Targeted photodynamic therapy of breast cancer cells using antibody–phthalocyanine–gold nanoparticle conjugates

TL;DR: Cellular experiments demonstrated that the nanoparticle conjugates selectively target breast cancer cells that overexpress the HER2 epidermal growth factor cell surface receptor, and that they are effective photodynamic therapy agents.

Abstract: A 4-component antibody-phthalocyanine-polyethylene glycol-gold nanoparticle conjugate is described for use as a potential drug for targeted photodynamic cancer therapy. Gold nanoparticles (4 nm) were stabilised with a self-assembled layer of a zinc-phthalocyanine derivative (photosensitiser) and a heterobifunctional polyethylene glycol. Anti-HER2 monoclonal antibodies were covalently bound to the nanoparticles via a terminal carboxy moiety on the polyethylene glycol. The nanoparticle conjugates were stable towards aggregation, and under irradiation with visible red light efficiently produced cytotoxic singlet oxygen. Cellular experiments demonstrated that the nanoparticle conjugates selectively target breast cancer cells that overexpress the HER2 epidermal growth factor cell surface receptor, and that they are effective photodynamic therapy agents.

Summary

Introduction

• Such nanoparticles show good biocompatibility as they are generally considered to be benign,1 possess a high surface area and are characterised by facile surface functionalisation through selfassembly of thiolates on the gold surface via formation of a Au–S bond.
• 2,3 A number of studies have shown that gold nanoparticles conjugated with antibodies are efficient in targeting and then can destroy cancerous tissue by conversion of near-infrared irradiation into heat.
• 5,9 In photodynamic therapy (PDT) of cancer, irradiation with visible and/or near-infrared light leads to activation of a photosensitiser drug.
• The authors have previously shown that the surface-bound Zn(II) phthalocyanine derivative (C11Pc) exhibits a remarkable enhancement of the singlet oxygen quantum yield in the presence of an associated phase transfer reagent, tetraoctylammonium bromide.
• 16 Incorporation of cell-targeting peptides or antibodies onto the nanoparticle surface is highly desirable for therapeutic applications, as it would enable selective cell and/or nuclear targeting.

Materials and methods

• All solvents and reagents were reagent-grade and were used as received.
• Gold(III) chloride tetrahydrate (4.8 mg in 4.8 ml of THF) was added to the solution with further stirring for 5 min.
• Attachment of the monoclonal antibody specific to the HER2 receptors was achieved via an amide linkage to the HS-PEG-COOH 3000.
• UV-visible absorption spectra were measured in the range 300–800 nm.

Cell culture

• Breast carcinoma cell lines (SK-BR-3 and MDA-MB-231) were routinely cultured in 75 cm3 tissue culture flasks in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% L-glutamine.
• The amount of cells for each cell-based assay and cell viability was monitored by counting viable cells in a hemocytometer by using the trypan blue exclusion method.
• The cells were observed using a confocal laser scanning microscope (Carl Zeiss, LSM 510 meta) using 633 nm HeNe laser excitation with fluorescence emission measured between 650– 720 nm. Differential Interference Contrast (DIC) images were collected simultaneously with transmitted light from the 633 nm excitation.
• After incubation, the cells were washed with PBS buffer 3 times to remove non-bound nanoparticle conjugates, and the medium was replaced.

MTT assay

• Then the medium was removed and the resultant formazan crystals were washed with 200 ml PBS (3 times) and then dissolved in 200 ml of dimethyl sulfoxide.
• The absorbance intensity of each well was measured at 550 nm.
• Each variable (concentration of nanoparticle conjugates, incubation time and irradiation time) was assessed through MTT assay in triplicate.

ApoTox-GloTM Triplex Assay

• The ApoTox-GloTM triplex assay enables assessment of cell viability, cytotoxicity and caspase activation events within a single assay well.
• The apoptotic response was reflected by an increase in caspase-3/7 activity, whereas the cytotoxic responses were marked by decreases in AFC (live cell) fluorescence and increases in R110 (dead cell) fluorescence.
• The reaction can be performed directly in a homogeneous format in assay wells containing a mixed population of viable and damaged cells.
• The fluorescence signal monitoring the cell proliferation was recorded with 560Ex/590Em filters.
• Lysis solution (Triton X 100 9%) was used as a positive control for maximum LDH release.

Results and discussion

• In order to develop multifunctional gold nanoparticles as a potential drug delivery system for PDT, a 4-component system consisting of a phthalocyanine derivative as the photosensitiser together with a heterobifunctional polyethylene glycol (HS-PEGCOOH) was self-assembled onto the gold nanoparticle surface.
• To deliver this photosensitiser for PDT, previously the authors synthesised gold nanoparticles using the Brust method2 which incorporated a phase transfer reagent, tetraoctylammonium bromide, together with the phthalocyanine as a stabilising self-assembled monolayer.
• To ensure that the decreasing absorbance intensity of the ADPA was not due to changes of the absorption spectrum of the C11Pc phthalocyanine photosensitiser, the nanoparticle conjugates were irradiated without the singlet oxygen probe.
• Further evidence of increased cell mortality following photodynamic treatment with the nanoparticle conjugates was provided through a series of cell-based assays in vitro.
• Only a moderate rise of cytotoxicity was observed when the cells were treated under the same conditions (Fig. 7(A) and Fig. 7(B)).

Conclusions

• In summary, the authors have developed a 4-component nanoparticle conjugate which is capable of specifically targeting and then photodynamically treating cancer cells.
• To achieve the specificity, the authors have combined both a phthalocyanine photosensitiser drug and a primary antibody, specific to cell surface receptors, on a gold nanoparticle.
• Attachment of the anti-HER2 antibodies to the gold nanoparticle conjugates leads to enhanced drug targeting of breast cancer cells that overexpress the HER2 receptor on the cellular surface.
• Results from several in vitro cell-based assays suggests that the targeting capability of the 4-component nanoparticle conjugates enhances the efficacy of PDT cell death when tumourassociated antigens are present on the cytoplasmic membrane of the malignant cells.
• These first results of the treatment of breast cancer cells are very promising, and demonstrate that the 4-component nanoparticle conjugates have a great potential for targeted photodynamic therapy.

Figures

Fig. 3 Visible absorption spectrum of the Zn–phthalocyanine photosensitiser together with HS-polyethylene glycol-COOH supported on gold nanoparticles and conjugated with anti-HER2 antibody; absorption at 698 nm and 644 nm corresponds to monomeric and dimeric form of the phthalocyanine photosensitiser respectively. Inset: Visible absorption spectrum of the Zn–phthalocyanine photosensitiser together with the HS-polyethylene glycol-COOH on the gold nanoparticles without antibody attached. The ratio between monomeric and dimeric form of the phthalocyanine was not affected by antibody attachment, thus preserving the high rate of reactive singlet oxygen production.

Fig. 4 (A) Time-dependent decay of ADPA absorption spectrum as a result of photobleaching after irradiation in the presence of the 4-component gold nanoparticle conjugates. (B) Time-dependent decay of the C11Pc–PEG–gold nanoparticle absorption spectrum after irradiation (C) Singlet oxygen production as monitored by the photobleaching of ADPA absorption band at 400 nm after irradiation (HeNe laser, 633 nm) of the 4-component nanoparticle conjugates antibody–C11Pc–PEG–gold nanoparticles (dots) and antibody–PEG–nanoparticles (with no C11Pc) (squares, control).

Fig. 1 The complete 4-component system of photosensitiser–nanoparticle conjugates. The conjugates incorporate the photosensitiser (a derivative of zinc(II) phthalocyanine, C11PC), the heterobifunctional HS-polyethylene glycol-COOH and the HER2 (ErbB2) mouse monoclonal antibody, all on the gold nanoparticle surface.

Fig. 5 Combined confocal fluorescence and DIC images of non-treated cells (A, SK-BR-3 cell line) and cells after incubation with the 4-component gold nanoparticle conjugates, [C11Pc] = 0.4 mM: (B, SK-BR-3 cell line) and (C, MDA-MB-231 cell line). The presence of the nanoparticle conjugates within the cells can be readily seen from the red fluorescence emission following laser excitation at 633 nm. Scale bars are 5 mm in all images.

Fig. 7 Response to PDT treatment for the breast cancer cell lines with (SK-BR-3) and without (MDA-MB-231) HER2 amplification: (A) increase in cytotoxicity, (B) decrease in cell viability, (C) increase in apoptosis as a function of time after treatment of the SK-BR-3 and MDA-MB-231 cell lines with the 4-component nanoparticle conjugates (10 nM) and illumination at 633 nm (HeNe laser, 10.2 J cm-2). Data from the ApoTox-GloTM Triplex Assay, represented in relative fluorescent units (RFU) or relative luminescent units (RLU) normalised to the fluorescence or luminescence signal of non-treated cells with background signal from growth media subtracted. Each experiment was performed in triplicate, and each experiment was performed on 3 separate occasions. Error bars represent the relative standard error.

Full text

This article is published as part of a themed issue of
Photochemical & Photobiological Sciences on
Drug delivery technologies and immunological aspects of
photodynamic therapy
Guest edited by Kristian Berg, Jakub Golab, Mladen Korbelik
and David Russell
Published in issue 5, 2011
Perspectives
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Photobiol. Sci., 2011, 10, 653
Cancer vaccines generated by photodynamic
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664
DAMPs and PDT-mediated photo-oxidative
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A. D. Garg, D. V. Krysko, P. Vandenabeele and P.
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670
Antitumor immunity promoted by vascular
occluding therapy: lessons from vascular-
targeted photodynamic therapy (VTP)
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Photobiol. Sci., 2011, 10, 681
On the cutting edge: protease-sensitive
prodrugs for the delivery of photoactive
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Photochem. Photobiol. Sci., 2011, 10, 689
Combination of photodynamic therapy and
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Z. Huang, Photochem. Photobiol. Sci., 2011, 10,
704
Nanoparticles: their potential use in
antibacterial photodynamic therapy
S. Perni, P. Prokopovich, J. Pratten, I. P. Parkin
and M. Wilson, Photochem. Photobiol. Sci., 2011,
10, 712
Photosensitiser–antibody conjugates for
photodynamic therapy
A. J. Bullous, C. M. A. Alonso and R. W. Boyle,
Photochem. Photobiol. Sci., 2011, 10, 721
The immunosuppressive side of PDT
P. Mroz and M. R. Hamblin, Photochem.
Photobiol. Sci., 2011, 10, 751
Synthetic approaches for the conjugation of
porphyrins and related macrocycles to
peptides and proteins
F. Giuntini, C. M. A. Alonso and R. W. Boyle,
Photochem. Photobiol. Sci., 2011, 10, 792
Combination approaches to potentiate
immune response after photodynamic therapy
for cancer
T. G. St. Denis, K. Aziz, A. A. Waheed, Y.-Y.
Huang, S. K. Sharma, P. Mroz and M. R. Hamblin,
Photochem. Photobiol. Sci., 2011, 10, 802
Clinical and immunolgical response to
photodynamic therapy in the treatment of
vulval intrepithelial neoplasia
S. Daayana, U. Winters, P. L. Stern and H. C.
Kitchener, Photochem. Photobiol. Sci., 2011, 10,
810
Papers
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Ding, Z. Zhang and G. Zheng, Photochem.
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Preliminary safety and efficacy results of laser
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breast cancer patients
X. Li, G. L. Ferrel, M. C. Guerra, T. Hode, J. A.
Lunn, O. Adalsteinsson, R. E. Nordquist, H. Liu
and W. R. Chen, Photochem. Photobiol. Sci.,
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Targeted photodynamic therapy of breast
cancer cells using antibody–phthalocyanine–
gold nanoparticle conjugates
T. Stuchinskaya, M. Moreno, M. J. Cook, D. R.
Edwards and D. A. Russell, Photochem.
Photobiol. Sci., 2011, 10, 832
Methylene blue covalently loaded
polyacrylamide nanoparticles for enhanced
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potential photosensitizers in photodynamic
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Photochemical &
Photobiological Sciences
Dynamic Article Links
Cite this:
Photochem. Photobiol. Sci.
, 2011, 10, 822
www.rsc.org/pps
PAPER
Targeted photodynamic therapy of breast cancer cells using
antibody–phthalocyanine–gold nanoparticle conjugates†
Tanya Stuchinskaya,
a
Miguel Moreno,
a
Michael J. Cook,
a
Dylan R. Edwards
b
and David A. Russell*
a
Received 11th January 2011, Accepted 7th March 2011
DOI: 10.1039/c1pp05014a
A 4-component antibody–phthalocyanine–polyethylene glycol–gold nanoparticle conjugate is
described for use as a potential drug for targeted photodynamic cancer therapy. Gold nanoparticles
(4 nm) were stabilised with a self-assembled layer of a zinc–phthalocyanine derivative (photosensitiser)
and a heterobifunctional polyethylene glycol. Anti-HER2 monoclonal antibodies were covalently
bound to the nanoparticles via a terminal carboxy moiety on the polyethylene glycol. The nanoparticle
conjugates were stable towards aggregation, and under irradiation with visible red light efficiently
produced cytotoxic singlet oxygen. Cellular experiments demonstrated that the nanoparticle conjugates
selectively target breast cancer cells that overexpress the HER2 epidermal growth factor cell surface
receptor, and that they are effective photodynamic therapy agents.
Introduction
There has been a growing interest in the use of gold nanoparticles
in biomedical research due to a combination of unique properties.
Such nanoparticles show good biocompatibility as they are
generally considered to be benign,
1
possess a high surface area and
are characterised by facile surface functionalisation through self-
assembly of thiolates on the gold surface via formation of a Au–S
bond.
2,3
It has been demonstrated that gold nanoparticles can be
used as efficient drug delivery vehicles both in cancer diagnostics,
e.g., intracellular imaging, and in cancer therapy.
1,4
For anti-cancer therapy, molecular recognition towards specific
cell surface receptors can be achieved by conjugation with an
antibody. A number of studies have shown that gold nanoparticles
conjugated with antibodies are efficient in targeting and then can
destroy cancerous tissue by conversion of near-infrared irradi-
ation into heat.
1,5,6
Gold nanostructures, including nanocages,
5
nanorods
6
and nanoshells,
7
have all served as photothermal
therapeutic agents following illumination with light of wavelengths
between 700–800 nm. Typical ‘spherical’ gold nanoparticles of ca.
16 nm have a surface plasmon absorption maximum around 514–
520 nm. However, structural modification of the gold nanostruc-
tures during synthesis provides a shift of the absorption band
to the desirable ‘therapeutic window’ of 650–900 nm, i.e.,the
near-infrared region where blood and soft tissue are relatively
transparent.
5–7
In photothermal experiments, gold nanoparticles
with anti-VEGF antibodies adsorbed on the surface proved to be
a
School of Chemistry, University of East Anglia, Norwich, Norfolk, NR4
7TJ, UK. E-mail: d.russell@uea.ac.uk
b
School of Biological Sciences, University of East Anglia, Norwich, Norfolk,
NR4 7TJ, UK
† This article is published as part of a themed issue on immunological
aspects and drug delivery technologies in PDT.
more efficient for the induction of apoptosis in b-chronic lympho-
cytic leukemia cells as compared to gold nanoparticles without
antibodies.
8
Disadvantages of photothermal cancer treatment can
be the high power density of irradiation of up to 35 J cm
-2
and a
poor selectivity, as both normal and tumour cells can be destroyed
in the path of the laser light.
9
However, it has been also reported
that specifically designed gold nanocages as well as agglomerated
spherical gold nanoparticles can induce cancer cell death at a
poweraslowas1.5Jcm
-2
using short-pulse irradiation with a
femtosecond laser.
5,9
In photodynamic therapy (PDT) of cancer, irradiation with
visible and/or near-infrared light leads to activation of a photo-
sensitiser drug. Upon illumination, the photosensitiser generates
reactive oxygen species, which in turn react rapidly with biological
substrates, initiating an apoptotic or necrotic response and eventu-
ally leading to oxidative damage and cell death.
10–12
Since the light
is specifically chosen to coincide with the maximum absorption
wavelength of the photosensitiser, initial damage following PDT
is limited to the site of concentration of the photosensitiser drug
molecule without destruction of healthy tissue.
4
Among some of the promising second-generation photosensi-
tisers are phthalocyanines (Pc), which have a strong absorption
band in the far-red region of the visible spectrum (l
max
= ca.
680 nm) and can efficiently absorb light (Pc molar extinction
coefficient (e) = ca. 10
5
M
-1
cm
-1
).
11
The efficacy of phthalocyanine
derivatives as a photosensitiser can be significantly enhanced
by attachment to the surface of gold nanoparticles. We have
previously shown that the surface-bound Zn(
II) phthalocyanine
derivative (C11Pc) exhibits a remarkable enhancement of the
singlet oxygen quantum yield in the presence of an associated
phase transfer reagent, tetraoctylammonium bromide.
13,14
This
enhancement in singlet oxygen generation is possibly due to
the phase transfer agent stabilising an active monomeric form
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of the photosensitiser. Cellular experiments using the HeLa
cervical cancer cell line demonstrated that irradiation of the
conjugates within the cells causes high cell mortality through
the photodynamic effect.
13
An extensive photodynamic effect
using the C11Pc nanoparticle conjugates has been demonstrated
recently in vivo where a significant decrease in the rate of growth
of a subcutaneously implanted amelanotic melanoma tumour was
observed.
15
Similarly, experiments on 5RP7 rat fibroblasts cancer
cells showed promising results in vivo by suppressing the tumour
growth after treatment with zinc phthalocyanine-loaded single-
wall carbon nanohorns.
16
Incorporation of cell-targeting peptides or antibodies onto the
nanoparticle surface is highly desirable for therapeutic applica-
tions, as it would enable selective cell and/or nuclear targeting.
Direct physisorption of fluorescently tagged antibodies to the
surface of gold nanoparticles has been used for some time
to produce ‘immunogold’. However, such simple methods of
nanoparticle functionalisation results in poor orientation of the
recognition component (the F(ab)
2
region) of the antibody, non-
specific binding of the nanoparticle conjugates and agglomeration
of small gold nanoparticles.
17
These drawbacks can be overcome
by modification of the nanoparticle’s surface with polyethylene
glycol (PEG), which has been approved for human intravenous
application and which stabilises the nanoparticles by steric repul-
sion to inhibit colloidal aggregation in physiological conditions.
18
The use of a heterobifunctional PEG with two different terminal
moieties allows covalent attachment of an antibody to the outer
end of the polyethylene glycol chain, thus maintaining availability
of antibody binding sites to cell surface receptors. It has been
demonstrated previously that nanoparticles modified with mixed
receptor-mediated endocytosis peptide and bifunctional HS-PEG-
COOH 5000 monolayers were efficiently internalised by HeLa
cells.
19,20
Despite the significant efforts to develop modified nanostruc-
tures for efficient PDT treatment, it still remains a challenge to
develop a nanostructured system based on surface-functionalised
gold nanoparticles that combines molecular recognition with
effective production of reactive singlet oxygen under irradia-
tion. In this study we show that it is possible t o combine an
efficient singlet oxygen generating hydrophobic photosensitiser
with cancer-specific targeting antibodies on the surface of gold
nanoparticles. Importantly, these multifunctional gold nanoparti-
cle conjugates are water-soluble, a characteristic which is essential
for antibody stability and for the facile delivery of conjugates for
photodynamic treatment, but still retain the ability to generate
significant levels of singlet oxygen. The preparation and prop-
erties of the 4-component (anti-HER2 antibody–phthalocyanine
photosensitiser–PEG–gold nanoparticle) nanoparticle conjugates
(Fig. 1) are described. Further, it is shown that the nanoparticle
conjugates can be used to specifically target and then photody-
namically destroy breast cancer cells which overexpress the HER2
cell surface receptor.
Experimental
Materials and methods
All solvents and reagents were reagent-grade and were used as
received. Breast carcinoma cell lines (SK-BR-3 and MDA-MB-
Fig. 1 The complete 4-component system of photosensitiser–nanoparti-
cle conjugates. The conjugates incorporate the photosensitiser (a derivative
of zinc(
II) phthalocyanine, C11PC), the heterobifunctional HS-polyethy-
lene glycol-COOH and the HER2 (ErbB2) mouse monoclonal antibody,
all on the gold nanoparticle surface.
231) were obtained from the Cancer Research UK bank. Normal
mammary epithelial cells (MCF-10A) were purchased from the
American Type Cell Culture (ATCC) together with Mammary
Epithelial Growth Media. Cell culture growth medium for the SK-
BR-3 and MDA-MB-231 cell lines was purchased from GIBCO.
Cell-based assays (ApoToxGlo Triplex assay, Caspase-Glo 3/7
and CytoTox-One homogeneous membrane integrity assay) were
purchased from Promega. MTT [(3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] was obtained from Sigma
Aldrich. The monoclonal antibody against ErbB-2 (HER2) was
purchased from Invitrogen.
UV-Visible absorption spectra were recorded on a Hitachi U-
3000 UV-visible spectrophotometer, while fluorescence spectra
were obtained on a Hitachi F-4500 fluorescence spectrometer.
All laser excitation experiments for the cell-based assays were per-
formed using a 632.8 nm HeNe laser (Uniphase 1125P) at a fluence
rate of 34 mW cm
-2
and a light dose of 10.2 J cm
-2
. The particle
size of the antibody–photosensitiser nanoparticle conjugates was
measured on a transmission electron microscope (JEOL JEM-
2000EX). A typical TEM image of the nanoparticle conjugates
is shown in Fig. 2(A). Centrifugation of the nanoparticles was
performed using a Beckman Coulter Allegra
TM
X-22R Centrifuge.
Elemental analysis of the photosensitiser-bound nanoparti-
cles to determine the number of phthalocyanine molecules per
nanoparticle was achieved using inductively coupled plasma-mass
spectrometry (ICP-MS). The amount of gold and zinc (as a marker
for the phthalocyanine photosensitiser) within the nanoparticle
conjugates was measured on an Agilent 7500ce ICP-MS, fitted
with a concentric Micromist nebuliser and water-cooled Scott
double-pass spray chamber. The standards used for calibration
were from Merck (Merck VI Multi Element Standard) for zinc
and from CPL International (Single Element Gold 1000 ± 3 mg
ml
-1
) for gold.
Measurements of the cell-based assays were performed on En-
Vision (Wallac) 2103 Multilabel Reader (Perkin Elmer) equipped
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Fig. 2 (A) A TEM image of the gold nanoparticles stabilised with the Zn phthalocyanine photosensitiser and the
HS-polyethylene glycol-COOH and with the HER2 antibody conjugated to the PEG (scale bar = 10 nm), (B) structure of
1,1¢,4,4¢,8,8¢,15,15¢,18,18¢,22,22¢-tetradecakisdecyl-25,25¢-(11,11¢dithiodiundecyl) diphthalocyanine zinc (C11Pc).
13–15
with an EnVision work station. Luminescence was measured with
a 700 nm low-pass luminescence filter. Fluorescence was measured
using excitation and emission filters recommended by the cell-
based assay provider. The absorbance intensity for the colorimetric
MTT assay was measured on a Dynatech MRX plate reader at
550 nm.
The cellular uptake of the nanoparticle conjugates was observed
using a confocal laser scanning microscope (Carl Zeiss, LSM
510 meta). Differential Interference Contrast (DIC) images were
obtained using 633 nm HeNe laser excitation with fluorescence
emission measurement between 650–720 nm. A 63¥,1.4NA
objective was used to obtain high-resolution images.
Preparation and characterisation of the 4-component nanoparticle
conjugates [C11Pc–PEG–antibody–gold nanoparticles]
The photosensitiser used in this study was 1,1¢,4,4¢,8,8¢,15,15¢,18,
18¢,22,22¢-tetradecakisdecyl-25,25¢-(11,11¢dithiodiundecyl) diph-
thalocyanine zinc (C11Pc). The structure of this photosensitiser
is shown in Fig. 2(B). This photosensitiser was prepared as
previously reported.
13–15
Gold nanoparticles of ca. 4nmin
diameter were synthesised using the method originally described
by Brust et al.,
2
albeit with modifications. In a typical experiment,
the C11Pc phthalocyanine derivative (8 mg) was dissolved in
tetrahydrofuran (THF) (4 ml). PEG-COOH 3000 Da (a-thio-w-
carboxy polyethylene glycol, 30 mg, Iris Biotech GmbH (3274
Da)) in THF (8 ml) was added, and the mixture was stirred
vigorously for 5 min at room temperature. Gold(
III) chloride
tetrahydrate (4.8 mg in 4.8 ml of THF) was added to the
solution with further stirring for 5 min. An aqueous solution of
sodium borohydride (6 mg in 4.8 ml) was added dropwise with
continuous stirring, and the solution was further stirred at room
temperature overnight in the dark. The solvent was removed by
rotary evaporation at 60
◦
C, and the particles were re-suspended in
MES (2-(N-morpholino)ethanesulfonic acid) buffer, pH 5.5 with
0.05% Tween 20 (Polyoxyethylene (20) sorbitan monolaurate, a
non-toxic polysorbate surfactant, obtained from Sigma–Aldrich).
The particles were then centrifuged in polypropylene Eppendorf
tubes (1 ml) at 14 000 rpm for 30 min. The supernatant containing
the particles was removed from the pellet with a micropipette
and then characterised using UV-visible spectrophotometry. For
control experiments, PEGylated gold nanoparticles were also
prepared using the same method as described above for the C11Pc–
PEG–gold nanoparticles, but without addition of the C11Pc
phthalocyanine derivative.
Attachment of the monoclonal antibody specific to the
HER2 receptors was achieved via an amide linkage to
the HS-PEG-COOH 3000. The amide bond was achieved
through EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride, obtained from Sigma–Aldrich) and NHS (
D-
hydroxysulfosuccinimide sodium salt, from Fluka) two-step cou-
pling activation of the carboxylic group of the PEG derivative.
The activation converted the carboxylic moiety of the PEG to
a succinimidyl ester, which then reacts with an amine group on
the antibody forming a stable amide bond between the PEG and
the antibodies.
21
EDC (0.4 ml) and NHS (1.1 mg) were added
to 1 ml of the photosensitiser–PEG nanoparticle conjugates, the
mixture was stirred for 30 min at room temperature, centrifuged
at 9000 rpm for 30 min in ultra-filtration spin columns, and the
particles were re-suspended in PBS (phosphate saline buffer pH =
7.4, filter sterilised). Then 1 ml of the HER2 antibody (1 mg
ml
-1
) was added to 1 ml of the activated nanoparticles, stirred
for 2.5 h, mixed by orbital shaking for 15 h at 1400 rpm, placed
into ultrafiltration spin columns and then centrifuged at 14 000
rpm for 30 min. The particles were re-suspended in PBS (pH = 7.4,
filter sterilised, 400 ml per column) and characterised using UV-
visible spectrophotometry. The stock solution of nanoparticles in
PBS was adjusted to a concentration of 5 mM with respect to the
C11PC phthalocyanine derivative, and diluted for the cell line tests
as required.
Measurement of singlet oxygen production
The generation of intracellular reactive oxygen species by the anti-
HER2 antibody phthalocyanine–PEG–gold nanoparticle conju-
gates was determined using the molecular probe disodium, 9,10-
anthracenedipropionic acid (ADPA). Singlet oxygen converts the
ADPA into an endoperoxide which can be monitored by UV-
visible spectrophotometry.
22
100 ml of ADPA solution (1.2 mM
in methanol) was added to 900 ml of the nanoparticle conjugates
in a quartz cuvette and stirred thoroughly. UV-visible absorption
spectra were measured in the range 300–800 nm. The solution was
irradiated using the HeNe 632.8 nm laser. During irradiation the
solution was vigorously stirred. The production of singlet oxygen
was determined by the decay of the absorbance intensity of the
ADPA 400 nm absorption band every 5 min for 30 min.
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Cell culture
Breast carcinoma cell lines (SK-BR-3 and MDA-MB-231) were
routinely cultured in 75 cm
3
tissue culture flasks in Dulbecco’s
Modified E agle Medium (DMEM) supplemented with 10% fetal
calf serum (FCS) and 1%
L-glutamine. Normal mammary epithe-
lial cells (MCF-10A) were cultured in 75 cm
3
tissue culture flasks
in ATCC complete growth medium supplemented with 100 ng
ml
-1
cholera t oxin. The amount of cells for each cell-based assay
and cell viability was monitored by counting viable cells in a
hemocytometer by using the trypan blue exclusion method.
23
Analysis of nanoparticle conjugate uptake by confocal microscopy
Cells (25 ¥ 10
4
cells per well) were seeded onto 6-well plates on
round coverslips (diameter 18 mm) in 2 ml of DMEM growth
medium and left to attach for 24 h. The nanoparticle conjugates
(0.8 mM) were added and the cells were incubated for 3 h. The
cells were rinsed with 2 ml of phosphate-buffered saline (PBS)
3 times. The DMEM medium was replaced with phenol red
free medium, and the cells were mounted on a 37
◦
Cheated
stage. The cells were observed using a confocal laser scanning
microscope (Carl Zeiss, LSM 510 meta) using 633 nm HeNe
laser excitation with fluorescence emission measured between 650–
720 nm. Differential Interference Contrast (DIC) images were
collected simultaneously with transmitted light from the 633 nm
excitation. In order to monitor changes in cell viability, a dead cell
marker, propidium iodide, was used according to the published
protocol.
24
Cells were stained with propidium iodide (2 mgml
-1
,
from Invitrogen) for 10 min in the dark and then observed with
the confocal microscope combined with DIC using 543 nm HeNe
laser excitation with fluorescence emission measured between 620–
720 nm (excitation/emission maximum wavelengths for propidium
iodide are 536 nm/620 nm respectively). Cells incubated in the
absence of nanoparticle conjugates were used as a control. Images
were processed and composite images were obtained using Zeiss
LSM Image Browser Version 4.2.0.121.
Measurement of cell viability, apoptosis and proliferation
Cells were seeded onto 96-well plates, 1 ¥ 10
4
cells per well in 100 ml
of DMEM supplemented with 10% FCS and 1%
L-glutamine
(SK-BR-3 and MDA-MB-231 cell lines) or in ATCC complete
growth medium supplemented with 100 ng ml
-1
cholera toxin
(MCF-10A cell line). The cells were counted with a Neubauer
hemocytometer. The 96-well plates were obtained from Fisher
Thermo Scientific (Nunc) as either clear or solid white styles
depending on assay requirement. Plates were incubated overnight
at 37
◦
C under a 5% CO
2
atmosphere. The medium in the wells
was then replaced with fresh medium containing the antibody–
photosensitiser nanoparticle conjugates (final concentration was
10 nM of the phthalocyanine photosensitiser assembled on the
gold nanoparticles, ca. 0.03 m g of the nanoparticle conjugates
per 100 ml), and incubation continued for 2 h (unless specified
otherwise). After incubation, the cells were washed with PBS
buffer 3 times to remove non-bound nanoparticle conjugates, and
the medium was replaced. Each well was then irradiated with the
HeNe 632.8 nm laser for 5 min with a fluence rate of 34 mW cm
-2
,
light dose 10.2 J cm
-2
. The intensity of the laser light source was
measured using a power meter (Power Max 500AD Molectron).
MTT assay
The effect of the nanoparticles on cell viability was de-
termined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, Sigma) assay.
25,26
Cells at a density
of 1 ¥ 10
4
cells per well were seeded on clear 96-well plates
and treated with the nanoparticle conjugates as described above,
followed by light irradiation with the HeNe 632.8 nm laser for 5
min. 10 ml of the MTT solution (5 mg ml
-1
in PBS) was added
to each well together with 200 ml of DMEM or ATCC growth
medium, depending on the cell line. The plates were further
incubated for 4 h at 37
◦
C. Then the medium was removed and the
resultant formazan crystals were washed with 200 ml PBS (3 times)
and then dissolved in 200 ml of dimethyl sulfoxide. The absorbance
intensity of each well was measured at 550 nm. Each variable
(concentration of nanoparticle conjugates, incubation time and
irradiation time) was assessed through MTT assay in triplicate.
ApoTox-Glo
TM
Triplex Assay
The ApoTox-Glo
TM
triplex assay enables assessment of cell
viability, cytotoxicity and caspase activation events within a
single assay well. The first part of the assay simultaneously
measures two protease activities: one is a marker of cell viability
(glycylphenylalanyl-aminofluorocoumarin; GF-AFC), and the
other is a marker of cytotoxicity (bis-alanylalanyl-phenylalanyl-
rhodamine 110; bis-AAF-R110). The live- and dead-cell proteases
produce different products, AFC and R110 respectively, which
have different excitation and emission spectra, allowing them to
be detected simultaneously. The protocol is combined with the
luminescent Caspase-Glo 3/7 assay to monitor cell apoptosis.
The apoptotic response was reflected by an increase in caspase-3/7
activity, whereas the cytotoxic responses were marked by decreases
in AFC (live cell) fluorescence and increases in R110 (dead cell)
fluorescence.
27
Cells at a density of 1 ¥ 10
4
cells per well were seeded
on solid white 96-well plates and treated with nanoparticle
conjugates followed by irradiation as described above. 20 mlof
viability/cytotoxicity reagent containing both GF-AFC substrate
and bis-AAF-R110 substrate was added to all wells, and the
content was mixed by orbital shaking for one minute. The cells
were incubated for 1 h at 37
◦
C, and the two fluorescence
signals were recorded simultaneously. The fluorescence signal to
determine cell viability was recorded with 400
Ex
/505
Em
filters,
and the fluorescence signal monitoring the cell cytotoxicity was
recorded using 485
Ex
/520
Em
filters. After measurement of cell
viability and cytotoxicity, 100 ml of the Caspase-Glo 3/7 assay was
added to all wells, and the luminescence signal from the caspase-
3/7 activation was measured after 30 min of incubation. Each
plate had the cells without any treatment as negative controls.
Staurosporine (10 mM, 1 ml per 100 mlfrom1mMstockinDMSO)
was used as an apoptosis positive control. Lysis solution Triton
X 100 (2 ml per 100 ml from 9% stock in DMSO) was used as
a positive control to induce the cell death. Each experiment was
performed in triplicate.
CytoTox-ONE
TM
homogeneous membrane integrity assay
The CytoTox-ONE
TM
Assay is a rapid, fluorescent measure of
the release of lactate dehydrogenase (LDH) from cells with a
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The Royal Society of Chemistry and Owner Societies 2011 Photochem. Photobiol. Sci., 2011, 10, 822–831 | 825
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Frequently asked questions

Q1. What are the future works in this paper?

For future in vivo studies, an additional advantage of coating gold nanoparticles with polyethylene glycol is that it has been shown to increase the blood circulation time of the nanoparticles and subsequent accumulation within tumours. ICP-MS analysis of the gold nanoparticle conjugates suggests that approximately 10 molecules of the C11Pc phthalocyanine derivative are attached to each nanoparticle ( assuming that the ligand stabilised gold clusters are cuboctahedrons and that each nanoparticle consists of 309 gold atoms2,35,36 ). Further evidence of increased cell mortality following photodynamic treatment with the nanoparticle conjugates was provided through a series of cell-based assays in vitro. The cells were incubated for a further 18 h before proceeding with the MTT assay. 

Q2. What are the contributions in this paper?

The use of gold nanoparticles in biomedical research has attracted a growing interest due to a combination of unique properties, such as a high surface area and a facile surface functionalisation this paper.