A new porphyrin for the preparation of functionalized water-soluble gold nanoparticles with low intrinsic toxicity

Article abstract of DOI:10.1002/open.201402092

A potential new photosensitizer based on a dissymmetric porphyrin derivative bearing a thiol group was synthesized. 5-[4-(11-Mercaptoundecyloxy)-phenyl-10,15,20-triphenylporphyrin (PR-SH) was used to functionalize gold nanoparticles in order to obtain a potential drug delivery system. Water-soluble multifunctional gold nanoparticles GNP-PR/PEG were prepared using the Brust-Schiffrin methodology, by immobilization of both a thiolated polyethylene glycol (PEG) and the porphyrin thiol compound (PR-SH). The nanoparticles were fully characterized by transmission electron microscopy and ¹H nuclear magnetic resonance spectroscopy, UV/Vis absorption spectroscopy, and X-ray photoelectron spectroscopy. Furthermore, the ability of GNP-PR/PEGs to induce singlet oxygen production was analyzed to demonstrate the activity of the photosensitizer. Cytotoxicity experiments showed the nanoparticles are nontoxic. Finally, cellular uptake experiments demonstrated that the functionalized gold nanoparticles are internalized. Therefore, this colloid can be considered to be a novel nanosystem that could potentially be suitable as an intracellular drug delivery system of photosensitizers for photodynamic therapy.

Full text of DOI:10.1002/open.201402092

DOI: 10.1002/open.201402092
A New Porphyrin for the Preparation of Functionalized
Water-Soluble Gold Nanoparticles with Low Intrinsic
Toxicity
Oriol Penon,[a] Tania PatiÇo,[b] Lleonard Barrios,[b] Carme NoguØs,[b] David B. Amabilino,[c]
Klaus Wurst,[d] and Llu¨ısa PØrez-García*[a]
A potential new photosensitizer based on a dissymmetric por-
phyrin derivative bearing a thiol group was synthesized. 5-[4-
(11-Mercaptoundecyloxy)-phenyl-10,15,20-triphenylporphyrin
(PR-SH) was used to functionalize gold nanoparticles in order
to obtain a potential drug delivery system. Water-soluble mul-
tifunctional gold nanoparticles GNP-PR/PEG were prepared
using the Brust–Schiffrin methodology, by immobilization of
both a thiolated polyethylene glycol (PEG) and the porphyrin
thiol compound (PR-SH). The nanoparticles were fully charac-
terized by transmission electron microscopy and 1H nuclear
magnetic resonance spectroscopy, UV/Vis absorption spectros-
copy, and X-ray photoelectron spectroscopy. Furthermore, the
ability of GNP-PR/PEGs to induce singlet oxygen production
was analyzed to demonstrate the activity of the photosensitiz-
er. Cytotoxicity experiments showed the nanoparticles are non-
toxic. Finally, cellular uptake experiments demonstrated that
the functionalized gold nanoparticles are internalized. There-
fore, this colloid can be considered to be a novel nanosystem
that could potentially be suitable as an intracellular drug deliv-
ery system of photosensitizers for photodynamic therapy.
Introduction
Porphyrins and their derivatives are components present in
several complexes in nature such as haemoglobin, chlorophyll,
cobalamine, and bacterioclorophylls.[1] They are also one of the
most popular aromatic macrocycles in supramolecular chemis-
try because of their multiple functions.[2,3] Recently, the synthe-
sis of new porphyrin derivatives has been increasingly investi-
gated owing to their great potential in such diverse areas as
photochemistry, molecular recognition, sensors, and molecular
rotors, as well as in photodynamic cancer therapy.[4–8] Also, the
development of a synthetic porphyrin methodology for non-
symmetric systems is one of great interest because the existing
procedures to synthesize them are low yielding.[9–11] Especially,
the synthesis of dissymmetrical porphyrins presents an extra
difficulty because a mixture of porphyrins is generally ob-
tained, a fact that usually implies the need for complex purifi-
cation processes.[12–14]
Porphyrins are photosensitizers (PS) that can promote the
formation of singlet oxygen or free radicals that are highly
toxic locally at the cellular level.[15–17] The target tissue is typi-
cally irradiated with red light. PS can cause significant cellular
damage, destroy tumor blood vessels, and induce immunity
against the damaged tissues.[18] The accumulation of the PS in
the target region is one of the main difficulties associated with
photodynamic therapy (PDT) that makes use of the photosen-
sitizing capability of the porphyrins. More specifically, one of
the drawbacks of PDT is that the porphyrin structures that are
used generally are quite hydrophobic, which makes them diffi-
cult to formulate. However, the most serious disadvantage of
PDT is the nonspecific distribution of the PS in the body, which
leads to adverse effects upon a patient’s exposure to sunlight.
On the other hand, gold nanoparticles (GNPs) have raised
huge interest in the biomedical field because of their biocom-
patibility, their ready and versatile synthesis, as well as their
ability to act as drug delivery vehicles whereby suitable func-
tionalization can lead to site-specific accumulation.[19,20]
[a] Dr. O. Penon, Prof. L. PØrez-García
Departament de Farmacologia i Química Terap›utica,
and Institut de Nanoci›ncia i Nanotecnologia UB (IN2UB)
Universitat de Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona (Spain)
E-mail: mlperez@ub.edu
[b] T. PatiÇo, Prof. L. Barrios, Prof. C. NoguØs
Departament de Biologia Cellular
Fisiologia i Immunologia Universitat Autònoma de Barcelona
Campus de la UAB, 08193 Bellaterra (Spain)
[c] Prof. D. B. Amabilino+
Institut de Ci›ncia de Materials de Barcelona (ICMAB-CSIC)
Campus de la UAB, 08193 Bellaterra (Spain)
[d] Prof. K. Wurst
Institut für Anorganische Chemie
Innrain 80/82, 6020 Innsbruck (Austria)
[+] Present address: School of Chemistry, The University of Nottingham
University Park, Nottingham NG7 2RD (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/open.201402092.
ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
Therefore, photosensitizer-coated nanoparticles (NPs) are
a promising platform currently investigated to improve specif-
icity in PDT, while at the same time allowing the hydrophobici-
ty of the photosensitizer to be circumvented.[21] Despite the in-
terest in such systems, only a few examples combine NPs and
ChemistryOpen 2015, 4, 127 – 136
127
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

photosensitizers, specifically phtalocyanines and porphyr-
ins,[22–27] as well as incorporate an antibody against a receptor
present in breast cancer cells.[28] In particular, monopodal por-
phyrins containing a thiol group are amenable to be linked co-
valently to gold surfaces[29–31] as well as be coated on
GNPs.[32–34] Depending on the solubility of the photosensitizer,
either the Brust–Schiffrin[35] or the Turkevich[36] methods can be
used for GNPs preparation. Systems based on porphyrin-func-
tionalized GNPs for PDT have been reported where porphyrin-
coated GNPs were prepared, and their PDT efficacy was dem-
onstrated in vitro and in vivo.[37–39] In these published works,
protoporphyrin, hematoporphyrin, and brucine–porphyrin de-
rivatives were used to functionalize GNPs, indicating that these
porphyrin derivatives are more efficient for PDT when they are
immobilized on the GNPs, in comparison with the free ligands.
The Turkevich method was used in these examples, because
the porphyrin derivatives used are water-soluble.
assessed by inductively coupled plasma mass spectrometry
(ICP-MS) and TEM, respectively.
Results and Discussion
Synthesis and characterization of the photosensitizer PR-SH
The formation of the dissymmetrical porphyrin PR-OH, incor-
porating three phenyl groups and one hydroxyphenyl group
at the meso positions, was achieved by reaction of pyrrole
with a mixture of benzaldehyde and 4-hydroxybenzaldehyde
in propionic acid (Scheme 1), that gives a mixture of all possi-
ble meso-substituted porphyrins.
Two factors facilitated the isolation of PR-OH: first, acetoni-
trile washing allowed the elimination of all the impurities and
pyrrole secondary products, and secondly, two rounds of silica
gel column chromatography using dichloromethane and con-
sequently chloroform, gave good separation of the desired
porphyrin PR-OH (5.4%).[43]
PR-OH was isolated as a purple solid, and this material was
further purified by crystallization from acetonitrile, giving crys-
tals suitable for single-crystal X-ray characterization to know
As an alternative to the method that generated these earlier
particles, the Brust–Schiffrin method has been employed in
this work due to the possibility to immobilize non-water-solu-
ble ligands onto the GNPs, because a biphasic solution is used.
Thus, the capability to incorporate a lipophilic PS onto the
GNP surface was proven by preparing a double coating with
a porphyrin derivative and a polyethylene glycol
(PEG) that increases the water solubility of the nano-
system.
In this context, this study describes first the syn-
thesis of a new dissymmetrical porphyrin composed
of four phenyl groups at the meso positions, with
one of them incorporating an alkyl chain with a thiol
end group. Thus, 5-[4-(11-mercaptoundecyloxy)-
phenyl-10,15,20-triphenylporphyrin (PR-SH) was pre-
pared, and its capability to coat and stabilize water-
soluble GNPs have been investigated. The porphyrin
was chosen because of its similarity to second gener-
ation photosensitizers, previously studied in solution
for PDT,[40–42] and the thiol group allows its incorpora-
tion onto GNPs. Therefore, water-soluble GNPs (GNP-
PR/PEG) have been synthesized, coadsorbing the
thiol-modified porphyrin and a PEG-modified thiol
chain to the surface of the metal. GNPs were pre-
pared by the Brust–Schiffrin method providing a po-
tential water-soluble drug delivery system in order to
localize the activity of the photosensitizer. The same
methodology was used to synthesize GNP-PEG,
which were used for the control experiments. The
GNPs were characterized using a variety of tech-
niques such as proton nuclear magnetic resonance
spectroscopy (1H NMR), UV/Vis absorption spectros-
copy, transmission electron microscopy (TEM), and
X-ray photoelectron spectroscopy (XPS). Furthermore,
the production of singlet oxygen after irradiation was
measured, and the cytotoxicity of the GNPs was also
analyzed in human mammary epithelial cell cultures
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
Scheme 1. Stepwise synthesis of PR-SH. Reagents and conditions: a) propionic acid,
reflux, 2 h, 5%; b) 11-bromoundecyl thioacetate, K2CO3, CH3CN, 828C, 24 h, 67%; c) KOH,
tetrazolium bromide (MTT) proliferation assay kit. In-
ternalization and GNP localization inside cells were MeOH/H2O, rt, 3 h, 80%.
ChemistryOpen 2015, 4, 127 – 136
www.chemistryopen.org
128
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

the order and packing of the structure (see Supporting Infor-
mation).
and PEG, were isolated from the aqueous phase and were puri-
fied by sequential washing and centrifugation. In order to pro-
vide a model colloid for the biological control experiments,
GNP-PEG colloids were synthesized using the same methodol-
ogy described above, but without the incorporation of the
photosensitizer.
The thiolated chain was incorporated by the reaction of PR-
OH with 11-bromoundecyl thioacetate under basic conditions
yielding PR-SAc (67%). Subsequently, thioacetate PR-SAc was
deprotected using potassium hydroxide in a mixture of metha-
nol and water, giving the desired compound PR-SH as
a purple oil (80%) (Scheme 1). The thiol chain incorporated
into the porphyrin acts as a linker between the photosensitizer
and the GNP.
UV/Vis absorption spectroscopy
GNP formation and the presence of the porphyrin on the GNP
were confirmed by UV/Vis absorption spectroscopy. The GNPs
prepared here have a characteristic peak near 520 nm originat-
ing from their surface plasmon absorption.[35] Moreover, free-
base porphyrins also present five characteristic absorption
bands: the Soret band (near 420 nm) and four Q bands be-
tween 500 and 700 nm.[2] Figure 1 shows the UV/Vis spectra of
the free-base porphyrin PR-SH and GNP-PR/PEG. PR-SH shows
the typical porphyrin bands: the Soret band at 421 nm and the
Q bands at 515, 551, 593, and 646 nm. In the case of the GNPs,
the Soret band still appears at 421 nm, and although the spec-
tra are quite broad, weak absorption bands corresponding to
the four Q bands of the porphyrin can be identified. The typi-
cal surface plasmon resonance (SPR) peak of the GNP at
520 nm coincides with the lowest wavelength absorption peak
of the Q bands of the porphyrin. UV/Vis absorption spectrosco-
py results also demonstrate that the core of the porphyrin is
still metal-free once attached to the GNPs. During the synthesis
of the GNPs, gold atoms might be incorporated into the pyr-
role ring because it can complex the gold(III) ion.[44] When the
porphyrin complexes with gold, only one Q band appears; al-
though in the UV/Vis spectra for GNP-PR/PEG the four Q
bands were identified, demonstrating a metal-free porphyrin
The free porphyrins were identified and characterized using
1H NMR spectroscopy. Deprotection of porphyrin PR-SAc to
obtain PR-SH was monitored in deuterated chloroform. The
peak corresponding to the methyl group of the protected thiol
at 2.32 ppm was not present in the spectrum of the deprotect-
ed porphyrin (PR-SH), confirming the efficiency of the depro-
tection protocol. Furthermore, a shift of the peak correspond-
ing to the SÀCH2 (from 2.88 to 2.48 ppm) was observed, indi-
cating the presence of the SH group in PR-SH (Figure S1 in the
Supporting Information).
Preparation of gold nanoparticles (GNP-PR/PEG)
The preparation of water-soluble GNPs was performed using
the biphasic method described by Brust and Schiffrin because
of the low hydrophilicity of the porphyrin PR-SH (Scheme 2).
Tetraoctyl ammonium bromide (TOAB) was used as a phase-
transfer agent in order to allow the interaction between the
PR-SH, soluble in toluene, and the gold dissolved in water.
Thiol-terminated PEG was also incorporated to the aqueous
phase to increase the polarity of the final colloidal system.
GNP-PR/PEG, water-soluble NPs functionalized with the PR-SH
Scheme 2. Synthesis of GNP-PR/PEG. Reagents and conditions: a) HAuCl4·3H2O, NaBH4, TOAB, toluene/H2O, rt, 16 h.
ChemistryOpen 2015, 4, 127 – 136
www.chemistryopen.org
129
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

most intense peaks correspond to elements C and O, indicat-
ing the presence of the PEG chain on the GNPs. Furthermore,
typical peaks for the presence of Au appear at 88.5 and 85 eV
corresponding to the double peaks of Au 4f5/2 and Au 4f7/2.
These are typical values for Au0. Also, very-low-intensity peaks
appear in the S and the N1s regions, showing the presence of
pyrrolic nitrogen atoms (NÀH) that confirm the presence of the
porphyrin core.[48]
Transmission electron microscopy analysis
A morphologic and size study of the GNPs described here was
done using TEM. A drop of the GNP solutions was placed on
a carbon-coated copper grid for the analysis. Figure 2a shows
a representative TEM image of the porphyrin-bearing GNP-PR/
PEG. Spherical NPs with a narrow distribution, in terms of size
polydispersity and polymorphism, were observed. The corre-
sponding histogram shows diameters in the range of 3.5 to
4 nm (Figure 2b). The water-soluble NPs containing only PEG
(GNP-PEG) give a practically identical average core size be-
tween 3.5 and 4 nm (Figure S6 in the Supporting Information).
In both cases, the particles are well separated and in very few
Figure 1. UV/Vis absorption spectra of solutions of PR-SH (0.25 mm) (light
grey) and GNP-PR/PEG (0.4 mgmLÀ1) (dark grey). The spectra of PR-SH and
GNP-PR/PEG were recorded in CH2Cl2 and H2O, respectively. For PR-SH
(0.5 mm), magnification of the Q band region of the spectrum is also
shown.
pyrrole ring. Fluorescence of the GNP-PR/PEG was also exam-
ined, indicating a low-intensity emission.
Quantification of the amount of porphyrin per GNP was at-
tempted using the absorption spectra and taking into account
the diameter of the colloid determined by TEM (see section
below). First, a calibration line of PR-SH was obtained using
a range of concentrations between 25 mm and 0.5 mm (Fig-
ure S3 in the Supporting Information) in order to calculate its
extinction coefficient (e418 =38940). The wavelength selected
was that corresponding to the Soret band (418 nm) because it
was the most intense peak confirming the porphyrin that ap-
peared in the GNP spectrum.[45] The UV/Vis absorption spec-
trum of GNP-PR/PEG shows quite broad absorption bands,
and in order to normalize the absorbance value of the Soret
band, the background signal of the GNP core at 442 nm was
subtracted from the Soret band peak (Figure S4 in the Sup-
porting Information).
Accordingly, we calculated that the molarity of the porphyrin
present on the surface of the GNPs corresponds to 7.30 mm for
GNP-PR/PEG. In order to find the porphyrin to NP ratio, the
concentration of GNP was calculated using the diameter ob-
tained by TEM (3–4 nm) and its absorbance value at 450 nm
(0.56) obtaining a mean value of about 0.22 mm.[46] Using
a volume of 2 mL and Avogadro’s number, we get a ratio of
about 33 porphyrin adducts per GNP for GNP-PR/PEG.
X-ray photoelectron spectroscopy analysis
To confirm the formation of the GNPs and their functionaliza-
tion with PR-SH, XPS experiments were performed. Initially, in
order to detect the gold atoms, a sputtering of the sample of
GNP-PR/PEG was necessary to identify the peaks clearly.[47] The
sputtering is normally used when the NP is completely coated,
and the photoelectrons need to penetrate to detect the gold
atoms. Figure S5 in the Supporting Information shows the
binding energies of the XPS spectrum of GNP-PR/PEG, and the
Figure 2. Transmission electron microscopy (TEM) images of a) GNP-PR/PEG
and b) their corresponding size-distribution histogram.
ChemistryOpen 2015, 4, 127 – 136
www.chemistryopen.org
130
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

cases show short distances between particles, a good indica-
tion that they are well dispersed in water and can be used as
essentially single-particle delivery systems.
Measure of singlet oxygen production
In order to demonstrate the promising use of the GNP-PR/PEG
in PDT, its capacity to induce the production of reactive
oxygen species after laser irradiation was tested. The singlet
oxygen production was examined by measuring the fluores-
cence decay of 9,10-anthracenedipropionic acid (ADPA), be-
cause it is photobleached in the presence of singlet
oxygen.[49–51]
Thus, ADPA was added to a water solution of GNP-PR/PEG
(0.25 mgmLÀ1) and the sample was irradiated at 647 nm for
30 min. The sample was irradiated at 647 nm because it corre-
sponds into the porphyrin Q band nearest to the red region,
which is a wavelength region commonly used in PDT.[16]
Figure 3 shows the fluorescence spectra of ADPA, taken in
5 min intervals over 30 min, as well as the corresponding fluo-
Figure 4. Toxicity of GNP-PEG (dark grey) and GNP-PR/PEG (light grey). Cells
were incubated in the presence of different concentrations of GNPs for
24 h (top) or 72 h (bottom). The discontinuous line corresponds to 50% of
viable cells. Data represent the mean Æ SEM (n=3).
toxic than the GNP-PR/PEG after incubation at both 24 and
72 h. After 24 h of incubation, the IC50 was found at 15 mgmLÀ1
for GNP-PEG whereas for GNP-PR/PEG, it was at 200 mgmLÀ1
.
At 72 h, the IC50 was found at 15 mgmLÀ1 for GNP-PEG, and at
20 mgmLÀ1 for GNP-PR/PEG. According to literature,[54–56] the
cytotoxicity of GNPs depends on their size and surface proper-
ties (charge, ligand density, hydrophobicity) and on their con-
centration. As each published study has been done with differ-
ent GNP sizes, surface properties, concentrations, and different
cell lines, it is extremely difficult to compare the results ob-
tained in the present article with the published ones in a mean-
ingful way, although some trends can be found.
Figure 3. a) Fluorescence emission spectra of ADPA after irradiation of GNP-
PR/PEG at 647 nm for 30 min, and b) decay of ADPA fluorescence intensity
at 407 nm.
rescence intensity decay in the typical 407 nm peak of the
ADPA. After 10 min of irradiation, a significant decay in fluores-
cence intensity started, demonstrating the capacity of porphy-
rin-modified GNP to induce singlet oxygen production, making
it a promising candidate for PDT.
Regarding the size of the GNPs, on the one hand, citrate-sta-
bilized GNPs of 5 nm have been shown to be cytotoxic at
a concentration higher than 50 mm (10 mgmLÀ1), when incubat-
ed for 72 h with Balb/3T3 cells in an assay for colony forming
efficiency. However, using the same concentration of GNPs of
15 nm did not show cytotoxicity even at the highest concen-
tration tested—300 mm (58.8 mgmLÀ1).[57] On the other hand, it
has been described that cell viability of MCF-7 and PC-3 cells
was not decreased when incubated with citrate-capped GNPs
of different sizes (5, 6, 10, 17 and 45 nm) and only slightly de-
creased when incubated with similar sizes (3, 8 and 30 nm).
Thus, in that work GNP size was not related with viability at
least in a size-dependent manner.[58] In the present study, the
IC50 for GNP-PR/PEG is 200 mgmLÀ1 at 24 h and 20 mgmLÀ1 at
Cytotoxicity of GNP-PEG and GNP-PR/PEG
To assess the suitability of GNP-PEG and GNP-PR/PEG for PDT,
it is necessary to evaluate their potential cytotoxicity in living
cells. For this reason, MCF10A cells were chosen. The MTT
assay has been widely used[52,53] and recommended for the
evaluation of the cytotoxicity of NPs.[54]
After 24 and 72 h of culture, there was a clear and significant
dose-dependent cytotoxic effect for both types of NPs, starting
at 5 mgmLÀ1 (Figure 4). GNP-PEG was significantly more cyto-
ChemistryOpen 2015, 4, 127 – 136
www.chemistryopen.org
131
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

72 h in contrast with the GNP-PEG which is more cytotoxic
(IC50 =15 mgmLÀ1 at 24 and 72 h). In this case, the size of the
particles are similar, although GNP-PEG is slightly smaller than
GNP-PR/PEG; consequently, in the present study the differen-
ces in cytotoxicity are not related to GNP size.
3–5 nm GNP-PEG.[60,61] In both cases, as in the present work, in-
ternalization was analyzed by ICP-MS and TEM, demonstrating
that these GNPs can be internalized by different types of cells:
HeLa,[60] CT26,[61] and MCF10A (present work). The amount of
GNPs internalized varies within studies, probably due to type
of GNP-PEG used and its concentration.
Taking into account the surface properties, it must be point-
ed out that GNP-PEG has been used in several studies. It has
been described in literature that 30, 50, and 90 nm plain GNPs
stimulated cell proliferation, whereas PEGylated GNP spheres
of the same size did not interfere with proliferation of PC-3
cells at a concentration of 1.5 nm for 88 h.[59] Other works,
using GNP@MPA-PEG of 3.5 nm diameter at different concen-
trations (0.08–10 mm) found that cytotoxicity was dose-depen-
dent, obtaining a 70% viability when incubated with HeLa
cells for 72 h using the highest concentration.[60] On the other
hand, no cytotoxicity was observed when CT26 cells were incu-
bated with 4.7 nm GNP-PEG for 48 h at high concentrations
(500–3000 mm).[61] Finally, GNPs functionalized with different PS
have also been tested for cell viability. GNP-porphyrin–brucine
(14.7 nm) was not cytotoxic at 1 or 2.5 mm using PE/CA-PJ34
cells,[32] whereas GNP-hematoporphyrin (15 and 45 nm) incu-
bated with MT-4 cells was cytotoxic in a dose-dependent
manner (different percentages of hematoporphyrin and Au
were tested).[33] Our results are in agreement with a dose-de-
pendent cytotoxicity of GNPs. However, a lower cytotoxicity
was found for GNP-PR/PEG when compared with GNP-PEG,
that could be explained, at least in part, by the presence of
porphyrin which reduces the number of PEG molecules and
probably their cytotoxicity, because it is known that photosen-
sitizers are not toxic by themselves.[22] Taking all these results,
we can confirm that GNP-PR/PEG does not affect cell viability
at low concentrations of NPs (2–10 mgmLÀ1), and thus it is
a good candidate for PDT.
Analysis of the ultrathin sections of MCF10A cells incubated
with 100 mgmLÀ1 GNPs for 24 h has shown that both kinds of
NPs, GNP-PEG and GNP-PR/PEG, have been taken up by the
cells, and that, once inside, they are not aggregated. This is rel-
evant because it has been described that the cytotoxicity of
GNPs biofunctionalized with polyvinyl pyrrolidone (GNP-PVP)
on HeLa cells was caused by NP aggregation rather than parti-
cle size.[62] Small size (2 nm) NPs were not cytotoxic and did
not aggregate inside HeLa cells in contrast with 25 nm GNP-
PVP, which caused 50% cell death after 1 day in culture and
presented large aggregates in TEM sections. NP clustering is
an added problem when internalization uptake is compared
since it is not known if aggregates are formed prior to or
during interaction with the membrane.[55]
On the other hand, it has been suggested that it is necessary
to produce enough free energy to bind NPs to the surface of
the cell membranes; NPs smaller than 14 nm need to aggre-
gate with other NPs in order to be able to wrap the mem-
brane, while NPs larger than 50 nm can reach the membrane
and be endocytosed individually by cells.[63] That would explain
why GNP-PEG and GNP-PR/PEG, which are smaller than
50 nm, are internalized less efficiently. Owing to the small size
and nonaggregation of the NPs of the present study, it was dif-
ficult to observe these particles inside the cells, but both
types, GNP-PEG and GNP-PR/PEG, were found either in the cy-
toplasm, in vesicles, in the Golgi, and also in the nucleus
(Figure 5). Some authors have found large aggregates of 5 nm
and 15 nm GNPs inside vesicles, but not in other subcellular or-
ganelles.[57] Others have found that 3.5 nm GNP@MPA-PEG can
Uptake of GNP-PEG and GNP-PR/PEG
The uptake of GNP-PEG and GNP-PR/PEG was analyzed by
means of TEM and ICP-MS. Whereas TEM determines the locali-
zation of NPs inside cells, ICP-MS enables quantification of NPs
internalized by the cells. The total amount of Au present in the
cells (pellet) and in the culture medium (together with phos-
phate-buffered saline washes) was quantified using ICP-MS.
When cells were incubated for 24 h with 200 mgmLÀ1 of GNP-
PEG, 1.97% of the total amount of Au was detected in the
pellet, indicating that some GNP-PEG was internalized by cells.
In the same conditions, the cells were able to internalize
0.72% of the total amount of the gold present in 200 mgmLÀ1
of GNP-PR/PEG. Thus, the cells internalized more Au from
GNP-PEG than from GNP-PR/PEG. This could explain, in part,
the higher cytotoxicity of GNP-PEG in relation to GNP-PR/PEG.
However, the IC50 of GNP-PEG was 15 mgmLÀ1, whereas the
IC50 of GNP-PR/PEG was more than 10 times this value
(200 mgmLÀ1).
Concerning cell uptake of GNP-PEG, it has been described
that PEG prevents nonspecific interactions of serum proteins
with NPs and, in turn, their recruitment and internalization,[46]
but at least two studies have reported the internalization of
Figure 5. Transmission electron microscopy (TEM) images of MCF10A cells
incubated with GNP-PEG (a,b) or GNP-PR/PEG (c,d) at 24 h. Intracellular dis-
tribution: Golgi (a), cytoplasm (b), vesicles (c,d). Bar=200 nm.
ChemistryOpen 2015, 4, 127 – 136
www.chemistryopen.org
132
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

madzu, Kyoto, Japan). Matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was per-
formed using a Voyager-DE-RP (Appplied Biosystems, Framingham,
USA) mass spectrometer, and high-resolution mass spectra (HRMS)
were obtained with electrospray ionization (ESI) on a liquid chro-
matograph/mass-selective detector time-of-flight LC/MSD-TOF
mass spectrometer (Agilent Technologies, Santa Clara, USA) at the
Centres Cientifics i T›cnològics de la Universitat de Barcelona
(CCiT-UB). MS analysis was operated in the delayed-extraction
mode using 2,5-dihydroxybenzoic acid as a matrix. TEM experi-
ments were performed at the CCiT-UB. The samples were observed
with a Tecnai SPIRIT microscope (FEI Co., Hillsboro, USA) at 120 kV.
The images were captured by a Megaview III camera and digital-
ized with the iTEM program. The size of the gold core of the NPs
was measured with the analysis software (Olympus). XPS experi-
ments were performed in a PHI 5500 Multitechnique System (Phys-
ical Electronics, Chanhassen, USA) with a monochromatic X-ray
source (aluminum K-alpha line of 1486.6 eV energy and 350 W),
placed perpendicular to the analyzer axis and calibrated using the
3 d5/2 line of Ag with a full width at half maximum (FWHM) of
0.8 eV. The analyzed area was a circle of 0.8 mm diameter. The se-
lected resolution for the spectra was 187.5 eV of pass energy and
0.8 eV/step for the general spectra and 23.5 eV of pass energy and
0.1 eV/step for the spectra of the different elements. All measure-
ments were made in an ultra-high vacuum (UHV) chamber with
a pressure between 5”10À9 and 2”10À8 torr. Fluorescence spec-
troscopy measurements were performed using a Jobin–Yvon SPEX
Nanolog-TM spectrofluorometer (Horiba, Kyoto, Japan).
reach the nuclear envelope forming large aggregates, clusters
of few NPs, and even some individual ones.[60] It has also been
described that plain spherical GNPs of 30 nm are internalized
mainly in clusters, and, once inside the cells, NPs can be seen
as single particles or as clusters inside vesicles, free in the cyto-
sol, or even in the nucleus.[59] Finally, individual particles and
aggregations of GNP-PVP of 2, 10, and 25 nm in the cyto-
plasm, mitochondria, and vesicles have been also described.[62]
Overall, we can conclude that the surface properties of the
GNP-PEG and GNP-PR/PEG prevent their clustering, hamper-
ing their uptake by MCF10A cells. However, we have demon-
strated that they can be internalized, and that some particles
of GNP-PR/PEG were individually localized in the cytoplasm.
Conclusions
A new thiolated porphyrin (PR-SH) has been synthesized in
only a few steps. The capability of the porphyrin to be at-
tached to gold nanoparticles (GNPs) as well as to stabilize
them has been demonstrated. The corresponding GNP-PR/PEG
gold colloid was prepared using the Brust–Schiffrin method in-
corporating the photosensitizer and a polyethylene glycol
(PEG) chain in order to make the nanoparticles water soluble.
Transmission electron microscopy images of GNP-PR/PEG have
been obtained showing well-dispersed spherical nanoparticles
with a size near 3–4 nm. The coverage of the functionalized
tetrapyrrole on the GNPs is approximately 30 porphyrins per
nanoparticle. The capability of the porphyrin-modified GNP to
induce singlet oxygen production was tested by observing
9,10-anthracenedipropionic acid (ADPA) photobleaching upon
irradiation.
5-[4-Hydroxyphenyl]-10,15,20-triphenylporphyrin (PR-OH): Dry
pyrrole (4.57 mL, 65.6 mmol) was added to propionic acid (100 mL,
1408C) at reflux. Subsequently, p-hydroxybenzaldehyde (2 g,
16.4 mmol) and benzaldehyde (5 mL, 49.2 mmol) were added to
the solution at reflux, and the reaction mixture was stirred for 2 h.
Then, propionic acid was evaporated in vacuo, and the crude mix-
ture was redissolved in EtOAc and washed with H2O (3”100 mL)
and with a solution of 5% NaHCO3 (3”100 mL). Then the organic
phase was dried with MgSO4, filtered, and the solvent was evapo-
rated in vacuo. CH3CN (70 mL) was added and a purple solid was
obtained by filtration. The mixture of porphyrins was separated by
silica gel column chromatography using CH2Cl2 as the eluent, fol-
lowed by column chromatography in CHCl3 giving PR-OH as
a purple solid (560 mg, 5.4%);[43] mp>3008C; 1H NMR (300 MHz,
CDCl3): d=8.87 (d, J=4.8 Hz, 2H, pyr’.), 8.83 (s, 6H, pyr.), 8.21 (dd,
J=7.3, 1.9 Hz, 6H, H2,6), 8.05 (d, J=8.1 Hz, 2H, H2’,6’), 7.78–7.70 (m,
9H, H3,4,5), 7.15 (d, J=8.0 Hz, 2H, H4’,6’), À2.69–À2.83 ppm (m, 2H,
NH); UV/Vis (CH2Cl2): lmax =412.5, 515, 550.0, 590, 647.5 nm; MS-
MALDI-TOF: m/z=631.2 [M+H]+.
Cytotoxicity experiments were carried out with the GNP-PR/
PEG with GNP-PEG as a control. IC50 for GNP-PR/PEG and
GNP-PEG after 24 h were 200 mgmLÀ1 and 15 mgmLÀ1, respec-
tively, demonstrating that they are nontoxic. Finally, uptake ex-
periments using MCF10A cells demonstrated the internaliza-
tion of the modified GNPs. Therefore, GNP-PR/PEG is novel
nanosystem that could be potentially suitable as a drug deliv-
ery system for photodynamic therapy.
Experimental Section
Solvents and reagents: propionic acid, EtOAc, hexane, distilled H2O,
CH2Cl2, CHCl3, CH3CN, MeOH, toluene, and absolute EtOH were an-
alytical grade. Commercial compounds: 9,10-anthracenedipropion-
ic acid (ADPA), pyrrole, p-hydroxybenzaldehyde, benzaldehyde,
S-(11-bromoundecyl) thioacetate, HAuCl4·3 H2O, tetraoctyl ammoni-
um bromide (TOAB), and NaBH4 were purchased from Sigma–
Aldrich. NaHCO3, K2CO3, and KOH were purchased from Panreac. a-
Thio-w-hydroxy polyethylene glycol (487 Da) (PEG) was purchased
from Prochimia.
5-[4-(11-Undecyloxythioacetate)-phenyl]-10,15,20- triphenylpor-
phyrin (PR-SAc): Dry CH3CN (60 mL) was heated to 828C in an Ar
atmosphere, and K2CO3 (55.2 mg, 0.8 mmol) was added. The solu-
tion was left to stir, and after 30 min, PR-OH (165 mg, 0.262 mmol)
and 11-bromoundecyl thioacetate (243 mL, 0.8 mmol) were added.
After 24 h, the reaction was cooled to rt, filtered, and evaporated
in vacuo. The crude mixture was dissolved in CH2Cl2 and washed
with H2O (3”100 mL). Then, the organic phase was dried with
MgSO4 and after filtration the solvent was evaporated in vacuo.
The product was isolated by silica gel column chromatography
using hexane/EtOAc (7:3) as eluent, obtaining PR-SAc as a purple
Instrumentation: 1H NMR and 13C NMR spectra were recorded
using a 400 MHz spectrometer (Varian, Palo Alto, USA). Chemical
shifts (d) are expressed in ppm relative to the solvent peak (CDCl3).
Thin-layer chromatography (TLC) was performed on F254-silica-gel-
coated plates from Merck. Column chromatography was carried
out on silica gel 60 (Merck 9385, 230–400 mesh).UV/Vis absorption
spectra were obtained using a UV-1800 spectrophotometer (Shi-
1
oil (151 mg, 67%); H NMR (400 MHz, CDCl3): d=8.89 (d, J=4.8 Hz,
2H, pyr’.), 8.84 (d, J=2.4 Hz, 6H, pyr.), 8.23–8.20 (d, J=7.2, 6H,
H2,6), 8.13–8.09 (d, J=8.5 Hz, 2H, H2’,6’), 7.79–7.74 (m, 9H, H3,4,5),
7.29–7.26 (d, J=8.4 Hz, 2H, H4’,6’), 4.25 (t, J=6.5 Hz, 2H, O-CH2),
ChemistryOpen 2015, 4, 127 – 136
www.chemistryopen.org
133
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

2.88 (t, J=7.3 Hz, 2H, CH2-S), 2.32 (s, 3H, SCOCH3), 2.03–1.93 (m,
2H, CH2CH2O-), 1.60 (m, 4H), 1.40–1.32 (m, 12H, -CH2-), À2.76 ppm
(s, 2H, NH); UV/Vis (CH2Cl2): lmax =412.5, 517, 556.5, 597.5,
649.5 nm; MS-MALDI-TOF: m/z=859.4 [M+H]+.
(Sigma), 100 ngmLÀ1 cholera toxin (Sigma), and 10 mgmLÀ1 insulin
(Gibco) at 378C and 5% CO2. Cells were split 1:3 every 72 h.
Cytotoxicity of GNP-PEG and GNP-PR/PEG: Cytotoxicities of GNP-
PEG and GNP-PR/PEG were determined using the Vybrant MTT
Cell Proliferation Assay Kit (Molecular Probes), following the manu-
facturer’s instructions. MCF10A cells were seeded in 96-well dishes
at a density of 5000 cells/well and grown for 72 h when confluence
was reached. Then, cells were incubated with GNP-PEG or GNP-
PR/PEG at six different concentrations: 2, 5, 10, 15, 20, or
200 mgmLÀ1. After 24 or 72 h, the medium was replaced with fresh
culture medium (100 mL) and a stock solution of MTT (10 mL of
12 mm) and incubated for 2 h. Afterwards, the medium was re-
moved leaving only 25 mL, and then DMSO (50 mL) was added.
Cells were incubated for 10 min at 378C and 5% CO2 to solubilize
the formazan crystals. The measurement of absorbance was done
at 570 nm using a Victor 3 Multilabel counter (PerkinElmer, Wal-
tham, USA). For each treatment, viability was calculated as the ab-
sorbance of treated cells divided by the absorbance of untreated
control cells. IC50 values (concentration of NPs that resulted in 50%
of cell viability) were obtained from three independent experi-
ments.
5-[4-(11-Mercaptoundecyloxy)-phenyl-10,15,20-triphenylpor-
phyrin (PR-SH): Porphyrin PR-SAc (151 mg, 0.175 mmol) was dis-
solved in a mixed solution of CHCl3 (50 mL) and MeOH (15 mL).
KOH (1 g) was dissolved in H2O/MeOH solution (5 mL, 50% MeOH),
and the basic solution was added to the porphyrin solution and
was left to stir at rt. The degree of hydrolysis was monitored by
TLC. After the completion of the hydrolysis (2 h), H2O was added
to the solution, and the reaction was stirred for 1 h. The organic
phase was then washed with H2O until the pH of the aq phase
became neutral. The final product was obtained by evaporating
the solvent from the organic phase, giving PR-SH as a purple oil
(114 mg, 80%); 1H NMR (400 MHz, CDCl3): d=8.90 (d, J=4.8 Hz,
2H, pyr’.), 8.84 (s, 6H, pyr.), 8.22 (d, J=7.2, 6H, H2,6), 8.12 (d, J=
8.5 Hz, 2H, H2’,6’), 7.76 (m, 9H, H3,4,5), 7.29 (d, J=8.4 Hz, 2H, H3’,5’),
4.25 (t, J=6.3 Hz, 2H, O-CH2), 2.56 (t, J=7.3 Hz, 2H, CH2-SH), 2.05–
1.92 (m, 2H, CH2CH2O-), 1.68–1.35 (m, 16H), À2.75 ppm (s, 2H,
NH); 13C NMR (101 MHz, CDCl3): d=136.58, 133.51, 129.58, 126.41,
125.62, 125.43, 124.12, 117.56, 67.28, 48.42, 36.08, 33.26, 31.73,
30.91, 29.60, 29.01, 28.68, 28.64, 28.56, 28.47, 28.34, 26.95, 26.06,
ICP-MS analysis: Cells were seeded in 24-well dishes at a density
of 3”104 cells/well. After 48 h, GNP-PR/PEG or GNP-PEG was
added to the culture medium at a concentration of 200 mgmLÀ1
and incubated for 24 h. After co-incubation, the cell culture
medium containing GNPs was collected, and the attached cells
were washed with phosphate-buffered saline (PBS) twice to
remove the GNPs that adhered to the plasma membrane. Then,
cells were harvested by trypsinization and collected in a separate
tube. Cells were collected by centrifugation, and the cell pellet was
digested in aqua regia (HNO3:HCl 1:3) at 1508C for 20 min. After
that, the amount of gold in the cells and culture medium was de-
termined on an Agilent 7500ce ICP-MS (Agilent, Santa Clara, USA).
23.44, 21.67, 18.70, 18.64, 16.62, 13.09 ppm; UV/Vis (CH2Cl2): lmax
=
412.5, 514.5, 551, 592.5, 645.5 nm; MS-MALDI-TOF: m/z=817.4
[M+H]+; HRMS-ESI m/z [M+H]+ calcd for C55H52N4OS: 817.3856,
found: 817.3935.
Water-soluble porphyrin/PEG-functionalized GNPs (GNP-PR/
PEG): Water-soluble GNPs were synthesized using the Brust–Schif-
frin method.[35] HAuCl4·3 H2O (63 mg, 0.16 mmol) was dissolved in
H2O (5 mL) and added to a stirred solution of tetraoctyl ammonium
bromide (202 mg, 0.37 mmol) in toluene (10 mL). Next, PR-SH
(20 mg, 0.021 mmol) was dissolved in toluene (1.5 mL) and added
to the stirred solution, and subsequently thiol-PEG (20 mg,
0.042 mmol) was added to the biphasic solution. An excess of
aqueous reducing agent NaBH4 (45 mg, 1.26 mmol) in H2O (5 mL)
was also added to the mixture dropwise. The reaction was allowed
to occur while stirring at rt overnight. The aq phase of the result-
ing dark red solution was separated using an extraction funnel and
was subjected to H2O removal in a rotary evaporator, followed by
multiple washings with EtOH (3”5 mL) using centrifugation. Final-
ly, a red solid (137 mg) was obtained which was dissolved in H2O
(5 mL) giving GNP-PR/PEG that was characterized by UV-Vis, TEM,
TEM analysis: To analyze the intracellular location of GNPs, 1”106
MCF10A cells were seeded in a 9 cm2 plate and grown for 48 h. Af-
terwards, GNP-PEG or GNP-PR/PEG (100 mgmLÀ1) was added to
the culture. After 24 h in the presence of the GNPs, cells were fixed
with 2.5% glutaraldehyde in phosphate buffer (PB) for 1 h at 48C,
washed with PB, and postfixed with 1% OsO4 in PB containing
0.8% K3[Fe(CN)6] at 48C. Then, samples were dehydrated in ace-
tone, infiltrated with Epon resin (Electron Microscopy Sciences) for
48 h, embedded in the same resin, and polymerized at 608C for
48 h. Ultrathin sections were obtained using a Leica Ultracut UC6
ultramicrotome (Leica Microsystems, Wetzlar, Germany) and
mounted on Formvar-coated copper grids. They were gently
stained with 2% uranyl acetate in H2O for 10 min. Finally, sections
were observed in a Jeol-J1010 electron microscope (Jeol Ltd,
Tokyo, Japan) equipped with a SIS Megaview III CCD camera and
with a Jeol-JEM 2011 electron microscope (Jeol Ltd, Tokyo, Japan)
(operated at 200 kV) equipped with energy dispersive X-ray (EDX)
compositional analysis.
1
XPS, and H NMR. Synthesis of the GNP-PEG was performed as was
described before for the GNP-PR/PEG but the porphyrin was not
added.
Singlet oxygen experiments: Singlet oxygen formation was moni-
tored using fluorescence spectroscopy determining the fluorescent
decay of ADPA upon irradiation. ADPA (100 mL of a 1.2 mm solution
in MeOH) was added to GNP-PR/PEG (3 mL of 0.25 mgmLÀ1 in
H2O). The mixture was added to a quartz cuvette with a length of
1 cm, and it was irradiated at 647 nm using the laser source of the
fluorimeter (5 slits) for a duration of 30 min. Every 5 min, fluores-
cence emission spectra of the mixture was recorded between 365
and 600 nm (lex =355 nm).
Statistical analysis: Data were analyzed using IBM SPSS Statistics
(Ver. 19.0; Oregon, USA). First, all data were tested for normality
and homoscedasticity by using the Kolmogorov–Smirnov and
p
Levene tests. In the case of data at 24 h, a previous arcsin
x
transformation on viability percentages was applied to accomplish
the parametric assumptions. Then, one-way analysis of variance
(ANOVA) was run followed by a post-hoc ScheffØ test for pairwise
comparisons. For data at 72 h, no linear transformation remedied
the heteroscedasticity, and in this case the Kruskal–Wallis and
Mann–Whitney tests were applied as an alternative to parametric
Cell culture: The MCF10A nontumorigenic epithelial cell line was
purchased from the American Type Culture Collection (ATCC) and
maintained in Dulbecco’s modified eagle medium F12 (DMEM/F12)
(Gibco) supplemented with 5% horse serum (Gibco), 20 ngmLÀ1
epidermal growth factor (Gibco), 0.5 mgmLÀ1 hydrocortisone
ChemistryOpen 2015, 4, 127 – 136
www.chemistryopen.org
134
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

[22] D. K. Chatterjee, L. S. Fong, Y. Zhang, Adv. Drug Delivery Rev. 2008, 60,
1627–1637.
[23] L. BekalØ, S. Barazzouk, S. Hotchandani, J. Mater. Chem. 2012, 22, 2943–
2951.
statistics. In all the cases, the level of significance was set at
p <0.05, and data are shown as means Æ standard error of the
mean (SEM).
[24] D. Bechet, P. Couleaud, C. Frochot, M.-L. Viriot, F. Guillemin, M. Barberi-
Heyob, Trends Biotechnol. 2008, 26, 612–621.
[25] Y. N. Konan, M. Berton, R. Gurny, E. AllØmann, Eur. J. Pharm. Sci. 2003,
18, 241–249.
Acknowledgements
[26] G. Obaid, I. Chambrier, M. J. Cook, D. A. Russell, Angew. Chem. Int. Ed.
2012, 51, 6158–6162; Angew. Chem. 2012, 124, 6262–6266.
[27] M. Camerin, M. Magaraggia, M. Soncin, G. Jori, M. Moreno, I. Chambrier,
M. J. Cook, D. A. Russell, Eur. J. Cancer 2010, 46, 1910–1918.
[28] T. Stuchinskaya, M. Moreno, M. J. Cook, D. R. Edwards, D. A. Russell, Pho-
tochem. Photobiol. Sci. 2011, 10, 822–831.
[29] J. Yang, M. Li, H. Li, Y. Yang, Y. Kashimura, C. Wang, K. Torimitsu, X. Lu,
W. Hu, J. Phys. Chem. C 2010, 114, 12320–12324.
[30] G. Zuo, X. Lu, Z. Xue, B. Lv, Y. Wang, J. Kang, Synth. React. Inorganic,
Met. Nano-Metal Chem. 2006, 36, 589–594.
The authors acknowledge financial support from the Ministerio
de Ciencia e Innovación (MICINN), Spain (project TEC2011–29140-
C03–02 and 03) and the Generalitat de Catalunya, Spain (2009
SGR 158, 2009 SGR 00282). T.P. thanks the Generalitat de Catalu-
nya for the AGAUR FI-DGR 2011 predoctoral grant. The authors
wish to thank the staff at the Servei de Microscòpia of the Uni-
versitat Autònoma de Barcelona and the staff at the Unitat de
Microscòpia Electrònica i Reconeixement Molecular in Situ. Spe-
cial thanks goes to Carmen López at the Centres Cientifics i T›c-
nològics de la Universitat de Barcelona (CCiT-UB) and Dr. Laura
Rodríguez from the Universitat de Barcelona.
[31] Y.-H. Chan, A. E. Schuckman, L. M. PØrez, M. Vinodu, C. M. Drain, J. D.
Batteas, J. Phys. Chem. C 2008, 112, 6110–6118.
[32] J. Ohyama, Y. Hitomi, Y. Higuchi, M. Shinagawa, H. Mukai, M. Kodera, K.
Teramura, T. Shishido, T. Tanaka, Chem. Commun. 2008, 6300–6302.
[33] F. Canitez, M. Yavuz, R. Ozturk, J. Nanopart. Res. 2011, 13, 7219–7228.
[34] T. Hasobe, H. Imahori, P. V. Kamat, T. K. Ahn, S. K. Kim, D. Kim, A. Fujimo-
to, T. Hirakawa, S. Fukuzumi, J. Am. Chem. Soc. 2005, 127, 1216–1228.
[35] M. Brust, M. Walker, D. Bethell, D. J. Schiffrin, R. Whyman, J. Chem. Soc.
Chem. Commun. 1994, 801–802.
Keywords: drug delivery · gold nanoparticles · photodynamic
therapy · photosensitizers · porphyrins
[36] J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, A. Plech, J. Phys.
Chem. B 2006, 110, 15700–15707.
[1] R. Lemberg in Fortschritte Der Chemie Org. Naturstoffe/Prog. Chem. Org.
Nat. Prod./ProgrØs Dans La Chim. Des Subst. Org. Nat. (Ed.: L. Zechmeis-
ter), Springer, Vienna, 1954, pp. 299–349.
[2] K. M. Kadish, K. M. Smith, R. Guilard, Handbook of Porphyrin Science:
Supramolecular Chemistry, World Scientific Publishing Company, Singa-
pure, 2010.
ˇ
ˇ
[37] K. Zµruba, J. Krµlovµ, P. Rezanka, P. Pouckovµ, L. Veverkovµ, V. Krµl, Org.
Biomol. Chem. 2010, 8, 3202–3206.
[38] N. F. Gamaleia, E. D. Shishko, G. A. Dolinsky, A. B. Shcherbakov, A. V. Usa-
tenko, V. V. Kholin, Exp. Oncol. 2010, 32, 44–47.
[39] S. C. Hayden, L. A. Austin, R. D. Near, R. Ozturk, M. A. El-Sayed, J. Photo-
chem. Photobiol. A 2013, 269, 34–41.
[3] I. Beletskaya, V. S. Tyurin, A. Y. Tsivadze, R. Guilard, C. Stern, Chem. Rev.
2009, 109, 1659–1713.
[40] R. R. Allison, G. H. Downie, R. Cuenca, X.-H. Hu, C. J. H. Childs, C. H.
Sibata, Photodiagnosis Photodyn. Ther. 2004, 1, 27–42.
[41] M. G. H. Vicente, J. Osterloh, J. Porphyrins Phthalocyanines 2002, 6, 305–
324.
[42] S. Banfi, E. Caruso, L. Buccafurni, R. Murano, E. Monti, M. Gariboldi, E.
Papa, P. Gramatica, J. Med. Chem. 2006, 49, 3293–3304.
[43] E. BrulØ, Y. R. de Miguel, K. K. Hii, Tetrahedron 2004, 60, 5913–5918.
[44] A. Antipas, D. Dolphin, M. Gouterman, E. C. Johnson, J. Am. Chem. Soc.
1978, 100, 7705–7709.
[4] S. M. B. Costa, S. M. Andrade, D. M. Togashi, P. M. R. Paulo, C. A. T. Laia,
M. Isabel Viseu, A. M. GonÅalves da Silva, J. Porphyrins Phthalocyanines
2009, 13, 509–517.
[5] C. M. Drain, K. C. Russell, J.-M. Lehn, Chem. Commun. 1996, 337–338.
[6] R. Purrello, S. Gurrieri, R. Lauceri, Coord. Chem. Rev. 1999, 190–192,
683–706.
[7] J. Puigmartí-Luis, W. J. Saletra, A. Gonzµlez, D. B. Amabilino, L. PØrez-
García, Chem. Commun. 2014, 50, 82–84.
[8] K. Lang, J. Mosinger, D. M. Wagnerovµ, Coord. Chem. Rev. 2004, 248,
321–350.
[45] H. Imahori, H. Norieda, Y. Nishimura, I. Yamazaki, K. Higuchi, N. Kato, T.
Motohiro, H. Yamada, K. Tamaki, M. Arimura, Y. Sakata, J. Phys. Chem. B
2000, 104, 1253–1260.
[9] G. R. Geier, Y. Ciringh, F. Li, D. M. Haynes, J. S. Lindsey, Org. Lett. 2000, 2,
1745–1748.
[46] W. Haiss, N. T. K. Thanh, J. Aveyard, D. G. Fernig, Anal. Chem. 2007, 79,
4215–4221.
[10] F. Li, Y. Kexin, J. S. Tyhonas, K. A. MacCrum, J. S. Lindsey, Tetrahedron
1997, 53, 12339–12360.
[47] D. R. Baer, D. J. Gaspar, P. Nachimuthu, S. D. Techane, D. G. Castner, Anal.
Bioanal. Chem. 2010, 396, 983–1002.
[11] P. Moonsin, K. Sirithip, S. Jungsuttiwong, T. Keawin, T. Sudyoadsuk, V.
Promarak, Tetrahedron Lett. 2011, 52, 4795–4798.
[48] N. Katsonis, J. Vicario, T. Kudernac, J. Visser, M. M. Pollard, B. L. Feringa,
J. Am. Chem. Soc. 2006, 128, 15537–15541.
[12] L. Xiaoquan, G. Zaixin, K. Jingwan, W. Zhihua, G. Jinzhang, Synth.
Commun. 2000, 30, 2213–2221.
[49] B. A. Lindig, M. A. J. Rodgers, A. P. Schaap, J. Am. Chem. Soc. 1980, 102,
5590–5593.
[13] C.-L. Peng, P.-S. Lai, C.-C. Chang, P.-J. Lou, M.-J. Shieh, Dye. Pigment.
2010, 84, 140–147.
[50] K. Knop, A.-F. Mingotaud, N. El-Akra, F. Violleau, J.-P. Souchard, Photo-
chem. Photobiol. Sci. 2009, 8, 396–404.
[51] Y. Guo, M. Kumar, P. Zhang, Chem. Mater. 2007, 19, 6071–6072.
[52] S. Fraga, H. Faria, M. E. Soares, J. A. Duarte, L. Soares, E. Pereira, C.
Costa-Pereira, J. P. Teixeira, M. de Lourdes Bastos, H. Carmo, J. Appl. Toxi-
col. 2013, 33, 1111–1119.
[14] P. Iavicoli, H. Xu, L. N. Feldborg, M. Linares, M. Paradinas, S. Stafstrçm, C.
Ocal, B. Nieto-Ortega, J. Casado, J. T. López Navarrete, R. Lazzaroni, S. De
Feyter, J. Am. Chem. Soc. 2010, 132, 9350–9362.
[15] R. Bonnett, G. Martínez, Tetrahedron 2001, 57, 9513–9547.
[16] . Juarranz, P. JaØn, F. Sanz-Rodríguez, J. Cuevas, S. Gonzµlez, Clin.
Transl. Oncol. 2008, 10, 148–154.
[17] S. Swavey, M. Tran, Porphyrin and Phthalocyanine Photosensitizers as PDT
Agents: A New Modality for the Treatment of Melanoma, InTech, Manhat-
tan, NY, 2013.
[18] I. J. Macdonald, T. J. Dougherty, J. Porphyrins Phthalocyanines 2001, 5,
105–129.
[19] E. Boisselier, D. Astruc, Chem. Soc. Rev. 2009, 38, 1759–1782.
[20] P. Ghosh, G. Han, M. De, C. K. Kim, V. M. Rotello, Adv. Drug Delivery Rev.
2008, 60, 1307–1315.
[53] I. M. Martinez Paino, V. S. Marangoni, R. de Cµssia Silva de Oliveira,
L. M. G. Antunes, V. Zucolotto, Toxicol. Lett. 2012, 215, 119–125.
[54] Y. Pan, M. Bartneck, W. Jahnen-Dechent in Methods Enzymol. (Ed.: D.
Nejat), Academic Press, Waltham, 2012, pp. 225–242.
[55] R. LØvy, U. Shaheen, Y. Cesbron, V. SØe, Nano Rev. 2010, 1, 4889.
[56] N. Khlebtsov, L. Dykman, Chem. Soc. Rev. 2011, 40, 1647–1671.
[57] R. Coradeghini, S. Gioria, C. P. García, P. Nativo, F. Franchini, D. Gilliland,
J. Ponti, F. Rossi, Toxicol. Lett. 2013, 217, 205–216.
[21] Y. Cheng, A. C. Samia, J. D. Meyers, I. Panagopoulos, B. Fei, C. Burda, J.
Am. Chem. Soc. 2008, 130, 10643–10647.
[58] S. Vijayakumar, S. Ganesan, Toxicol. Environ. Chem. 2013, 95, 277–287.
[59] Arnida, A. Malugin, H. Ghandehari, J. Appl. Toxicol. 2010, 30, 212–217.
ChemistryOpen 2015, 4, 127 – 136
www.chemistryopen.org
135
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

[60] Y.-J. Gu, J. Cheng, C.-C. Lin, Y. W. Lam, S. H. Cheng, W.-T. Wong, Toxicol.
Appl. Pharmacol. 2009, 237, 196–204.
[63] B. D. Chithrani, W. C. W. Chan, Nano Lett. 2007, 7, 1542–1550.
[61] C.-J. Liu, C.-H. Wang, C.-C. Chien, T.-Y. Yang, S.-T. Chen, W.-H. Leng, C.-F.
Lee, K.-H. Lee, Y. Hwu, Y.-C. Lee, C.-L. Cheng, C.-S. Yang, Y. J. Chen, J. H.
Je, G. Margaritondo, Nanotechnology 2008, 19, 295104.
[62] W. Cui, J. Li, Y. Zhang, H. Rong, W. Lu, L. Jiang, Nanomedicine 2012, 8,
46–53.
Received: October 24, 2014
Published online on December 1, 2014
ChemistryOpen 2015, 4, 127 – 136
www.chemistryopen.org
136
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim