Photophysicochemical properties and photodynamic therapy activity of chloroindium(III) tetraarylporphyrins and their gold nanoparticle conjugates

Article abstract of DOI:10.1142/S1088424618501146

Novel chloroindium(III) complexes of tetra(4-methylthiophenyl)porphyrin (2a) and tetra-2-Thienylporphyrin (2b) dyes have been synthesized and characterized. The main goal of the project was to identify fully symmetric porphyrin dyes with Q-band regions th

Full text of DOI:10.1142/S1088424618501146

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2019; 23: 1–12
DOI: 10.1142/S1088424618501146
Published at http://www.worldscinet.com/jpp/
Photophysicochemical properties and photodynamic
therapy activity of chloroindium(III) tetraarylporphyrins
and their gold nanoparticle conjugates
Rodah C. Soya, Balaji Babua, David O. Oluwolea, Njemuwa Nwajia◊◊, James Oyimb◊◊,
Edith Amuhayab, Earl Prinslooc, John Mack*a◊ and Tebello Nyokonga◊
a Centre for Nanotechnology Innovation, Department of Chemistry, Rhodes University, Makhanda 6140, South Africa
b School of Pharmacy and Healthy Sciences, USIU-Africa, Nairobi, Kenya
c Biotechnology Innovation Centre, Rhodes University, Makhanda 6140, South Africa
This Paper is part of the 2019 Women in Porphyrin Science special issue.
Received 21 October 2018
Accepted 20 November 2018
ABSTRACT: Novel chloroindium(III) complexes of tetra(4-methylthiophenyl)porphyrin (2a) and tetra-
2-thienylporphyrin (2b) dyes have been synthesized and characterized. The main goal of the project was
to identify fully symmetric porphyrin dyes with Q-band regions that lie partially in the therapeutic window
that are suitable for use in photodynamic therapy (PDT). 2a and 2b were found to have fluorescence
quantum yield values ≤ 0.01 and moderately high singlet oxygen quantum yields (0.54−0.73) due to heavy
atom effects associated with the sulfur and indium atoms. The dark toxicity and PDT activity against
-1
.
epithelial breast cancer cells (MCF-7) were investigated over a dose range of 3.0−40 mg mL . The
-1
.
in vitro dark cytotoxicity of 2a is significantly lower than that of 2b at ≤ 40 mg mL . 2a was conjugated
with gold nanoparticles (AuNPs) to form a nanoconjugate (2a-AuNPs), which exhibited a higher singlet
oxygen quantum yield (FD) value and PDT activity than was observed for 2a alone. The results suggest
that the AuNPs nanoconjugates of readily synthesized fully symmetric porphyrin dyes are potentially
suitable for PDT applications, if meso-aryl substituents that provide scope for nanoparticle conjugation
can be introduced that shift the Q bands into the therapeutic window.
KEYWORDS: porphyrins, photophysics, singlet oxygen, gold nanoparticles, photodynamic therapy,
dark toxicity.
a PS, which selectively targets the tumor cells, and
INTRODUCTION
irradiation of the PS with laser light of appropriate
Photodynamic therapy (PDT) has emerged as a non-
wavelength to initiate a series of photochemical
invasive clinical treatment modality for cancer which is
reactions. Typically, this involves the excitation of the
a promising alternative to conventional cancer treatment
PS to a long-lived triplet state, which upon interaction
protocolssuchasradiotherapy,chemotherapyandsurgery
with the intracellular molecular dioxygen subsequently
that are known for their harmful side effects including
results in the generation of singlet oxygen known as a
the indiscriminate destruction of both healthy and tumor
reactive oxygen species (ROS) which is cytotoxic to
cells [1–5]. A prerequisite requirement for PDT is a
tumor cells [1–3]. Photosensitizers intended for PDT
light absorbing chromophore known as a photosensitizer
applications should fulfil some basic requirements
(PS). The technique involves the administration of
such as efficient generation of singlet oxygen, low
dark toxicity, photostability and absorption in the near
infrared therapeutic window among others [6].
◊ SPP full and ◊◊ꢀstudent member in good standing
*Correspondence to: John Mack, tel.: +27 46-603-7234, email:
j.mack@ru.ac.za.
Metalloporphyrin derivatives have gained consider-
able attention as photosensitizers in many fields such
Copyright © 2019 World Scientific Publishing Company

2
R. C. SOY ET AL.
as in photoelectric devices, catalytic reactions, solar
cells and PDT applications [6–11]. The applicability of
metalloporphyrins as PS in PDT is due to their efficient
photosensitization ability, particularly their ability to
absorb visible light, which excites the PS to an excited
singlet state, leading to intersystem crossing to the triplet
manifold. Energy transfer from the lowest excited triplet
stateofthePStomolecularoxygenresultsinthegeneration
of singlet oxygen, which is the chief cytocidal agent in
the selective destruction of tumor cells [1–3]. Recently
we reported the photophysicochemical properties of a
Sn(IV) complex of meso-tetra-2-thienylporphyrin with an
unusually high singlet oxygen quantum yield (FD) value,
and we demonstrated that the presence of bulky pyridyloxy
axial ligands limits aggregation through p–p stacking,
resulting in promising photodynamic therapy activity
properties during irradiation with a 625 nm light emitting
diode (LED), by enhancing the solubility of the dyes in
polar solvents [12]. A significant shift of the Q00 band
to the red relative to the analogous tetraphenylporphyrin
so that it lies further into the therapeutic window also
contributed to the enhanced PDT properties [12]. In
this study, we extend this work to the chloroindium(III)
complexes of meso-tetra[4-(methylthio)phenyl]porphyrin
(2a) and meso-tetra-2-thienylporphyrin (2b). The choice
of 4-(methylthio)phenyl and 2-thienyl substituents was
based in part on the fact that these derivatives have been
found to contain antioxidants and to exhibit antifungal,
antibacterialandanticanceractivity[13–16]. Incorporation
of a heavy atom such as indium is also known to enhance
the triplet population resulting in better singlet oxygen
generation [17, 18], and the presence of axial ligands
helps to prevent aggregation in aqueous solvents [12].
One of the most significant issues faced when using
porphyrin dyes and their analogues in PDT is the need
for selective delivery of the PS to tumor cells to avoid
leaving the patient photosensitized for prolonged periods.
Conjugating nanoparticles to metalloporphyrins has been
found to be advantageous [19], due to their small size,
high stability and high surface area, allowing for specific
target localization and hence, selective accumulation
in the cancer cells due to an enhanced permeability
and retention (EPR) effect [19–23]. In addition, it
has been established that gold nanoparticles (AuNPs)
destroy tumors selectively through a photothermal
effect (PTT) [21–23]. PTT is an important anticancer
therapeutic strategy in which irradiation of nanoparticles
embedded in the tumors with laser light results in an
increase in temperature in the tumor tissues, which
selectively kills cancer cells [23]. The sulfur atom in
the 4-methylthiophenyl ring of 2a enables noncovalent
interactions with the AuNPs since gold is known to have a
strong affinity for the electron lone pairs of sp3 hybridized
sulfur atoms [24]. The photophysicochemical properties
of nanoconjugates prepared by conjugating 2a to gold
nanoparticles (2a-AuNPs) have been studied and their
photodynamic therapy activity has been investigated.
EXPERIMENTAL
Materials
Pyrrole, 1,3-diphenylisobenzofuran (DPBF), 2-thio-
phenecarboxaldehyde, 4-methylthiobenzaldehyde, chlo-
roform, methanol, petroleum ether, sodium acetate,
indium(III) chloride, Trypan Blue, trypsin, ethylene-
diaminetetraacetic acid (EDTA) and zinc(II) tetraphenyl-
porphyrin (ZnTPP) were purchased from Sigma-Aldrich.
Dimethyl sulfoxide (DMSO) and dichloromethane were
obtained from Merck. Cultures of the MCF-7 cell were
obtained from Cellonex®. 10% (v/v) heat-inactivated
-1
.
fetal calf serum (FCS), and 100 unit mL penicillin-
-1
.
100 mg mL streptomycin-amphotericin B were acquired
from Biowest®. Neutral red cell proliferation reagent
(WST-1), Dulbecco’s phosphate-buffered saline (DPBS)
and Dulbecco’s modified Eagle’s medium (DMEM) were
obtained from Lonza®. The syntheses of the chloroindium
complex of 5,10,15,20-tetraphenylporphyrin (ClInTPP)
[25], and free base 5,10,15,20-tetra-[4-(methylthio)-
phenyl]porphyrin (H2MTPP) [26, 27] and 5,10,15,20-
tetra-2-thienylporphyrin (H2TTP) [28, 29] were carried
out as reported in the literature.
Instrumentation
Mass spectrometry data were obtained from a Bruker
AutoFLEX III Smartbeam MALDI-TOF instrument,
and dithranol was used as the matrix in the positive ion
mode. The elemental analyses were carried out on a Vario
1
EL III Microcube CHNS Analyzer. H-NMR spectra
were recorded on a Bruker AVANCE II 600 MHz NMR
spectrometer using tetramethylsilane as the internal
reference standard. X-ray diffraction (XRD) patterns
were recorded on a Bruker D8 Discover diffractometer
using procedures described previously [30]. The ground
state electronic absorption spectra were measured
on a Shimadzu UV-2550 spectrophotometer, while
fluorescence excitation and emission were recorded on a
Varian Eclipse spectrofluorimeter. Fluorescence lifetimes
were measured using a time-correlated single photon
counting setup (TCSPC) (FluoTime 300, Picoquant®
GmbH) with a diode laser (LDH–P–670, Picoquant®
GmbH, 20 MHz repetition rate, 44 ps pulse width) in
a manner described previously [31]. The morphologies
of the nanoparticles and the conjugates were assessed
by transmission electron microscopy (TEM) with a
Zeiss Libra model 120 operated at 100 kV. Magnetic
circular dichroism (MCD) spectra [32] were recorded on
a Chirascan plus spectrodichrometer equipped with a 1
tesla permanent magnet by using both the parallel and
antiparallel fields and subtracting a solvent baseline.
Synthesis
The chloroindium complexes of H2MTPP and H2TTP
were synthesized using literature methods [33, 34].
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 2–12

PHOTOPHYSICOCHEMICAL PROPERTIES AND PHOTODYNAMIC THERAPY ACTIVITY OF CHLOROINDIUM(III)
3
Synthesisofchloroindium(III)meso-tetra-[4-(methy-
lthio)phenyl]porphyrin (2a). A mixture of glacial acetic
acid (30 ml) and 320 mg (0.4 mmol) of H2MTPP
was stirred and brought to reflux at 100 °C. 221 mg
(1.0 mmol) of InCl3 and 0.6 g (7.314 mmol) sodium
acetate were then added and the mixture was refluxed
for a further 16 h. UV-vis absorption spectroscopy
was used to monitor the completion of the reaction.
The reaction mixture was cooled in ice to obtain the
crude precipitate, which was filtered and washed with
Millipore water (3 × 100 mL) and dried in vacuo. The
crude product was then purified using silica gel column
chromatography with chloroform/methanol (2:1) as
the eluent to yield 2a as a green-purple solid. Yield:
309 mg (96.6%). UV-vis (DMSO): lmax nm (log e) 435
used to carry out TD-DFT calculations with the CAM-
B3LYP functional and SDD basis sets, since the CAM-
B3LYP functional is known to provide more accurate
results for transitions with significant charge transfer
properties [41].
In vitro dark cytotoxicity and PDT activity. In vitro
PDT studies were conducted using the illumination kit
of a Modulight® 7710−680 Medical Laser fitted with a
Thorlab M625L3 light emitting dioide that was found
-2
.
to provide an irradiance of 240 mW cm (measured
with a Coherent FieldmaxII TOP energy/power meter
fitted with a Coherent Powermax PM10 sensor), to
illuminate a 127.76 × 85.48 mm 96 well tissue culture
plate. The culturing of the MCF-7 cancer cell line was
carried out as described in the literature [42, 43]. The
MCF-7 cells were cultured using Dulbecco’s modified
1
(5.07), 568 (3.06), 612 (3.55). H NMR (600 MHz,
-1
.
DMSO-d6) d 9.09–8.97 (m, 8H, Ar-H), 8.36–8.14 (m,
8H, Ar-H), 7.90–7.68 (m, 8H, Ar-H), 2.85–2.60 (m,
12H, CH3).
Eagles’ medium (DMEM) containing 4.5 g L glucose
with L-glutamine and phenol red, supplemented with
10% (v/v) heat-inactivated fetal calf serum (FCS) and
-1
-1
.
Synthesis of chloroindium (III) meso-tetra-2-
thienylporphyrin (2b). 2b was synthesized in the same
manner described for 2a by using 255 mg (0.4 mmol)
of H2TTP. Yield: 249 mg (97.6%). UV-vis (DMSO): lmax
5% 100 unit mL penicillin 100 µg mL streptomycin
amphotericin B. The cells were grown in a 25 cm2
vented flask (Porvair) and incubated in a humidified
5% CO2 atmosphere at 37°C until 70% confluence
was achieved. Standard trypsinization and cell seeding
were undertaken as described in the literature [42, 43].
1
nm (log e) 438 (5.50), 570 (2.51), 618 (1.88). H NMR
(600 MHz, DMSO-d6) dH ppm 9.32–9.12 (m, 8H, Ar-H),
8.35–8.27 (m, 4H, Ar-H), 8.26–8.04 (m, 4H, Ar-H),
7.73–7.60 (m, 4H, Ar-H).
Doses of 2a, 2a-AuNPs and 2b were administered
-1
.
over a concentration range of 3–40 µg mL by adding
Conjugation of 2a to gold nanoparticles (2a-AuNPs).
AuNPs were synthesized according to a reported
literature method [35]. The 2a-AuNPs nanoconjugate
was synthesized by adding 2a (10 mg, 0.01 mmol)
dissolved in 5 ml of chloroform into 20 mL of refluxing
toluene, followed quickly by AuNPs (4 mg) in 2 mL of
toluene. After 1 h of further heating at reflux, the solution
was stirred at room temperature for 24 h. The conjugate
was precipitated out of solution using methanol by
centrifugation for 10 min at 5000 rpm, then washed with
methanol and ethanol to remove unreacted 2a.
appropriate aliquots of stock solution prepared by
dissolving the drugs in DMSO and making up the volume
with supplemented DMEM as described in literature
[42, 43]. No irradiation was performed on the treated
cells for the in vitro dark cytotoxicity studies while the
PDT study involved 10 min irradiation with the Thorlabs
LED at 625 nm. A Zeiss AxioVert.A1 Fluorescence LED
inverted microscope was used for routine examination
of the cells. After 24 h of drug treatment, the cells
were washed with 100 µL DPBS and re-incubated in
fresh culture media. Post-treatment cell viability was
measured using the cell proliferation neutral red reagent
(WST-1 assay) on a Synergy 2 multi-mode microplate
reader (BioTek®) at a wavelength of 450 nm. The
percentage cell viability was determined as a function
of absorbance sample (drug treated) over absorbance
control (culture media only); both determined at 450 nm.
This is described in Equation 1:
Photophysical parameters
Fluorescence and singlet oxygen quantum yields.
The fluorescence quantum yield (FF) values for ClInTPP,
2a, 2b and the 2a-AuNPs nanoconjugate were determined
in DMSO using a comparative method described
previously in the literature [36]. Zinc(II) meso-substituted
tetraphenylporphyrin (ZnTPP) was used as the standard
(FF = 0.0397) [36]. The singlet oxygen quantum yield
(FD) values were also calculated using a comparative
method [37, 38] by using DPBF as a singlet oxygen
quencher in DMSO and Rose Bengal as the standard
(FD = 0.76) [39].
Theoretical calculations. Geometry optimizations of
the structures of the ClInTPP parent porphyrin complex,
2a and 2b were carried with the Gaussian 09 software
package [40] using the B3LYP functional with SDD
basis sets. The optimized B3LYP geometries were then
Absorbance of samples at 450nm
% CellViability =
× 100
Absorbance of control at 450nm
(1)
The experimental data obtained for in vitro and
photodynamic therapy studies were analyzed statistically.
Each experiment was carried out in triplicate, and the
analysis of variance (ANOVA) was evaluated for the
in vitro and photodynamic therapy data of the drugs
against MCF-7 cell line.
Copyright © 2019 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2019; 23: 3–12

4
R. C. SOY ET AL.
RESULTS AND DISCUSSION
Synthesis and characterization of 2a and 2b
Scheme 1 provides the synthetic routes for 2a and
2b. The complexes were characterized using UV-vis and
1H-NMR spectroscopy, elemental analysis and MALDI-
TOF mass spectrometry. The results obtained were
in close agreement with the proposed structures. The
1H-NMR spectrum of 2a has methyl proton peaks from
the meso-aryl substituents at 2.77 and 2.09 ppm, which
afforded 12 protons upon integration, while the aromatic
ring proton peaks for 2a and 2b lie in the 7.6−9.2 ppm
region and integrate to the expected number of protons.
Electronic absorption spectra of 2a and 2b
The p-MOs associated with the 16 atom 18 p-electron
inner ligand perimeter are arranged in an ML = 0, 1, 2,
3, 4, 5, 6, 7, 8 sequence in ascending energy terms
due to the angular nodal properties associated with the
porphyrin ring. Since the HOMO and LUMO have ML
values of 4 and 5, there are four and five angular nodal
planes (Fig. 1), respectively. Gouterman’s 4-orbital model
[44] predicts the presence an allowed B transition (DML =
1) at high energy and a forbidden Q transition (DML =
9) at lower energy on this basis. In order to facilitate a
comparison of the MOs of different complexes, Michl
[45] referred to p-MOs with angular nodal planes that lie
on the y-axis as the a and -a MOs, respectively, while the
corresponding MOs that have significant MO coefficients
on the y-axis are referred to as the s and -s MOs (Fig. 1).
The introduction of different meso-aryl groups to form
2a and 2b makes only relatively minor changes to the
electronic structure of the parent ClInTPP complex.
A stabilization is predicted for the frontier p-MOs of 2a
and 2b relative to those of ClInTPP. The smaller average
HOMO−LUMO gap of 2b when all four frontier p-MOs
Fig. 1. Angular nodal patterns of the a, s, -a and -s MOs of the
parent ClInTPP complex (TOP). MO energies of ClInTPP, 2a
and 2b (BOTTOM). The HOMO−LUMO gap energies and the
energy of the Q00 band are highlighted with red diamonds and
blue circles are plotted against the secondary axis
are taken into account arises primarily from there being
a larger stabilization of the -a and -s MOs and this makes
this dye potentially more suitable for use in PDT since
there is greater absorption in the therapeutic window.
The UV-vis absorption spectra of 2a and 2b (Fig. 2) in
DMSO are typical of metalloporphyrin spectra with four-
fold symmetry and are characterized by an intense B (or
Soret) band in the 400−450 nm region and weaker Q00
and Q01 bands further to the red for porphyrin complexes
[46]. The B band for 2a is observed at 435 nm, and the
two Q bands lie at 567 and 607 nm, while for 2b, these
bands are observed at 439, 573 and 618 nm, respectively.
There is a consistent red shift of the bands observed for
2b relative to those of 2a, due to the differing effects of
the meso-aryl substituents on the HOMO−LUMO gap
(Fig. 1). This has been attributed to the smaller size of the
meso-2-thienyl groups when compared to six-membered
phenyl rings [47, 48]. The fluorescence emission spectra
of 2a and 2b in DMSO are shown as insets in Fig. 2. The
emission spectra are typical of metalloporphyrins with
R
O
N
+
HN
N
Propionic Acid
N
H
R
H
R
R
120 °C 2 h
NH
Cl
R
1
R
InCl3 salt
Acetic acid/NaOAc
Reflux 16 h
N
N
In
R
R
S
2a =
2b =
N
N
R =
S
2
R
Scheme 1. The synthetic pathway for chloroindium (III) com-
plexes of tetra(4-methylthio)phenylporphyrin (2a) and tetra-2-
thienylporphyrin (2b)
Copyright © 2019 World Scientific Publishing Company
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PHOTOPHYSICOCHEMICAL PROPERTIES AND PHOTODYNAMIC THERAPY ACTIVITY OF CHLOROINDIUM(III)
5
inner perimeter of the porphyrin ring. MCD
spectroscopy has proven to be an important
technique for identifying the electronic
structures and state degeneracies of porphyrins
and related macrocycles, which cannot be
obtained from UV-vis absorption spectroscopy
alone [50, 51]. The MCD spectra of the
complexes showed cross-over points between
the negative and positive lobes of intensity at
564 nm and 607 nm for 2a and 574 nm and
618 nm for 2b, essentially corresponding to
the Q-band maxima observed in corresponding
UV-vis absorption spectra (Fig. 3). This is the
pattern that would normally be anticipated for
the Faraday A1 terms associated with the main
transitions of metal porphyrins with four-fold
symmetry, since the derivative shaped signals
arise from the Zeeman splitting of the orbitally
degenerate excited states that are predicted
in the TD-DFT calculations (Table 1) for the
Q and B transitions [32, 51].
Synthesis and characterization of AuNPs
and the 2a-AuNPs nanoconjugate
Scheme 2 illustrates the synthetic route for
conjugation of AuNPs to complex 2a. The
linkage of nanoparticles to the surface of 2a
is expected to occur via non-covalent Au−S
interactions due to the high affinity of these
atoms [23]. Porphyrins are approximately 1 nm in
diameter; hence, it is likely that more than one
porphyrin will be bonded onto the surface of
AuNPs (ca. 16 nm). The number of porphyrins
bonded to the AuNPs was estimated according
to the reported literature methods using
absorption instead of fluorescence [52]. This
Fig. 2. UV-vis absorption spectra of (a) H2MTPP, 2a and ClInTPP,
(b) H2TTP, 2b and ClInTPP in DMSO. Fluorescence spectra are provided
as insets with the same line types used as for the absorption spectra
involves comparing the absorbance intensity of
the Q bands of the porphyrin in the conjugate
with that of the porphyrin before conjugation
[53]. The loading of porphyrins (Ps) to the
nanoparticles (NPs) in mg (P)/mg (NP) was
determined to be 30 on this basis.
two bands of different intensities [49]. For 2a, the bands
were observed at 625 and 670 nm, while those of 2b are
observed at 643 and 689 nm. In a similar manner to the
absorption spectra, the fluorescence emission bands for
Electronic absorption spectra of 2a-AuNPs
The UV-vis absorption spectra of the AuNPs alone, 2a
and its nanoconjugate (2a-AuNPs) are shown in Fig. 4.
The surface plasmon resonance (SPR) band of theAuNPs
was observed at 536 nm. However, upon conjugation of
2a to the AuNPs, the shoulder of intensity arising from
the SPR band was observed at 523 nm, thus indicating an
apparent blue shift. The absorption spectra of 2a-AuNPs
nanoconjugate showed a significant blue shift of 6 nm
of the B band to 429 nm when compared to that of 2a
at 435 nm (Table 2). This could be due to close packing
attributed to the orientation of metalloporphyrins on
the surface of the nanoparticles [53–56]. A typical
2b are hence shifted to longer wavelengths.
TD-DFT calculations were carried out to analyze the
trends in the electronic structures and optical spectra of
2a and 2b (Fig. 3 and Table 1). The trends observed in the
calculated spectra are similar to those in the experimental
data, but there is a significantly smaller red shift
predicted for 2a relative to the parent In ClTPP complex
than was observed experimentally. This may be related
to there being a fixed geometry for the freely rotating
meso-aryl substituents in the calculations, which results
in an inaccurate prediction of their mesomeric effects
on the frontier MOs that are localized primarily on the
Copyright © 2019 World Scientific Publishing Company
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6
R. C. SOY ET AL.
Fig. 3. Absorption and MCD spectra of 2a (a) and 2b (b) in DMSO. The calculated TD-DFT spectra of 2a and 2b plotted against
secondary axis. The red diamonds highlight the Q and B bands associated with Gouterman’s 4-orbital model [44]. The details of
calculations are provided in Table 1
Table 1. Calculated UV-vis absorption spectra of the B3LYP optimized geometries of the parent
ClInTPP complex, 2a and 2b obtained using the CAM-B3LYP functional with SDD basis set
ClInTPP
b
Banda
#
Calcc
Expd
Wavefunctione =
Ground state
—
1
—
—
—
—
—
Q
2,3
4,5
17.8
563
(0.02)
16.6
603
60% s → -a/-s; 40% a → -a/-s; …
60% a → -a/-s; 40% s → -a/-s; …
B
26.2
367
(1.43)
23.4
428
2a
—
1
—
—
—
—
—
Ground state
Q
2,3
17.7
564
(0.03)
16.3
23.0
612
60% s → -a/-s; 40% a → -a/-s; …
B
4,5
27.1
369
(1.59)
435
59% a → -a/-s; 40% s → -a/-s; …
2b
—
Q
B
1
—
—
—
—
—
Ground state
2,3
17.5
572
(0.03)
16.2
618
59% s → -a/-s; 40% a → -a/-s; …
4,5
26.7
374
(1.40)
22.8
438
58% a → -a/-s; 38% s → -a/-s; …
a Band assignment described in the text. b The number of the state assigned in terms of ascending energy
c
3
-1
.
within the TD-DFT calculation. Calculated band energies (10 cm ), wavelengths (nm) and oscillator
d
3
-1
e
.
strengths in parentheses (f). Observed energies (10 cm ) and wavelengths (nm), The wave functions
based on the eigenvectors predicted by TD-DFT. One-electron transitions associated with the a, s, -a
and -s MOs are highlighted in bold.
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PHOTOPHYSICOCHEMICAL PROPERTIES AND PHOTODYNAMIC THERAPY ACTIVITY OF CHLOROINDIUM(III)
7
S
Cl
S
N
N
In
N
AuNPs
N
S
+
S
Toluene
24
h
S
S
Cl
S
Cl
N
S
N
N
In
N
In
N
AuNPs
N
N
N
S
S
S
S
Scheme 2. The synthetic pathway for 2a-AuNPs
Fig. 4. Absorption spectra of 2a-AuNPs (black) and 2a (red) and AuNPs (blue) in DMSO. Fluorescence spectra are provided as an
inset with the same line types used as for the absorption spectra
metalloporphyrin fluorescence emission spectrum was
observed for the 2a-AuNPs conjugate (Fig. 4 insert) with
bands at 617 and 662 nm. There is a 9 nm blue shift of the
emission bands relative to those in the spectrum of 2a.
Upon conjugation to form 2a-AuNPs, aggregation was
observed, and there was an increase in the average size
to 26 nm. This can be attributed to the interactions of
metalloporphyrins on adjacent nanoparticles via p–p
stacking [55].
Transmission electron microscopy
X-ray diffraction
The TEM micrographs of AuNPs and the 2a-AuNPs
nanoconjugate are shown in Fig. 5. The AuNPs were
monodispersed, and the average size was ca. 16 nm.
The X-ray diffraction (XRD) patterns of the AuNPs,
2a and 2a-AuNPs nanoconjugate are shown in Fig. 6.
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R. C. SOY ET AL.
Table 2. The photophysicochemical parameters of 2a, 2b and 2a-AuNPs conjugate in DMSO
l
Abs (nm)
SPR
Sizea
(nm)
P loading
(µg/mg)
lem
(nm)
FF
tF
(ns)
FD
B
Q01
Q00
2a
2b
435
438
428
—
566
573
561
—
607
618
601
—
—
—
—
—
—
16
26
—
—
—
—
30
625
643
646
—
0.010
0.007
0.05a
—
0.51
0.44
0.8a
—
0.54
0.73
0.72a
—
ClInTPP
AuNPs
—
536
523
2a-AuNPs 429
aValue from Ref. 47.
573
618
617
0.005
0.41
0.63
Fig. 5. Representative TEM micrographs of (a) AuNPs and (b) 2a-AuNPs
The XRD diffraction patterns for AuNPs showed well-
defined crystalline peaks which correspond to 111, 200,
220, 311, and 222 planes of the face centered-cubic
structures of metallic gold [57]. Peak broadening was
observed for 2a between 10 and 20° as would normally
be anticipated for amorphous porphyrin samples [57].
A similar peak broadening is observed between 10
and 20° for 2a-AuNPs along with the presence of the
crystalline peaks corresponding to AuNPs that are
consistent with the formation of a nanoconjugate.
Fluorescence quantum yields and lifetimes
Table 2 provides a summary of the photophysico-
chemical parameters of 2a, 2b, the parent ClInTPP
porphyrin complex and the 2a-AuNPs conjugate. The
fluorescence quantum yield and lifetime (tF) values were
measured in DMSO. A lower FF value was obtained for
2b than for 2a (Table 2). This can be attributed to the
Fig. 6. XRD diffractograms of 2a, 2a-AuNPs, and AuNPs
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PHOTOPHYSICOCHEMICAL PROPERTIES AND PHOTODYNAMIC THERAPY ACTIVITY OF CHLOROINDIUM(III)
9
Fig. 8. Representative spectra for 2a-AuNPs in DMSO during
the determination of the FD value with DPBF as a scavenger
Singlet oxygen quantum yields
Singlet oxygen is produced through an energy transfer
process between the excited triplet state of the PS and
ground state molecular oxygen [9]. In this study, the
FD value was determined by monitoring the chemical
Fig. 7. Representative fluorescence decay (black), c2 fitting
(red) and instrumental response function (blue) of 2a in DMSO
photodegradation of DPBF as a singlet oxygen quencher
in DMSO. Figure 8 shows the spectral changes observed
for the 2a-AuNPs nanoconjugate. The FD values ranging
from 0.54 for 2a to 0.73 for 2b (Table 2) reflect the very
low FF values of 0.010 for 2a and 0.007 for 2b, due to
the enhanced rate of intersystem crossing related to the
presence of the central In(III) ion. This, in turn, increases
the interaction between ground state molecular dioxygen
with the excited triplet state of the photosensitizer. There
is an increase in the FD value of 2a-AuNPs compared to
that of 2a as would be anticipated based on an external
heavy atom effect [60].
meso-2-thienyl substituents which enhance the rate of
intersystem crossing to the triplet manifold due to the
presence of sulfur atoms [28, 29]. Upon conjugation
of 2a to the AuNPs a further decrease in the FF value
was observed (Table 2). This is probably due to the
deactivation of the singlet excited state of 2a by the
AuNPs due to the external heavy atom effect, hence,
enhancing the rate of intersystem crossing to the triplet
state [58]. A typical fluorescence decay curve for 2a
is shown in Fig. 7. Mono-exponential curves were
used to derive the tF values for 2a and 2b. 2b was
found to have a shorter lifetime than 2a (Table 2). In
contrast, a biexponential decay curve was observed
for the 2a-AuNPs nanoconjugate. An average lifetime
is provided in Table 2, which is shorter than that of 2a
alone. The biexponential fluorescence decay could be
due to the presence of different orientations of 2a with
respect to the nanoparticles, which results in differing
interactions between the fluorophore and the free
electrons of the metallic surface [59]. This alters the
electric field around the molecules and may result in a
decrease or increase in fluorescence lifetimes depending
on the distance between and the relative orientations of
the molecules and metallic nanoparticles [54, 59]. These
data suggest that the observed reduction in the FF and tF
values following conjugation of 2a is related to external
heavy atom effect from the AuNPs [60].
Cell studies
In vitro dark cytotoxicity. In vitro dark cytotoxicity
investigations were carried out for 2a, 2b and the
2a-AuNPs conjugate using a range of concentrations
-1
.
from 3.0−40 mg mL . Histograms showing the PS
concentrations against percentage cell viability are
shown in Fig. 9. The photosensitizers considered in this
-1
.
study showed over 50% cell viability at 3.0−20 mg mL ,
suggesting that they were relatively innocuous against
MCF-7 breast cancer cells in the absence of irradiation.
The in vitro dark cytotoxicity of 2a and its 2a-AuNPs
nanoconjugate showed minimal cytotoxicity when
-1
.
compared to 2b at ≤ 40 mg mL (Fig. 10). Treatment
-1
.
with 2a resulted in cell viabilities ≥ 67% at ≤ 40 mg mL
while 2a-AuNPs exhibited cell viabilities ≥ 89% and
2b showed a slight cytotoxic effect with a cell viability
-1
.
value of 49% at 40 mg mL . This suggests that the
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10
R. C. SOY ET AL.
Fig. 9. Histograms showing percentage cell viability in the dark
and after irradiation of (a) 2a and (b) 2b
conjugation of 2a to AuNPs reduces the dark cytotoxicity
significantly at higher concentrations. Essentially, low
cell viability indicates higher dark cytotoxicity, which is
an undesirable feature for the PDT application since the
photosensitizer should only be cytotoxic in the presence
of light [9].
Fig. 10. Histograms showing percentage cell viability in the dark
and after irradiation of 2a-AuNPs (TOP) and 2a (CENTER), and
a comparison of the values obtained at 40 mg mL . (BOTTOM)
Photodynamic therapy activity. The investigation of
the PDT activities of 2a, 2b and the 2a-AuNPs conjugate
was carried out over the same range of concentrations
-1
.
-1
.
(3.0−40mg mL )thatwasusedforthedarktoxicitystudies
of the former. Although 2b has a higher FD value,
significant dark toxicity is observed during cell studies
with the MCF-7 cancer cell line. 2a exhibits an enhanced
FD value upon conjugation to AuNPs, and improved
dark cytotoxicity and phototoxicity properties are also
observed in this context. These results demonstrate the
potential utility of the 2a-AuNPs nanoconjugate for
application in PDT, since the nanoparticles also enhance
the delivery of the photosensitizer dye and its selective
accumulation in cancer tumors due to enhanced solubility
in aqueous solvents and the EPR effect.
and involved excitation with a Thorlabs 625 nm LED for
10 min (625 nm, dose of 144 J cm ). Treatment with 2a
resulted in greater than 79% cell death at ≤ 40 µg mL ,
-2
.
-1
.
while the use of 2a-AuNPs nanoconjugate and 2b resulted
in ≥ 89% and ≥ 82% cell death, respectively (Fig. 10).
Overall, the PDT activity of the 2a-AuNPs nanoconjugate
outperformed that of 2a as would be anticipated based on
the higher FD value (Table 2).
CONCLUSION
In this work, the chloroindium(III) complexes of
tetra(4-methylthiophenyl)porphyrin and tetra-2-thienyl-
porphyrin have been successfully synthesized and
characterized along with the AuNPs nanoconjugate
Acknowledgment
This work was supported by National Research Found-
ation (NRF) of South Africa SA-Kenya collaborative
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J. Porphyrins Phthalocyanines 2019; 23: 10–12

PHOTOPHYSICOCHEMICAL PROPERTIES AND PHOTODYNAMIC THERAPY ACTIVITY OF CHLOROINDIUM(III)
11
grant (UID: 105809) to JM and EA, the Department of
Science and Technology (DST) Nanotechnology (NIC)/
NRF) of South Africa through DST/NRF South African
Research Chairs Initiative to Professor of Medicinal
Chemistry and Nanotechnology (UID: 62620), Rhodes
University and the Organization for Women in Science
for the Developing World (OWSD) scholarship to RS.
The theoretical calculations were carried out at the
Centre for High Performance Computing in Cape Town.
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