, 1a and 1b) to 89 nm (functionalized PLLA block copolymer micelles, 2b)
(Table 1), and was consistent with our previous studies of Pluronic28 and poly(L-lactide) block copolymer
micelles.24 Moreover, the obtained nanocarriers had a narrow size distribution (PdI < 0.3) (Table 1), which
could make them good candidates as nanocarriers useful for example in cancer treatment. AFM imaging
confirmed the DLS data of the obtained polymeric micelles (Figure S2), since spherical and moderately uniform
particles were observed. The dimensions of the obtained nanosystems are amenable to avoiding clearance by
first pass renal filtration as well as detection by the phagocytic system and consequently they may achieve a
longer circulation time in the bloodstream.
Table 1. Characteristics of the polymeric micelles composed of block copolymers and functionalized with
covalently bound hydrophilic zinc(II) phthalocyanine (ZnPc)
a
Copolymer (mg/ml)
Micelle
system
DH
PdIb
(nm)
Pluronic P123
PLLA
1a
1b
2a
2b
10.2
14
14
24
75
0.16
0.26
0.23
0.09
10.2 (+ZnPc)
4.0 (+PEG)
4.0 (+ZnPc)
a DH hydrodynamic diameter. b PdI polydispersity index.
ZnPc-conjugated polymeric micelles were designed to evaluate their possible role as therapeutic
nanocarriers containing a fluorescent marker, chemically attached to their corona. Fluorescence spectroscopy
was utilized to confirm the presence of the fluorescent marker (ZnPc) and its activity in the studied
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nanosystems (Figure S3). The results obtained clearly indicate that the polymeric micelles studied are good
candidates as multifunctional transport agents, due to their ability to give a fluorescence response.
Biological studies
The results of biological studies are presented in Figure 1. The viability, measured by two independent assays,
indicated that polymeric micelles with and without fluorescent marker (ZnPc) are not cytotoxic to the
melanoma cells (MeWo). There was observed an insignificant decrease of cellular viability for micelle systems
1a and 1b (Figure 1a). Micelle systems 2a and 2b did not exhibit any destructive effect on mitochondrial
activity (Figure 1a) or ATP production (Figure 1b). The evaluated functionalized micellar systems induced
activity of caspase 3/7 on the level of control cells activities (Figure 1c). The results obtained indicate that
micelles functionalized with ZnPc are safe for in vitro study and did not affect crucial cellular parameters.
Additionally, an intracellular distribution of PMs (system 2b) in MeWo cells was shown after 24 hours
incubation (Figure 1d), as we could observe ZnPc localized mainly in the cytoplasm of melanoma cells.
Figure 1. The evaluation of biological effect of functionalized micelles measured after 24 hours of incubation in
MeWo cells: a. cellular viability measured by MTT assay; b. cellular viability measured by fluorescent ATP
assay; c. activity of caspase-3/7 measured by luminescent assay; d. intracellular distribution of PMs (system
2b) after 24h of incubation. See descriptions for the systems 1a-b and 2a-b in Table 1.
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The results obtained indicate that polymeric micelles based on Pluronic 123 and PLLA conjugates with
fluorescent marker (ZnPc) are safe and the nanosystems do not cause cell death. Those functionalized
polymeric micelle systems may act as an effective drug delivery system. Our previous study on polymeric
micelles loaded with hydrophobic zinc(II) phthalocyanine indicated that these micelles can be effective
nanocarriers in photodynamic reactions in metastatic melanoma cells (Me45). Additionally, the mentioned
micelles, both empty and loaded with anticancer cargo, appear to be haemato-biocompatible and safe for
normal keratinocytes, macrophages and endothelial cells.24
Conclusion
The present contribution is focused on the synthesis and characterization of hydrophilic derivatives of zinc(II)
phthalocyanine conjugates with Pluronic P123 and PLLA. The hydrophilic ZnPc was prepared conveniently in a
multistep process including cyclotetramerization of 4-nitrophthalimide, reduction of the tetranitrophthalo-
cyanine and coupling with previously synthesized carboxy-terminated poly(ethylene oxide). For the synthesis
of two different ZnPc-conjugated block copolymers, Steglich esterification was utilized giving the desired
products in good yields. The fabricated polymeric micelles, grafted with the zinc(II) phthalocyanine-type
function, exhibited good physical stability and appropriate size (less than about 100 nm) combined with low
polydispersity indexes (PdI < 0.3). Biological investigation of the nanocarriers obtained, including MTT assay
cytotoxicity in human melanoma cells (MeWo) as well as the proapoptotic potential by the detection of
caspase 3/7 activity, confirmed a lack of their cytotoxicity, while intracellular distribution – localization of PMs
(system 2b) in cytoplasm. These results indicate the ZnPc-functionalized polymeric micelles constitute a
promising addition to the inventory in cancer therapy and diagnostics, and additionally to monitor the
efficiency of chemotherapy.
Experimental Section
Chemistry
General. 1H NMR spectra were recorded on Bruker AMX600 spectrophotometer (Rheinstetten, Germany) and
chemical shifts were referenced to TMS signal as an external standard (in CDCl3), while IR spectra on a Fourier
transform, Bruker VERTEX 70 V vacuum spectrometer (Rheinstetten, Germany) with samples placed on the
diamond crystal of the ATR accessory. Melting points were determined on a Boetius melting point apparatus
and have not been corrected. Chemicals used were purchased from Sigma-Aldrich (including deuterated
solvents), Fluka (N,N'-dicyclohexylcarbodimide, ethyl bromoacetate, potassium), Avantor Performace
Materials (solvents and urea), Schuchart (tert-amyl alcohol) and Akina, inc (methoxypoly(ethylene oxide)-b-
poly(L-lactide) block copolymer, PEG-b-PLLA) were of analytical grade. tert-Amyl alcohol was dried over CaH2
and distilled before use, while other solvents and reagents were used as received.
Synthetic procedure for carboxyl terminated poly(ethylene glycol) (1). Potassium (2.40 g, 60 mmol) was
dissolved in tert-amyl alcohol (50 mL) under a protecting atmosphere of nitrogen. After complete dissolution
of the potassium, poly(ethylene glycol) (PEG) (12.00 g, 3 mmol, MW 4 000) was added and the mixture stirred
at 50 °C for 8 h in a nitrogen atmosphere. Ethyl bromoacetate (6.64 mL, 60 mmol) was added dropwise, then
stirring at 50 °C was continued for 24 h. After completion of reaction, excess tert-amyl alcohol was
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evaporated. The residue was dissolved in 0.1 M NaOH (70 mL) and stirred for 8 h at ambient temperature.
After adjusting the pH to 3 with 0.1 M HCl, the reaction mixture was extracted with CH2Cl2 (3 20 mL), dried
×
over anhydrous MgSO4, filtered and evaporated in vacuum. The residue was dissolved in minimal amount of
CH2Cl2 and precipitated from cold Et2O to obtain 1.
White solid, yield 90%, 10.07 g; IR (film, νmax, cm-1): 2882 (C-H, stretching), 1749 (C=O, stretching), 1466 (CH2,
scissoring), 1359 and 1341 (CH2, wagging), 1279 (CH2, twisting), 1104 (C-O-C, twisting), 958 (C-O-C, vibration
1
and CH2, rocking), 841 (CH2, rocking), 529 (C-O-C, bending). H NMR (600 MHz, CDCl3): δH 3.65 (360H, s,
(OCH2CH2)90), 4.15 (4H, s, 2xOCH2COOH).
4-Nitrophthalimide (2). To fuming, 100% HNO3 (20 mL), conc H2SO4 (80 mL) was slowly added and the mixture
cooled in an ice bath. To the obtained mixture phthalimide (15 g, 0.1 mol) was added in portions over a 15-
min interval with stirring at a temperature under 15 °C, then the temperature was raised to about 35 °C and
held for 45 min. After completion of the reaction the mixture was cooled to 0 °C and slowly added to ice (375
g) at such a rate that the temperature remained below 15 °C. The precipitated crude product was collected by
vacuum filtration, washed with cold water, dried at rt and purified by recrystallization from anhydrous EtOH.
1
Pale yellow crystals, yield 61%, 11.70 g, mp 194.5-195 °C (lit.27 m.p. 194.5-195 °C). H NMR (600 MHz, DMSO-
d6): δH 8.00 (1Harom, d, 2JHH 5.7 Hz), 8.34 (1Harom, s), 8.54 (1Harom, d, 2JHH 6.9 Hz), 11.76 (1H, s, NH).
Zinc tetraaminophthalocyanine (4). 4-Nitrophthalimide (2) (1.95 g, 10 mmol), urea (3 g, 50 mmol), ZnCl2·6H2O
(0.43 g, 2.5 mmol) and ammonium molybdate (0.15 g, 0.76 mmol) were finely ground, transferred into a round
bottom flask and heated to 180 and 200 °C for 3 h under a protecting atmosphere of nitrogen. The obtained
solid was treated with HCl (50 mL, 1 M) followed by NaOH (50 mL, 1 M), ground, washed with water (until Cl-
ions were not present in the filtrate) and dried in vacuum. To the obtained dark green solid (dissolved in N,N-
dimethylformamide (150 mL)) solution sodium sulfide nonahydrate (9 g, 28 mmol), dissolved in minimal
o
amount of water, was added and mixture was stirred for 3 h at 60 C under nitrogen. After completion of the
reaction, solvents were removed under reduced pressure followed by addition of water (100 mL) to the
residues. The product 4 was separated by filtration as a dark green solid and washed several times with
distilled water as well as repeatedly with MeOH/Et2O (1:10, v:v) mixture and EtOAc followed by drying in
vacuum at 60 C.
Dark green solid, yield 91%, 1.45 g; IR (νmax, cm-1): 3330 and 3206 (N-H, stretching), 1604 (N-H, bending), 1492
1
(C-N, stretching), 744 (plane, skeletal), 728 (C-H, out-of-plane, aromatic). H NMR (600 MHz, DMSO-d6): δH
8.05-9.65 (12Harom, m, CH).
Hydrophilic derivative of zinc(II) phthalocyanine (ZnPc) (5). Carboxylic acid terminated poly(ethylene glycol)
(6.44 g, 1.6 mmol) 1, zinc tetraaminophthalocyanine (0.1 g, 0.16 mmol) 4, N,N-dicyclohexylocarbodiimide
(1.29 g, 6.3 mmol), N-hydroxysuccinimide (0.72 g, 6.3 mole) and 4-dimethylaminopyridine (in catalytic
amount) were dissolved in dry THF (50 mL) and stirred in the dark for 48 h. After completion of the reaction
the precipited N,N-dicyclohexylurea was removed by filtration and this was followed by solvent evaporation
under reduced pressure. Water (150 mL) was added followed by solution dialysis (4
8000). Then obtained solution was additionally filtered and freeze-dried.
× 2 L, 3 days, MWCO
Pale green solid, yield 98%, 2.61 g; IR (film, νmax, cm-1): 2882 (C-H, stretching), 1749 (C=O, stretching), 1466
(CH2, scissoring), 1359 and 1341 (CH2, wagging), 1279 (CH2, twisting), 1104 (C-O-C, twisting), 958 (C-O-C,
1
vibration and CH2, rocking), 841 (CH2, rocking), 729 (C-H, out-of-plane, aromatic), 529 (C-O-C, bending). H
NMR (600 MHz, CDCl3): δH 3.65 (1440H, s, (OCH2CH2)90), 4.15 (16H, s, OCH2CO), 8.05-9.65 (12Harom, m, CH).
Zinc phthalocyanine modified Pluronic P123 (8). ZnPc (0.40 g, 0.0235 mmol) 5, Pluronic P123 (1.36 g, 0.047
mmol, MW 5 800) 6, N,N-dicyclohexylocarbodiimide (0.05 g, 0.135 mmol) and 4-dimethylaminepyridine (in
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catalytic amount) were dissolved in dry THF (50 mL) and stirred in the dark for 72 h. After completion of the
reaction, the precipited N,N-dicyclohexylurea was removed by filtration followed by solvent evaporation
under reduced pressure. The obtained solid was dissolved in THF, filtered and solvent was removed under
reduced pressure. To the obtained residue water (100 mL) was added followed by solution dialysis (4
days, MWCO 8 000). The obtained solution was additionally filtered and freeze-dried.
× 2 L, 3
Dark green, waxy solid, yield 86%, 0.545 g; IR (film, νmax, cm-1): 2875 (C-H, stretching), 1727 (C=O, stretching),
1466 (CH2, scissoring), 1360 and 1343 (CH2, wagging), 1280 (CH2, twisting), 1101 (C-O-C, twisting), 963 (C-O-C,
vibration and CH2, rocking), 842 (CH2, rocking), 744 (plane, skeletal), 528 (C-O-C, bending). 1H NMR (600 MHz,
Me2CO-d6): δH 1.24-1.25 (210H, m, (OCH(CH3)CH2)70), 3.40 (70H, m, (OCH(CH3)CH2)70), 3.55 (140H, m,
(OCH(CH3)CH2)70), 3.64 (1600H, s, 4x(OCH2CH2)90 + 2x(OCH2CH2)20), 4.15 (16H, s, OCH2CO), 8.05-9.65 (12Harom
,
m, CH).
Zinc phthalocyanine modified poly(L-lactide) (9). ZnPc (0.4 g, 0.0235 mmol) 5, poly(L-lactide) (0.235 g, 0.047
mmol, MW 5000) 7, N,N-dicyclohexylocarbodiimide (0.05 g, 0.135 mmol) and 4-dimethylaminopyridine (in
catalytic amount) were dissolved in dry THF/CH2Cl2 (50 mL) (1:1, v:v) mixture and stirred at dark for 72 h. After
completion of reaction the precipited N,N-dicyclohexylurea was removed by filtration followed by solvent
evaporation under reduced pressure. The solid obtained was dissolved in THF, filtered and solvent was
removed under reduced pressure.
Pale green, waxy solid, yield 60%, 1.02 g; IR (film, νmax, cm-1): 2887 (C-H, stretching), 1758 (C=O, stretching),
1455 (CH2, scissoring and CH3, bending), 1359 and 1343 (CH2, wagging), 1279 (CH2, twisting), 1188 (C-O-C,
vibration in poly(L-lactide) chain), 1104 (C-O-C, twisting), 962 (C-O-C, vibration and CH2, rocking), 842 (CH2,
rocking), 736 (C-H, out-of-plane, aromatic), 529 (C-O-C, bending). 1H NMR (600 MHz, CDCl3): δH 1.61 (210H, m,
(COCH(CH3)O)70), 3.65 (1440H, s, (OCH2CH2)90), 4.15 (16H, s, OCH2CO-), 5.20 (70H, m, (COCH(CH3)O)70), 8.05-
9.65 (12Harom, m, CH).
General procedures for polymeric micelles
Preparation of polymeric micelles. Polymeric micelles with covalently bound ZnPc were prepared by the thin
film method (for Pluronic micelles, System 1) and by modified interfacial polymer deposition-solvent
evaporation technique (for micelles of poly(L-lactide) derivatives, System 2). Briefly, block copolymers
(including their hydrophilic zinc phthalocyanine modified derivatives) were dissolved in appropriate organic
solvents: Me2CO/MeOH (4:1, v:v) mixture for Pluronic micelles and THF for poly(L-lactide) (PLLA) micelles
(PLLA micelles with no fluorescent marker were composed of PEG-b-PLLA instead of ZnPc-conjugated PLLA).
Then, in the thin film method, the obtained solution was placed in round bottom flask and the organic solvent
was removed under reduced pressure, followed by hydration of the resulting thin film. In interfacial polymer
deposition-solvent evaporation technique the obtained organic solutions were slowly added to stirred water
and THF was removed under reduced pressure at rt. In both methods, polymeric micelles solutions were
filtered through syringe filters to remove undissolved substances.
Characterization of polymeric micelles. The size distribution (expressed as the hydrodynamic diameter DH)
and polydispersity index (PdI) of the polymeric micelles were determined by dynamic light scattering (DLS)
measurements using a Zetasizer NanoZS Instrument (ZEM4228, Malvern Instruments, UK) equipped with a 4
mW He–Ne laser (λ 633 nm) and with noninvasive backscattering (NIBS) detection at a scattering angle of
o
173°. Samples were equilibrated for a minimum of 1 min at 25 C before measurements. The morphology and
dimensions of the polymeric micelles were examined by atomic force microscopy (AFM) using the Veeco
NanoScope Dimension V AFM with an RT ESP Veeco tube scanner. The scanning speed was 0.5 Hz and a low-
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resonance-frequency pyramidal silicon cantilever resonating at 250–331 kHz was employed (at a constant
force of 20–80 N/m). Before observations, the polymeric micelles solutions (system 1 - 10.2 mg/mL, system 2
– 4.0 mg/mL) were diluted 15 times in double-distilled water and placed on a cover glass surface and allowed
to dry at rt. Then the excess of micelles was removed by rinsing the surfaces in double-distilled water for 30
min and drying at rt. The fluorescence spectra were recorded on a Spectrofluorimeter F4500 (Hitachi, Japan)
with scan speed 240 nm/min and delay equal to 0.5 s. Properties of the obtained polymeric micelles systems
are presented in Table 1.
Biological evaluation
Cell human malignant melanoma cells (MeWo) line was obtained from European Collection of Authenticated
Cell Cultures (ECACC). DMEM medium, PBS, antibiotics, trypsin, 3(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide and EDTA were purchased form Sigma, Poland. Fetal bovine serum was
purchased from Biowhittaker, and ApoLive-GloTM Multiplex Assay was purchased from Promega, Poland.
Cell culture. The studies were performed on human malignant melanoma cells (MeWo). MeWo cells were
grown in DMEM medium with addition of 10% fetal bovine serum (FBS) and supplemented by antibiotics
(Penicillin/streptomycin). For the experiments cells were removed by trypsinization (trypsin 0.025% and EDTA
0.02% solution) and rinsed twice with PBS.
Cytotoxic activity. Cells survival was evaluated by the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. The cells were seeded into 96-well microculture plates at the density of 5 x 103
cells/well. Cell viability was determined after 24 h for standard cytotoxicity with polymeric micelles with and
without fluorescent marker. The MTT assay was performed according to the manufacturer’s protocol. The
absorbance was determined using a multiwell scanning spectrophotometer at λ 570 nm (EnSpire Perkin Elmer,
Poland). Mitochondrial function was expressed as a percentage of viable cells under treatment relative to
untreated control cells. The results are presented as the mean ± the standard deviation of the triplicates.
Caspase-3/7 activity. Caspase-3/7 activity was measured using the ApoLive-GloTM Multiplex Assay according
to the manufacturer's protocol. The applied assay enables measuring viability and caspase 3/7 activation in
the same sample well. MeWo cells were plated in triplicate in 96-well cell culture plates (Black/White Isoplate-
96 TC, Black Frame, White Well, Perkin Elmer, Poland) and left overnight at 37 C. Then the 24 h incubation
with polymeric micelles with and without fluorescent marker was performed. At that time the first stage was
evaluated - protease as a marker of cell viability was measured. The measurement was performed by
fluorescence detection at an excitation/emission wavelength of λ 400/505 nm with a multifunctional
microplate reader (EnSpire Perkin Elmer, Poland). Then caspase 3/7, substrate for luciferase, which contains
tetrapeptide sequence DEVD, was added and after 30 min of incubation at rt luminescence was measured.
Results are presented as the mean ± the standard deviation of the triplicates.
ZnPc distribution. MeWo cells were prepared for fluorescent studies. Cells were primarily harvested on cover
slides. The exemplary nanomicelles (system 2b) in concentration of 4µM were diluted in medium and added to
cells for 24 h incubation. Then cells were washed with PBS, fixed with 4% paraformaldehyde and washed with
PBS. For imaging with λexc 630 nm, Olympus BX53 fluorescent microscope with X-Cite 120PC Q adapter was
used.
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Acknowledgements
This work was supported by Wroclaw Research Center EIT+ under the project ‘Biotechnologies and advanced
medical technologies’ – BioMed (POIG 01.01.02-02-003/08-00) financed from the European Regional
Development Fund Operational Programme Innovative Economy 1.1.2, and was also financed by a statutory
activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of
Wrocław University of Technology.
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