Photodynamic inactivation of Staphylococcus aureus using low symmetrically substituted phthalocyanines supported on a polystyrene polymer fiber

Article abstract of DOI:10.1016/j.dyepig.2012.10.001

This work reports on the antimicrobial photo-Activities of a series of low symmetrically substituted phthalocyanine complexes in solution and in a fiber matrix. Phthalocyanine complexes were successfully electrospun into a polystyrene polymer. The fiber diameter ranged from 240 nm to 390 nm in average. The modified polymer fiber showed successful singlet oxygen production with the Ge monocarboxy phthalocyanine modified fiber giving the highest singlet oxygen quantum yield value of 0.46 due to lack of aggregation when in the polymer. All the unsymmetrically substituted complexes showed antimicrobial activity towards S. Aureus under illumination with visible light. The symmetrical ZnPc and ZnTPCPc showed no activity under illumination with light in the fiber matrix due to low singlet oxygen production.

Full text of DOI:10.1016/j.dyepig.2012.10.001

Dyes and Pigments 96 (2013) 500e508
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Photodynamic inactivation of Staphylococcus aureus using low symmetrically
substituted phthalocyanines supported on a polystyrene polymer fiber
Nkosiphile Masilela a, Phumelele Kleyi a, Zenixole Tshentu a, Georgios Priniotakis b, Philippe Westbroek c,
,
Tebello Nyokong a
*
a Department of Chemistry, Rhodes University, Grahamstown 6140, South Africa
b Technological Educational Institute of Piraeus, Textile Engineering Department, Petrou Ralli & Thevon 250, Aegaleo 12244, Athens, Greece
c Ghent University, Department of Textiles, Technologiepark 907, B-9052 Gent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
This work reports on the antimicrobial photo-activities of a series of low symmetrically substituted
phthalocyanine complexes in solution and in a fiber matrix. Phthalocyanine complexes were successfully
electrospun into a polystyrene polymer. The fiber diameter ranged from 240 nm to 390 nm in average.
The modified polymer fiber showed successful singlet oxygen production with the Ge monocarboxy
phthalocyanine modified fiber giving the highest singlet oxygen quantum yield value of 0.46 due to lack
of aggregation when in the polymer. All the unsymmetrically substituted complexes showed antimi-
crobial activity towards S. Aureus under illumination with visible light. The symmetrical ZnPc and
ZnTPCPc showed no activity under illumination with light in the fiber matrix due to low singlet oxygen
production.
Received 13 July 2012
Received in revised form
30 September 2012
Accepted 2 October 2012
Available online 23 October 2012
Keywords:
Low symmetry phthalocyanines
Germanium
Ó 2012 Elsevier Ltd. All rights reserved.
Titanium
Antimicrobial
Photodynamic therapy
Electrospun fiber
1. Introduction
The use of symmetrically substituted Pcs as photosensitizers for
photodynamic therapy is well established, however these
Phthalocyanines (Pcs) continue to receive attention for appli-
cations in a variety of scientific fields due to their unique physico-
chemical properties that arise from their aromatic structure [1,2].
A number of Pcs show potential as photosensitizers for photody-
namic therapy (PDT) of cancer [3e7]. PDT has been considered as
a low-cost, non-invasive and a gentle procedure for treatment of
various tumors [8]. PDT may be applied with high efficiency for
inactivation of pathogenic microorganisms which are resistant to
antibiotics [9], hence this work reports on a series of Pcs for the
inactivation of Staphylococcus aureus bacteria. Pcs show high
efficiency of generating reactive oxygen species, have low dark
toxicity and can easily be chemically modified [10]. Previous
reports have shown that introducing diamagnetic metals such as
Zn, Si, Al, Ga and In into the cavity of the Pc ring results in enhanced
triplet state parameters (triplet quantum yields and lifetimes) and
high singlet oxygen quantum yields [11e14].
complexes show low substrate binding selectivities [15]. It has also
been reported that decreasing the symmetry of phthalocyanines
improves the singlet oxygen quantum values [16], hence in this
work, low symmetry Pc complexes 1e5, symmetrical complex 6
and unsubstituted zinc phthalocyanine (Fig.1 and Schemes 1 and 2)
are reported for the inactivation of S. aureus bacteria. Cosimelli et al.
reported that low symmetry zinc phthalocyanine (in solution)
showed photo-inhibition of Candida albicans [17]. We have recently
reported on the inactivation of S. aureus using phthalocyanines in
solution and in the presence of nanoparticles [18]. The use of
supports for phthalocyanines during the inactivation of bacteria is
preferred since it allows for recyclability. The use of unsubstituted
zinc phthalocyanine (ZnPc) embedded in electrospun polyurethane
fibers has been reported by Mossinger et al. for the inactivation of
Escherichia coli [19]. In this work, low symmetrically substituted
phthalocyanine complexes embedded in electrospun polystyrene
(PS) polymer material are used for the inactivation of S. aureus
bacteria, a gram positive and drug resistant bacteria. We investigate
the photochemical behavior of MPc-PS fiber composite and their
antimicrobial activity towards S. aureus. The effects of asymmetrical
versus symmetrical substitution on the phthalocyanines are
* Corresponding author. Tel.: þ27 46 6038260; fax: þ27 46 6225109.
E-mail address: t.nyokong@ru.ac.za (T. Nyokong).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2012.10.001

N. Masilela et al. / Dyes and Pigments 96 (2013) 500e508
501
HOOC
N
COOH
N
O
S
O
S
N
N
M
N
N
N
N
N
Zn
N
N
N
N
N
N
N
N
N
O
O
S
O
HO
HOOC
N
COOH
(6)
M = (OH) Ge (1) (OH) GeMCPc
2
2
OTi
(2) OTiMCPc
(ac) Sn
(3) (ac) SnMCPc
2
2
N
N
N
N
N
Zn
N
N
N
(ZnPc)
Fig. 1. Molecular structures of the low symmetrically substituted complexes. MCPc ¼ monocarboxy phthalocyanine (complexes 1e3), TPCPc ¼ tetraphenylcarboxyphthalocyanine
(6) and ZnPc.
discussed. The results show that unsubstituted and symmetrically
substituted ZnPc embedded in polystyrene are not active towards
this bacteria due to low singlet oxygen production.
tetrahydrofuran (THF) were from MERCK Chemical Ltd. Anthra-
cene-9,10-bis-methylmalonate (ADMA),1,3-diphenylisobenzofuran
(DPBF), L-cysteine, dimethylacetamide (DMAc), 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU), zinc chloride and zinc phthalocyanine
(ZnPc) were purchased form SigmaeAldrich. Agar bacteriological
BBL Mueller Hinton broth and nutrient agar were purchased from
Merck. S. aureus (ATCC 6538) was purchased from Microbiologics.
The synthesis of complexes 1e3 [20] and 6 [21] (Fig. 1) have been
reported in literature. Complexes 4 and 5 (Schemes 1 and 2) are
reported here for the first time.
2. Experimental and methods
2.1. Materials
Polystyrene (PS, Mw ¼ 192,000 g/mol), N,N-dimethylfomamide
(DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM) and
COOH
COOH
H N
2
O N
2
CN
CN
K CO , DMF
3
2
S
CN
CN
+
H N
2
Argon, 72 hrs
SH
L-Cysteine
4-nitrophthalonitrile
7
H N
2
COOH
S
O
COOH
N
N
N
N
N
DBU, DMAc,
ZnCl
2
N
H N
2
Zn
N
130oC , 7hrs
O
CN
CN
S
CN
CN
N
O
+
O
8
7
4
Scheme 1. Synthesis of 4-cysteinyl phthalonitrile (10) and low symmetry monocysteinyl zinc (ZnMCsPc, 4).

502
N. Masilela et al. / Dyes and Pigments 96 (2013) 500e508
N
COOH
S
N
COOH
O
S
N
N
N
N
DBU, DMAc, ZnCl
N
2
Zn
N
N
130oC , 7hrs
N
S
S
CN
CN
O
CN
CN
N
S
S
+
N
N
S
S
N
N
N
9
10
5
Scheme 2. Synthesis of monophenoxy-carboxy zinc phthalocyanine (ZnMPCPc, 5) complex.
2.2. Equipment
Yield: 50%. IR [(KBr)-max/cmꢀ1]: 3428 (OH), 3102 (CeH), 2234
(C^N), 1610 (C]O), 1586, 1427, 1234, 862 (CeH), 742 (CeSeC). 1H
NMR (400 MHz, DMSO-d6)/ppm: 12.11 (1H, broad s, eCOOH), 7.24
(1H, d, aromatic CeH), 7.56 (2H, dd, aromatic CeH), 3.89 (1H, d, Ce
H), 3.46 (2H, dd, SeCH2), 2.12 (2H, broad, NH2). Calc. for C11
N3H9S1O2: C 53.46, H 3.64, N 17.00, S 12.97 Found: C 53.32, H 3.58, N
16.97, S 12.66.
UVevisible spectra were recorded on a Shimadzu 2550 UVeVis
spectrophotometer. Fluorescence excitation and emission spectra
were recorded on a Varian Eclipse spectrofluoremeter. IR spectra
(KBr pellets) were recorded on a PerkineElmer spectrum 2000 FTIR
spectrometer.
Photo-irradiations for antimicrobial photo-inhibition studies
and singlet oxygen determinations were performed using a General
electric Quartz line lamp (300 W). A 600 nm glass cut off filter
(Schott) and water were used to filter off ultraviolet and infrared
radiations, respectively. An interference filter (Intor, 670 and
700 nm with a band width of 40 nm) was additionally placed in the
light path before the sample. Light intensity was measured with
a POWER MAX5100 (Molelectron detector incorporated) power
meter. Scanning electron microscope (SEM) images of the fiber
alone or in the presence of MPc complexes were obtained using
a JOEL JSM 840 scanning electron microscope. The fiber diameters
were measured using Cell Dˇ software from Olympus. The average
diameter of seventy different fibers was taken.
Mass spectral data were collected with a Bruker AutoFLEX III
Smartbeam TOF/TOF Mass spectrometer. The spectra were acquired
using dithranol as the MALDI matrix.
All plate readings for the antimicrobial studies were obtained
using the LEDETECT 96 for in vitro diagnostic from LABXIM
PRODUCTS.
2.3.2. Tris{9 (10), 16 (17), 23 (24)-(4-phenoxy)-2-(4-cysteinyl)
phthalocyanine} Zn (Scheme 1, (ZnMCsPc (4)))
A mixture of 4-cysteinyl phthalonitrile (7) (0.32 g, 1.3 mmol) and
4-phenoxy phthalonitrile (8) (0.86 g, 3.9 mmol) was firstly finely
ground, homogenized and placed in a round bottom flask that con-
tained pre-heated DMAc. The mixture was then stirred under reflux
at 130 ꢁC for7 h in an argon atmospherein thepresence ofexcesszinc
chloride as a metal salt and DBU as a catalyst. Thereafter, the mixture
was cooled to room temperature and dropped in (1:1) water:
acetone. The green solid product which precipitated was collected by
centrifugation, washed with n-hexane and dried in air. Purification
was achieved using column chromatography with silica gel as
column material and MeOH (methanol):THF (2:10) as eluent fol-
lowed by the addition of (2:1) THF:DMF to elute the second fraction
which was our desired product. The product was dried in air and
further washed with ethanol, acetone, n-hexane and diethyl ether.
Yield: (12.50%). IR (KBr, cmꢀ1): 3452(OeH), 3128(CeH), 2977
(carboxylic acid OH), 1598 (C]O), 1534(C]C), 1447, 1348, 1300,
1235 (CeOeC) 878, 712 (CeSeC), 701,620, 553. NMR (DMSO-d6): d,
2.3. Synthesis
ppm 9.01e9.18 (12H, m, Pc-H), 7.27e7.88 (12H, m, aromatic-H),
7.10e7.21 (3H, dd, aromatic-H), 6.41e6.48 (1H, d, CeH), 4.53e
4.59 (1H, br, OH) 3.62e3.71 (2H,d, SeCH2), 2.41e2.47 (2H, broad,
4-Nitrophthalonitrile [22], 1,2 bis-(diethylaminoethylthiol)-4,5
dicyanobenzene (9) [23], 4-phenoxy phthalonitrile (8) [24] and 4-
(3,4-dicyanophenoxy)benzoic acid (10) [25] were synthesized
according to literature methods.
NH2). UV/Vis (DMF) lmax nm (log
3 ): 678 (5.09). Calc. for C53 H33 N9
S1 O5 Zn: C 65.45, H 3.39, N 12.96, S 3.29 Found: C 64.98, H3.41,
N13.01,
S 3.13 MALDI TOF MS m/z: Calcd: 973.36 Found:
[M ꢀ H]ꢀ ¼ 972.82.
2.3.1. Synthesis of 4-cysteinyl phthalonitrile (7, Scheme 1)
Compound 7 was synthesized as follows: to dry DMF (30 mL)
under argon, 3.5 g (25 mmol) of K2CO3 was added. L-cysteine (2.07 g,
2.3.3. Hexakis {9; 10, 16; 17, 23; 24-(1,2-bis-
(diethylaminoethylthiol))-2-(4-phenoxycarboxy) phthalocyanine}
Zn (Scheme 2, (ZnMPCPc (5)))
17.1 mmol) and 4-nitrophthalonitrile (2.0 g, 11.5 mmol) were added.
After 4 and 24 h, more K2CO3 (3.5 g, 25 mmol) was added to the
mixture. The mixture was stirred at room temperature for 72 h, after
this time the formed product was dissolved inwater: methanol (1:1),
and the pH of the solution adjusted to 2 by addition of HCl to give
a light reddish brown precipitate. The product was further purified
using size-exclusion column (Bio-Beads S-X1 from Bio-Rad).
Synthesis for
5 was as outlined for 4 except 4-(3,4-
dicyanophenoxy)benzoic acid (10) (0.34 g, 1.3 mmol) and 1,2-bis-
(diethylaminoethanethiol)-4, 5-dicyanobenzene phthalonitrile (9)
(1.52 g, 3.9 mmol) were employed instead of 7 and 8. Purification
was achieved using column chromatography with neutral alumina
as column material and DCM:MeOH:THF (10:5:5) as eluent,

N. Masilela et al. / Dyes and Pigments 96 (2013) 500e508
503
followed by drying. The desired product was further washed with
ethanol, acetone, n-hexane and diethylether.
dark at 37 ꢁC. The viable microorganisms were corrected with
controls containing no Pc. The optical density of the bacterial
viability was determined at 600 nm and expressed as percentage
growth inhibition. The experiments were done in triplicates.
Yield: (15.10%). IR (KBr, cmꢀ1): 3438(OeH), 3134(CeH), 2969
(carboxylic acid OH), 1610 (C]O) 1548(C]C), 1345, 1337, 1131, 854,
748 (CeSeC), 620, 553. 1H NMR (DMSO-d6):
d, ppm 10.01e10.09
(6H, m, Pc-H), 9.36e9.49 (3H, br, Pc-H), 7.35e7.23 (4H, m,
aromatic), 4.63e4.71 (1H, br, OH), 3.25e3.30 (12H, m, SeCH2),
2.98e3.01 (12H, m, NeCH2), 2.56e2.71 (24H, m, CH2-methyl)
2.6. Singlet oxygen quantum yield
Singlet oxygen quantum yield (FD) values were determined in
air using the relative method with DPBF acting as a singlet oxygen
chemical quencher in DMF. Equation (1) was employed for calcu-
lating singlet oxygen quantum yields:
1.73e1.79 (36H, q, CH3). UV/Vis (DMF) lmax nm (log
3 ): 695
(5.12).Calc. for C75 H98 N14 S6 O3 Zn þ H2O: C 59.31, H 6.45, N 12.91, S
12.67 Found: C 59.48, H 5.70, N 13.17, S 12.74. MALDI TOF MS m/z:
Calcd: 1500.5 Found: [M þ 2H]þ2 ¼ 1502.6.
Std
RI
RStdIabs
FD
¼
FSDtd
$
(1)
abs
2.4. Preparation of electrospun nanofibers
The electrospinning technique (with or without MPc
complexes) has been described before [26,27]. For the current
work, the MPc:PS electrospun fibers were formed as follows:
where FSDtd is the singlet oxygen quantum yield for the standard
(ZnPc, FSDtd ¼ 0:56 in DMF) [29]. R and RStd are the DPBF photo-
bleaching rates in the presence of the metallophthalocyanine
derivatives under investigation and the standard respectively. Iabs
a
solution containing 1.3
ꢂ
10ꢀ5 mol of polystyrene and
1 ꢂ10ꢀ6 mol of MPc in 10 mL DMF/THF(4:1) was stirred for 24 h to
produce a homogeneous solution. The solvent mixture of DMF/THF
was employed to allow both the PS and MPc to dissolve. The
solution was then placed in a cylindrical glass tube fitted with
a capillary needle. A potential difference between anode and
cathode of 20 kV (ꢀ5e15 kV) was applied to provide the charge for
the spinning process. The distance between the cathode (static
fiber collection point) and anode (tip of capillary needle) was
15 cm. The pump rate was maintained at 0.5 mL/h. The flow rate
Std
and I are the rates of light absorption by the MPc derivatives and
abs
the standard, respectively. To avoid chain reactions of the quencher
in the presence of singlet oxygen [29], the concentration of DPBF
was kept at w3 ꢂ 10ꢀ5 mol Lꢀ1
.
Solutions of the MPcs with an absorbance of w0.5 at the
irradiation wavelength were prepared in the dark and irradiated at
the Q band region in the presence of DPBF. The DPBF absorption at
417 nm was monitored with photolysis time. The error was w10%
from several values of FD
.
was increased to
1 mL/h in the case of the polystyrene/
For the modified fibers the absolute method was used. The
singlet oxygen quantum yield (FD) determinations for the MPc on
fibers were carried out in aqueous solutions using ADMA as the
quencher and its degradation was monitored at 380 nm. In each
case 10 mg of the modified fibers was suspended (as small pieces)
in an aqueous solution of ADMA and irradiated using the photolysis
set-up described above. The quantum yields (FADMA) were calcu-
lated using equation (2), using the extinction coefficient of AMDA in
phthalocyanine composite to avoid clogging of the needle.
2.5. Antimicrobial activity
S. aureus (S. aureus) was grown on a nutrient agar plate prepared
according to the manufacturer’s specifications. Electrospun fibers
modified with phthalocyanines were placed on the Baird Parker
agar base plates which were pre-inoculated with 100 mL of liquid
water, log (
3 ) ¼ 4.1 [30].
broth containing a suspension of S. aureus (w107e108 CFU/mL
(colony forming unit/mL)). This resulted in bacteria growing over
and around the fiber, hence completely covering it. One pair of
agar plates was kept in the dark for 90 min and the other pair
was kept under illumination with visible light (as described in
Section 2.2) for 90 min. All the samples were incubated for 16 h
at 37 ꢁC. A pair of agar plates containing the PS/Pc fiber without
bacteria was used as control both in the dark and under illumina-
tion with light. This was aimed at confirming that the changes
observed are due to bacteria inactivation and not phthalocyanine
leaching, even though the latter is not expected due to insolubility
of the complexes in water.
ðC0 ꢀ CtÞVR
FADMA
¼
(2)
I
abs$t
where C0 and Ct are the ADMA concentrations prior to and after
irradiation, respectively; VR is the solution volume; t is the irradi-
ation time per cycle and Iabs is defined by equation (3).
a
$A$I
NA
Iabs
¼
(3)
where
a
¼ 1ꢀ10ꢀA(l), A(
l
) is the absorbance of the sensitizer at the
irradiation wavelength, A is the irradiated area (2.5 cm2), I is the
The antimicrobial activity of the MPcs was also studied in
solution (not using a fiber mat). Firstly, single colony of S. aureus
from the agar plate was inoculated into 10 mL of Mueller Hinton
nutrient broth and allowed to grow at 37 ꢁC for 2e6 h until an
appropriate optical density (0.6e0.8 at 600 nm) was obtained.
intensity of light (4.54 ꢂ 1016 photons cmꢀ2
s
ꢀ1) and NA is Avoga-
dro’s constant.
The absorbances used for equation (3) are those of the phtha-
locyanines in the fibers (not in solution) measured by placing the
modified fiber directly on a glass plate. The light intensity measured
refers to the light reaching the spectrophotometer cells, and it is
expected that some of the light may be scattered, hence the FD
values of the phthalocyanines in the fiber are estimates. The singlet
oxygen quantum yields (FD) were calculated using equation (4) [31]
Separately, MPcs solutions (100
m
L with concentrations ranging
M in DMF) were added to the 96 well plates
L Mueller Hinton nutrient broth. Then, 5 L of
from 0.16 to 20
containing 100
m
m
m
the solution of S. aureus prepared on the first step was pipetted
into each 96 well plates containing the MPcs. The total amount of
DMF in each well was 50%. S. aureus is known to be resistant to
DMF [28].
A pair of 96 wells microplate containing the same Pc molecule
was then irradiated with visible light for 90 min, while another pair
was kept in the dark. Irradiated and non-irradiated cells were
incubated overnight on an incubator shaker (w200 rpm) in the
1
1
1
kd
1
¼
þ
$
$
(4)
FADMA
FD FD ka ½ADMAꢃ
where kd is the decay constant of singlet oxygen in respective
solvent and ka is the rate constant of the reaction of ADMA with

504
N. Masilela et al. / Dyes and Pigments 96 (2013) 500e508
O2(1Dg). The intercept obtained from the plot of 1/FADMA versus 1/
[ADMA] gives 1/FD
Table 1
Spectral, microscopic and photochemical properties of the low symmetry MPc
complexes in solution (DMF) and in the fiber matrix (water).
.
Sample
lQ/nm
(solution) (solid)
lQ/nm
FD
FD
Average
diameter (nm)
3. Results and discussion
(solution) (fiber)
(OH)2GeMCPc (1) 695
OTiMCPc (2) 734
(ac)2SnMCPc (3) 720
692
728
716
680
0.66
0.54
0.52
0.57
0.46
0.31
0.30
0.42
275 ꢄ 30
315 ꢄ 30
370 ꢄ 30
260 ꢄ 30
3.1. Spectroscopic and microscopic characterization
ZnMCsPc (4)
ZnMPCPc (5)a
ZnPc
678
The MPc complexes were mixed with polystyrene and electro-
spun into nanofibers as explained in the experimental section.
Scannining electron microscope images of the various MPc
complexes incorporated into polystyrene, Fig. 2 shows highly
branched cylindrical network of nanofibers. The fiber diameter
ranged from 240 ꢄ 45 nm to 390 ꢄ 30 nm, Table 1. The polystyrene
alone gave the smallest fiber diameter average of 240 ꢄ 45 nm
which is within the range of previously reported average diameter
of polystyrene [32]. An increase in fiber diameter was obtained in
the presence of phthalocyanines. The ZnMPCPc-PS composite gave
the highest average fiber diameter of 390 ꢄ 30 nm compared to all
the other complexes, followed by (ac)2SnMCPc with the average
diameter of 370 ꢄ 30 nm respectively, Table 1. The solution prop-
erties such viscosity, conductivity and surface tension may affect
the fiber formation hence the diameter of the fibers. Differences
and the conductivities of MPc complexes may have a large effect on
the electrospinning process. Differences in the nature of the central
metal atom, inter-planar distances between the molecules and
differences in stacking arrangements of the molecules all affect
conductivity in phthalocyanines [33], hence fiber diameter.
695 (683) 684 (680) 0.64 (0.45) 0.28 (0.12) 390 ꢄ 30
670
668
e
0.56
e
0.14
e
305 ꢄ 35
240 ꢄ 45
Polystyrene
e
a
Numbers in brackets are for the symmetrical ZnTPCPc(6).
(ac)2SnMCPc (3) and ZnMPCPc(5) on the fiber (curves ii in Fig. 3).
The blue shifting in the spectra of these complexes, which contain
sulfur bridges, suggests that there is interaction of the fiber with
the S groups, since S groups are known to result in red shifting. For
ZnTPCPc (6) containing no S groups there was no blue shifting but
broadening due to aggregation. Broadening in spectra was also
observed for ZnMPCPc (5). (ac)2SnMCPc (3) showed a clear split in
the Q band with the high energy band being due to the aggregate
and the low energy band due to the monomer. Aggregation of
phthalocyanines in general is expected in the solid state due to the
close proximity of PcePc rings, which results in very strong
pep
interactions. The (OH)2GeMCPc (1) and ZnMCsPc (4) complexes
did not show aggregation on the polymer fiber. ZnMCsPc (4)
showed a slight red shift in the solid state as opposed to blue
shifting. Red shifting of phthalocyanine Q-band in the polymer fiber
has been reported before [19].
The UVevisible absorption spectra of the complexes in solution
and in the polymer support are shown in Fig. 3. Blue shift in the Q
band spectra was observed for (OH)2GeMCPc (1), OTiMCPc (2),
Fig. 2. Scanning electron microscopic (SEM) images polystyrene electrospun fiber alone (a), (OH)2GeMCPc (1):PS (b), OTiMCPc (2):PS (c), (ac)2SnMCPc (3):PS (d), ZnMCsPc (4):PS (e)
and ZnMPCPc (5):PS (f), and. Scale ¼ 10 m.
m

N. Masilela et al. / Dyes and Pigments 96 (2013) 500e508
505
Fig. 3. Ground state electronic absorption spectra of (a) (OH)2GeMCPc (1), (b) OTiMCPc (2), (c) (ac)2SnMCPc (3), (d) ZnMCsPc (4) and (e) ZnMPCPc (5), in (dotted line) (polymer
fiber) and in solution (solid line) (DMF).
The IR spectra of polystyrene shows characteristic signals in the
aromatic finger print region which are due to the styrene subunits
(Fig. 4(ii)). The aromatic C]C bend from the styrene ring is
observed at the region w1470e1600 cmꢀ1. An increase in the
intensity of aromatic signals in the 1470e1600 cmꢀ1 was observed
on the IR spectra of MPc-PS composite (using (ac)2SnMCPc (3) as an
example) suggesting additional aromatic rings from the Pc ring
(Fig. 4(i)). The intense carbonyl C]O stretching around
w1650 cmꢀ1 was observed for all the complexes, and is due to the
carboxylic acid groups of the phthalocyanine complexes, serving as
a further confirmation of the presence of Pc in the polymer fiber.
C-H
(i)
C=O
(ii)
4000
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm
Fig. 5. Photodegradation of ADMA in the presence of ZnMCsPc (4):PS fiber (a) and
photodegradation of DPBF in the presence of ZnMCsPc (4) in DMF (b). Insert: Photo-
oxidative generation of triiodide in the presence ZnMCsPc (4) in aqueous media.
)
Fig. 4. Infrared spectra of (ac)2SnMCPc(3)/PS (i) and polystyrene fiber alone (ii).

506
N. Masilela et al. / Dyes and Pigments 96 (2013) 500e508
Fig. 6. Images of the antimicrobial inhibition test using phthalocyanine modified fibers, studies in the dark and under illumination with visible light. Unmodified polystyrene alone
employed as a control. The figure also shows PS/ZnPc fiber before use in agar plate for bacterial growth.
3.2. Singlet oxygen generating abilities of the MPc in solution and
on fiber
that can help minimize aggregation. Aggregation has been reported
to lower the triplet state behavior resulting in low singlet oxygen
quantum yields [35,36].
The ability of a photosensitizer to absorb light and produce
a cytotoxic singlet oxygen species serve as a selective tool for
photodynamic therapeutic applications. The singlet oxygen quantum
yields (FD) were determined by monitoring the photo-degradation
of ADMA (in aqueous solution for the fiber matrix (Fig. 5(a))) and
DPBF (in DMF for the MPc complexes (Fig. 5(b))), using ZnMCsPc (4)
complex as an example. Complimentary to the degradation of ADMA,
the polymer composite consisting of the MPcs was used for the
generation of triiodide ðI3ꢀÞ from iodide (Iꢀ) in the presence of light as
observed in Fig. 5(a) (insert), serving as a clear proof that singlet
oxygen is produced as the I3ꢀ absorption band increase with time
[34]. There was no absorption in the region between 600 and 800 nm
(Fig. 5(a)) where the main absorption band of the phthalocyanine is
located, suggesting no leakage of phthalocyanines from the fiber to
the solution in aqueous media. This is expected since the phthalo-
cyanines reported in this work are not soluble in water. The lack of
leaching in water is important for real biological applications of the
fibers in aqueous media as is the case in this work.
Higher singlet oxygen quantum yields were obtained for the
complexes in solution compared to fiber matrix (Table 1). The
decrease in the values of FD in the solid state might due to aggre-
gation in some cases. The (OH)2GeMCPc (1) complex gave the
highest (FD) value of 0.66 and 0.46 both in solution and in the fiber
matrix. In the fiber, ZnMCsPc (4) gave the second highest value of
FD ¼ 0.42 in the fiber. Higher FD values for the ZnMCsPc (4) and the
(OH)2GeMCPc (1) complexes in fiber may have been a result of the
less aggregation behavior of these complexes in the solid state, as
was discussed above for the UVeVis spectra. Of the asymmetrical
Pc complexes, ZnMPCPc (5) gave the lowest singlet oxygen value of
0.28 in the fiber matrix. The singlet oxygen quantum yield
production of low symmetry phthalocyanines in the solid stage
(fiber) are reported for the first time. Symmetrically substituted
ZnTPCPc (6) and unsubstituted ZnPc showed the lowest FD values
of all complexes both in solution and when embedded in the fiber,
Table 1. ZnTPCPc (6) gave lower FD values than ZnMPCPc (5), the
latter is the unsymmetrical tetrasubstituted derivative of former.
As stated in the introduction, metallophthalocyanines contain-
ing diamagnetic metals exhibit high photosensitizing abilities [35e
37], which enhance their singlet oxygen generation, hence are
employed in this work. In addition, some of the central metals used
in this work such as Ge (IV), Sn (IV) and Ti (IV) carry axial ligands
3.3. Photo-inhibition of S. aureus
The mechanism and the science behind photodynamic antimi-
crobial chemotherapy (PACT) is in its infancy, but it seems to follow

N. Masilela et al. / Dyes and Pigments 96 (2013) 500e508
507
the same mechanism and principles as PDT and hence involves
singlet oxygen. It is known that singlet oxygen reacts with inter-
cellular molecules (peptides and proteins etc) leading to oxidative
damage of the cell wall and cell membrane which cause cell death
[7]. A series of low symmetry phthalocyanines presented in this
study shows photo-activity in the presence of light hence their
PACT was investigated.
singlet oxygen quantum yield value. Thus even though unsub-
stituted ZnPc embedded in electrospun fiber was reported to show
inactivation of E. coli [19], its low singlet oxygen quantum yield
resulted in no activity for the more drug resistant S. Aureus. This
confirms the importance of reasonably large values of singlet
oxygen quantumyields for successful bacterial inactivation. The lack
of activity of the symmetrical ZnTPCPc (6), shows the importance of
unsymmetrical substitution with the carboxyphenoxy substituent.
The photo-inhibition studies were also investigated for all the
complexes in solution, as shown in Fig. 7. The minimum inhibitory
concentration (MIC) required to inhibit 50% bacterial growth was
used as a reference to determine the effectiveness of these prom-
ising antimicrobials photosensitizers towards the inactivation of
The photodynamic responses of the complexes were investi-
gated in the fiber matrix. Fig. 6 shows digital images of the nutrient
agar plates containing S. aureus in contact with electrospunfibers.
The Pcs embedded in the fiber are blue in color in the absence of
bacteria (in nutrient broth or as a separate fiber, Fig. 6, ZnPc as an
example). The bacteria grew on and around the fiber resulting in
the blue color of the Pcs on the fiber disappearing. The samples
irradiated with light in the presence of bacteria showed positive
growth inhibition as compared to those that were kept in the dark.
This is judged by the clearing around the fiber and the restoration of
the blue color of the fiber. The polystyrene fiber alone did not show
any bacterial inhibition both in the dark and under illumination
with light. Lack of growth of bacteria around the polymer fiber
modified with photosensitizers (Fig. 6) was obtained for all low
symmetry MPc complexes as a result of bacterial growth inhibition.
This serves as evidence that the monocarboxy phthalocyanine
complexes generate reactive oxygen species that prevent bacterial
growth.
S. aureus. The minimum concentration of w10
mM at which
approximately w50% bacterial inhibition was obtained for all the
MPc samples when kept in the dark, Fig. 7(a). A significant
improvement in bacterial inhibition was obtained for the samples
illuminated with light (Fig. 7(b)) giving w50% of bacterial inhibition
at a much lower concentration of w1.25 mM. This suggested that the
production of singlet oxygen had a strong contribution in photo-
inhibition of bacteria.
With the results presented in this work, the phthalocyanine
complexes studied showed a potential industrial application for
antimicrobial photo-inhibition of S. aureaus.
A pair of plates containing a nutrient agar with polymer fiber in
the absence of bacteria was used as a control both in the dark and
under illumination with light. The color of the Pc modified fiber was
blue both under illumination and in the dark (control images not
shown). The nutrient agar was prepared in aqueous media and Pcs
are insoluble in aqueous media, hence there was no possible
leaching or migration of phthalocyanine from the fiber matrix to
the nutrient agar from the control experiments both in the pres-
ence and in the absence of bacteria.
4. Conclusions
The photochemical behavior of a series of low symmetry
phthalocyanine complexes has been investigated. All the MPc
complexes showed the production of singlet oxygen both in the
fiber matrix and in solution. We report for the first time the anti-
microbial photo-inhibition activities of a series of low symmetry
phthalocyanine complexes supported on a polymer fiber and
compare the activity with symmetrically and unsubstituted
substituted ZnPc. The study shows positive results that can be
further engineered for industrial applications. Lack of bacterial
growth was observed on areas surrounding the modified fiber,
confirming successful bacterial growth inhibition. The minimum
inhibition concentration results showed w50% of the bacterial
The symmetrical ZnTPCPc (6) (Fig. 6) and ZnPc showed no
activity towards bacterial inactivation most likely due to the low
a
inhibition at w1.25 mM MPc concentration in DMF:broth mixture.
100
90
80
70
60
Acknowledgments
GeMCPc
50
This work was supported by the Department of Science and
Technology (DST) and National Research Foundation (NRF) of South
Africa through DST/NRF South African Research Chairs Initiative for
Professor of Medicinal Chemistry and Nanotechnology and Rhodes
University. The project was partially funded by FP7-PEOPLE-2009-
IRSES PROCOTEX.
40
SnMCPc
30
20
10
0
TiMCPc
ZnMCsPc
ZnMPCPc
MPc Concentration (µM)
b
100
90
80
70
60
50
40
30
20
10
0
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GeMCPc
SnMCPc
TiMCPc
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