BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine

8 (2012) 908 915

Research Article

nanomedjournal.com

Photothermal release of small molecules from gold nanoparticles in

live cells
Wesley F. Zandberg, PhD a , Amir Bahman Samsam Bakhtiari, MSc a , Zach Erno, BSc a ,
Dennis Hsiao, BSc a , Byron D. Gates, PhD a , Thomas Claydon, PhD b , Neil R. Branda, PhD a,
b

a
4D LABS, Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada
Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada
Received 10 January 2011; accepted 23 October 2011

Abstract
The ability of gold (Au) nanoparticles (NPs) to generate heat efficiently by absorbing visible and near-infrared (NIR) light holds great
promise as a means to trigger chemical and biochemical events near the NPs. Previous demonstrations show that pulsed laser irradiation can
selectively elicit the release of a fluorescent dye covalently anchored to the NP surface through a heat-labile linker without measurably
changing the temperature of the surroundings. This article reports that the authors demonstrate the biological efficacy of this approach to
photodelivery by showing that the decorated Au NPs are rapidly internalized by cells, are stable under physiological conditions, are nontoxic,
and exhibit nonlethal photorelease following exposure to pulsed laser radiation. These observations, further supported by the versatility of
our delivery motif, reaffirm the potential for further development of nonlethal photothermal therapeutics and their future relevance to such
fields as gene therapy and stem-cell differentiation.
From the Clinical Editor: The authors further refine previous observations suggesting that Au NP-s may be useful in targeted drug or gene
delivery systems. Due to a strong photothermal release effect and their generally low toxicity, Au NP-s may become an important subject of
choice in targeted delivery systems.
2012 Elsevier Inc. All rights reserved.
Key words: Gold nanoparticles; Photothermal effect; Drug delivery; Live cells

The challenge of developing pharmaceuticals capable of

distinguishing between healthy and diseased cells remains a
major impediment to the treatment of many illnesses and has
inspired the development of various strategies designed to facilitate
the delivery of therapeutic agents in a spatially and temporally
controlled manner. 1 These strategies benefit from the use of
delivery vehicles that are nontoxic, compatible with targeting

This research was supported by the Natural Sciences and Engineering

Research Council (NSERC) of Canada, the Canada Research Chairs Program
(Branda and Gates) and Simon Fraser University (SFU) through the
Community Trust Endowment Fund. This work made use of 4D LABS
shared facilities supported by the Canada Foundation for Innovation (CFI),
British Columbia Knowledge Development Fund (BCKDF), Western
Economic Diversification Canada, and Simon Fraser University.
The authors report that there are no commercial associations, current and
within the past five years, that might pose a potential, perceived, or real
conflict of interest.
Corresponding author: 8888 University Drive, Burnaby, B.C. V5A 1S6,
Canada.
E-mail address: nbranda@sfu.ca (N.R. Branda).

modalities, and able to circumvent biological barriers that would

otherwise render their therapeutic payloads ineffective. 2 Gold
nanoparticles (Au NPs) excel in many of these aspects. 3 They
exhibit low cytotoxicities, 4-6 are readily internalized by cells, 7-9
and are amenable to functionalization with biomolecules and
passivating agents that have been shown to confer enhanced celltargeting 10,11 and immunoevasive 12,13 properties, respectively.
Au NPs also possess intense absorbance bands that can be tuned
to specific regions throughout the visible and near-infrared (NIR)
spectrum by judicious control over their sizes and shapes. These
bands correspond to excitation of localized surface plasmons and
have extinction coefficients approaching 10 11 M 1 cm 1, which
are several orders of magnitude greater than archetypal organic
dyes. 14 Subsequent plasmonic relaxation is accompanied by
localized heating, a phenomenon that has been successfully
exploited to target and destroy cancer cells. 11,15 However, the
gradual dissipation of the heat with increasing distance from the NP
suggests an additional possibility: the use of the photothermal
effect as a means to selectively drive temperature-sensitive
chemical reactions at the particle surface without affecting the
temperature of the surroundings. The nearly ubiquitous

1549-9634/$ see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.nano.2011.10.012
Please cite this article as: W.F., Zandberg, et al, Photothermal release of small molecules from gold nanoparticles in live cells. Nanomedicine: NBM
2012;8:908-915, doi:10.1016/j.nano.2011.10.012

W.F. Zandberg et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 908915

909

temperatures generated during photothermal tumor ablation,

these conditions confine the heating to the surfaces of the NPs
without measurably increasing the temperature of the surroundings, offering the possibility of photorelease without killing cells.
However, several issues critical to the efficacy of our delivery
approach in biological settings were not addressed in the original
manuscript, 16 which include cellular uptake and cytoxicity of the
delivery vehicle, stability of the decorated Au NPs under cellular
physiological conditions, cell viability following laser irradiation, and the in vivo photothermal release of the NP's payload.
These outstanding issues are addressed within this article.

Methods
General preparations
Figure 1. Photothermolysis of a tailor-made tether anchoring a fluorescent
dye to the surface of a Au NP, resulting in a decrease in quenching efficiency
as the lumophore diffuses away from the particle surface. The two weak
bonds programmed into the linker are highlighted.

dependence of chemical reaction rates on local temperature implies

that such a universal approach could be used in conjunction with an
almost limitless variety of drug-delivery motifs.
A scant collection of reports describe photothermal chemicalrelease methodologies, either by eliciting direct dissociation of
surface ligands 16,17 or by enhancing the permeability of polymeric
vesicles encapsulating therapeutic agents. 18 Of these systems, the
studies reported by Lee 17 and Yavuz 20 are the only ones that
evaluate cellular uptake of the delivery vehicle and cell viability
following laser irradiation. However, these elegant examples have
some limitations. The former system, though potentially compatible
with a wide range of oligonucleic acid therapeutics, is not as well
suited to release small molecules. The composite delivery vehicles
developed by Yavuz are ultimately derived from silver nanocube
precursors whose preparation requires stringent optimization. 19
A more versatile photorelease system that overcomes these
limitations would be one capable of delivering almost any
molecular or macromolecular payload in response to nonlethal
photothermal stimuli using simple metal NPs. We recently
described two successful systems consisting of 16 nm Au or
200 nm silica-Au core-shell NPs decorated with a modified
fluorescein dye. 16 In these examples, the fluorescent dye was
tethered to the NP surface using a linker containing alkanethiol
and oxabicycloheptene moieties (Figure 1). The former acts to
anchor the dye to the Au surface to allow fluorescent quenching
by the NP whereas the latter (a versatile motif used in numerous
studies requiring the reversible assembly of covalent bonds) 20-24
provides the programming of the photothermal release due to its
readily undergoing predictable bond breaking when the
temperature is raised to above 6070C. Pulsed laser irradiation
of the delivery vehicle with 532 nm light (Au NPs) or 800 nm
light (core-shell NPs) led to the efficient photothermolysis of the
oxabicycloheptene motif via a retro-Diels-Alder reaction and
subsequent diffusion of the fluorescent dye away from the NP,
resulting in visible luminescence. In contrast to the lethal

Decorated Au NPs were prepared as previously described. 16 All

other chemicals were purchased from Sigma-Aldrich (Mississauga,
Ontario) unless otherwise stated. Oocytes were surgically isolated
from Xenopus frogs in accordance with the policies and procedures
of the Simon Fraser University Animal Care Committee (License
862K-08) and the Canadian Council of Animal Care.
Oocyte preparation and injection
Oocytes were acutely isolated from gravid Xenopus laevis
frogs following terminal anesthesia by immersion in tricaine
methanesulphonate (2 mg/mL). Stage V-VI oocytes were isolated
and defolliculated using a combination of collagenase treatment (1
hour in 1 mg/mL collagenase type 1A) and manual defolliculation.
Oocytes were injected with 50 nL of decorated GNPs using a
Drummond digital microdispenser (Fisher Scientific, Nepean,
Canada) and then incubated in Barth's medium (in mM: 88 NaCl,
1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, 20
HEPES, pH 7.4). Following microinjection, oocytes were
incubated at 19C for 1 hour prior to laser exposure.
Sample irradiation
A subset of the injected oocytes was irradiated with 532 nm
light (10 Hz, 90100 mW cm 2, 4 ns) emitted from a nanosecond
Nd:YAG (neodymium-doped yttrium aluminium garnet, Nd:
Y3Al5O12) PL8000 laser (Continuum). All samples were irradiated
over four 60-second intervals set 30 seconds apart, and all the
experiments subsequently described were carried out using the
same NPs and method of irradiation. A lens of 500 mm focal length
was used to ensure that oocytes were evenly irradiated; this was
omitted from the experiments described for tissue-cultured cells
and for capillary electrophoresis samples. After irradiation, some
oocytes were transferred onto glass slides and visualized by
widefield fluorescence microscopy at 5 magnification on an
Axio Imager upright microscope (Carl Zeiss Canada Ltd., Toronto,
Canada) equipped with a mercury lamp as a light source. Images
were acquired with an AxioCam MRc5 color camera (Zeiss) at
450490 nm and 515565 nm excitation and emission wavelengths, respectively, and 400-millisecond exposure times. Light
below 510 nm was excluded with a band pass filter.

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Electrophysiology
Following laser exposure, oocyte health and quality was assessed
electrophysiologically using the two-electrode voltage clamp
technique. Microelectrodes with a resistance of 0.52.0 M (tip
diameter, 15 m) filled with 3 M KCl were used. Resting
membrane potential and membrane currents were recorded under
voltage clamp using an Axoclamp 900 amplifier (Axon Instruments,
Foster City, California) with computer-driven voltage protocols
(Clampex software and Digidata 1440A interface, Axon Instruments). Currents were recorded during 200-millisecond voltage
pulses from a holding potential of 80 mV to a test potential of +60
mV. During experiments, oocytes were perfused with ND96
solution (mM: 96 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2 and 5 HEPES,
pH 7.4). Experiments were performed at room temperature (20
22C). Data are expressed as means standard deviation (SD).
Cell culture
Chinese hamster ovary (CHO)-K1 cells were routinely grown in
a 1:1 (v/v) mixture of Dulbeco's modified Eagle's medium (DMEM)
and Ham's F12 medium (Invitrogen, Carlsbad, California) supplemented with 5% fetal bovine serum (FBS; Gibco, Grand Island, New
York). Cells were maintained at 37C and 5 % CO2 unless indicated.
Cells were passed at a dilution of 1/20 every 5 or 6 days.
Au NP uptake, sample irradiation, and cell viability assay
CHO K1 cells were added to 3.5-cm tissue culture dishes
(Sarstedt, Numbrecht, Germany), each containing one flamesterilized 12-mm diameter glass coverslip (VWR, Radnor,
Pennsylvania), at a seeding density of 3 10 4 cells / plate. Then,
24 hours post seeding, plates were washed with 1 1 mL
phosphate buffered saline (PBS), pH 7.4, and 1 mL media
containing 5% FBS, decorated Au NPs (20 L Au NP stock/mL
media) and 100 ug/mL AlexaFluor 561 transferin-conjugate
(Invitrogen) was added. Cells were incubated in the presence of
Au NPs for 20 minutes, after which the media was removed and the
cells were washed with 1 1 mL 19-22 C PBS. Room temperature
DMEM:F12 without serum or the phenol red pH indicator (Gibco)
was then added to each sample. It was necessary to add the media at
room temperature to prevent the build-up of condensation droplets
under the lid of the tissue culture dish, which could scatter the laser
beam. Samples were irradiated with 532-nm laser light (see above).
Radiant flux through the samples was confirmed to be between
90100 mW cm 2 by placing a radiometer below the tissue culture
dish. Care was taken to ensure that each coverslip was directly
centered under the laser beam.
Cell viability, n = 8 for all conditions, was assessed with
AlamarBlue (Invitrogen) according to the manufacturer's instructions. Cells were seeded into 96-well plates (Corning, Inc.,
Corning, New York) 24 hours prior to Au NP or laser exposure.
All washes, Au NP/media concentrations, incubation times, and
irradiation protocols were identical to those described above
except that the volumes used were scaled down from 1 mL to 200
L. After irradiation, the media were replaced with fresh media
containing 5% FBS and the AlamarBlue reagent was added to
cells immediately or after 20 hours. For both groups, cells were
incubated for 3 hours before fluorescence was measured on a

fmax Microplate reader (Molecular Devices, Sunnyvale, California) at 544/590 nm (Ex/Em). Lactacystin (Toronto Research
Chemicals, Inc., North York, Canada) was used as a positive
control and included in the culture media (50 M) before and
during the viability assays.
Microscopy
Immediately after irradiation cells were washed with 1 1 mL
PBS and fixed in a solution of 4% paraformaldehyde in PBS
at 37C for 10 minutes. After washing with 4 1 mL PBS
(5 minutes/wash) coverslips were mounted in VectaShield
(Vector Laboratories) containing fluorescent 4,6-diamidino-2phenylindole (DAPI). Images were acquired with a WaveFX
spinning disc confocal microscope (QuorumTechnologies, Inc.,
Guelph, Canada), equipped with 491 and 561 nm solid state
lasers, using a 63 , 1.4 numerical aperture oil immersion
objective lens (Zeiss). Fluorescein (491/520 nm, Ex/Em),
AlexaFluor 561 (561/590 nm, Ex/Em) and DAPI (acquired
using an X-Cite series 120 UV lamp (Exfo, Quebec, Canada) in
wide-field mode) fluorescent signals were sequentially collected,
in the order listed, using an EMCCD camera (Hamamatsu
Photonics, Hamamatsu, Japan). All images are composed of a
series of XY planes acquired in increments of 0.25 m in the zaxis. The fluorescein and AlexaFluor 561 images were deconvoluted, and the fluorescence intensity was quantified using
Velocity 5.0 software (Improvision/Perkin-Elmer, Waltham,
Massachusetts). Parameters for quantification were selected so
that whole cells were defined as regions of interest. All images
were acquired and displayed using the same optical settings.
Capillary electrophoresis
Capillary electrophoresis was performed on a Beckman-Coulter
PA800 equipped with a laser-induced fluorescence detector (Ar laser
at 488 nm) using a boric acid buffer (10 mM, pH 9.2) containing
sodium dodecylsulfate (50 mM). The buffer was prepared by
dissolving analytical grade boric acid in 18 M water and adjusting
the pH with aqueous NaOH (1.0 M). Sodium dodecylsulfate was
then added, and the resulting solution was filtered before use. The
fused silica capillary was purchased from Beckman-Coulter and had
an internal diameter of 75 M and a total length of 60 cm; the length
from detection to injection was 50 cm. The capillary was conditioned
prior to sample injection by rinsing for 120 seconds with aqueous
NaOH (0.1 M), water (120 seconds), and then with buffer (120
seconds), each under an applied pressure of 20 psi. Samples were
injected into the cathode end of the capillary using hydrodynamic
pressure (0.5 psi) for 3 seconds, and electropheragrams were
acquired at a constant voltage of 30 kV at 22C.

Results
Wide-field fluorescence microscopy revealed little to no
observable difference in luminescence intensity between the
control, untreated oocytes (Figure 2, A) and those microinjected
with 50 nL of an aqueous dispersion containing 16 nm decorated
Au NPs 1 hour before imaging (Figure 2, B). Only a small
amount of background autofluorescence was observed in both

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911

Figure 2. Wide-field fluorescence micrographs of Xenopus laevis oocytes at 5 magnification (A) before and (B) after injection with the delivery vehicle, and
(C) after injection with the NP vehicle and exposure to pulsed laser irradiation. (D) Comparison of average resting membrane potentials and average holding
currents (required to maintain a 80 mV membrane potential) among samples of oocytes injected with the delivery vehicle and noninjected oocytes, measured
before and after pulsed laser irradiation (n = 19 for all samples except untreated cells, for which n = 20). The inset shows the number of cells (which were
not subjected to patch-clamp analysis) visually inspected to be alive after 48 hours. In addition, n = 20 for both irradiated (open circles) and control (closed
circles) oocytes.

cases. Bright fluorescence was visually observed only when the

cells were exposed to pulsed laser irradiation with 532 nm light
(10 Hz, 90100 mW/cm 2, 4 nanoseconds; Figure 2, C).
Oocyte viability was assessed based on resting membrane
potentials (RMPs) and leak current measurements in ND96
(Figure 2, D). The values measured for untreated oocytes prior to
irradiation (48 0.09 mV, 0.09 0.01 A, n = 20) were
similar to those acquired after exposure to the laser light (49
0.06 mV, 0.06 0.01 A, n = 19). Analogous results were
obtained from oocytes injected with 16 nm decorated Au NPs
before (44 1 mV, 0.15 0.03 A, n = 19) and after
irradiation (45 2 mV, 0.08 0.01 A, n = 19).
Internalization of the NP vehicle and subsequent photothermal
release were also studied using CHO cells. Confluent monolayers
of CHO cells grown on glass coverslips were incubated with
media containing 2% (v/v) of a decorated NP solution, 5% FBS,
and a fluorescent transferin (Tfn) derivative. Cells were exposed
to the NPs, under sterile conditions, for 20 minutes at 37C, after
which they were washed once and the media were replaced with
fresh, NP-free media. The cells were then kept in the dark or
irradiated over four 1-minute intervals separated by 30 seconds,
with 532 nm laser light (10 Hz, 90100 mW/cm 2 , 4

nanoseconds). Irradiation was performed directly through the

lid of the tissue culture dish as with the oocytes to maintain sterile
conditions. The cells were then fixed with paraformaldehyde and
visualized in three dimensions using a spinning-disc confocal
microscope (Figure 3). Cell nuclei were stained with DAPI and
appear in blue, and early endosomes stained with the luminescent
Tfn derivative are shown in red. Luminescence from the modified
fluorescein ligand, shown in green, is visible only in the group of
cells exposed to pulsed laser irradiation (Figure 3, bottom panel).
Quantification of the fluorescence microscope images based on
voxel intensities reveals similar amounts of emission from the Tfn
derivative in both irradiated and nonirradiated cells (Table 1). The
average voxel intensity per cell attributable to emission from
fluorescein, however, was 1.8 times greater among irradiated
CHO cells than values obtained from cells kept in the dark.
Before fixation with paraformaldehyde, CHO cells were
examined using an AlamarBlue viability assay in which
metabolic activity, used as a proxy for cell viability, was
quantified by measuring the reduction of the nonfluorescent dye,
resazurin, to the highly fluorescent dye, resorufin. Lactacystin (a
specific inhibitor of the 20S proteasome 21 that induces apoptosis
in some cell lines 22 by inhibition of cell cycle progression) 23,24

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W.F. Zandberg et al / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 908915

Figure 3. Cells were incubated with the functionalized NP vehicle for 20 min before pulsed laser irradiation (bottom panel); control cells (top panel) were not
irradiated. From left to right: DAPI-stained nuclei visualized in wide-field mode appear in blue; Tfn-AlexaFluor 561-stained early endosomes appear in red and
the released fluorescein derivative appears in green, both of which are visualized as stacks of multiple confocal planes; the final image is an overlay of the same
confocal plane acquired using each of the three separate filters.

Table 1
Quantification of fluorescence data acquired from CHO cells
Laser

Voxel count

Volume (m 3)

# of cells

AlexFluor 561 voxel intensity

min

max

avg

avg/cell

min

max

avg

avg/cell

2.15 10 7
1.99 10 7

129570
120089

22.5
21

0
0

789119
846329

7686
8755

342
417

664
490

127048
643952

3370
5616

150
267

Fluorescein voxel intensity

TFn and fluorescein fluorescence was quantified after image deconvolution. The average number of cells was visually estimated from the DAPI signal.

was utilized as a positive control for the viability assays. Four

hours after treatment with resazurin, cells exposed to the NP
vector and pulsed laser irradiation, independently or in
combination with one another, exhibited luminescence intensities similar to those observed in control populations (Figure 4).
When cells were exposed to resazurin and examined 24 hours
after laser irradiation, an increase in fluorescence was observed
in all populations except the ones treated with Lactacystin. To
eliminate the possibility that the fluorescence observed from
cells treated with the NPs and exposed to laser light was due to
free fluorescein, the fluorescence of a solution of the original NP
mixture added to the cells was measured before and after
irradiation using the AlamarBlue filter set (Figure 4, inset).
Although there was a slight increase in fluorescence upon
irradiation of the NP solution (0.92 0.13 increased to 1.62
0.04 RFU), the AlamarBlue signal in a sample treated with the
NPs and irradiated cells was much greater (5094 948 RFU).
Capillary electrophoretic analysis of the mixture generated by
pulsed laser irradiation of the decorated NPs revealed two peaks

(Figure 5, top). Comparison with authentic samples of furanfunctionalized fluorescein and free fluorescein (Figure 5,
bottom) enabled identification of the major product as the
former and the minor product as the latter (see Supplementary
Materials [available online at http://www.nanomedjournal.com]
for peak assignments).

Discussion
The ability of our delivery vehicle to function in live cells was
demonstrated in two systems. We selected Xenopus laevis
oocytes as a model cellular system because their large sizes
permit direct microinjection of the decorated Au NPs, allowing
us to measure the efficacy of the photothermal release under
cellular physiological conditions without the added complication
of addressing whether the delivery vehicles would undergo facile
endocytotic uptake. Furthermore, previous electrophysiological
characterization of the oocytes enables analysis of RMPs and

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Figure 4. Average fluorescence measurements acquired from populations of

Chinese hamster ovary cells (n = 8) during Alamar Blue viability studies
conducted 4 h (gray) and 24 h (white) after sample irradiation.

leak currents, using conventional patch-clamp procedures, as

indicators of membrane integrity and, more generally, cellular
health following injection of the delivery vehicle and exposure to
laser irradiation. 25
Photothermal release in oocytes was evaluated by comparing
wide-field fluorescence microscope images of untreated oocytes
(Figure 2, A), images of oocytes injected with decorated particles
(Figure 2, B), and images of injected oocytes that were
subsequently exposed to pulsed laser irradiation (Figure 2, C).
Similar levels of fluorescence shown in both samples prior to
irradiation suggest that the luminescent ligands remain bound to
the Au NPs under cellular physiological conditions, a feature
ultimately essential to targeted delivery applications where
premature chemical release is undesirable. The observed increase
in luminescence following pulsed laser irradiation of oocytes
injected with the NP vector indicates successful photothermal
release of the lumophore within the cells.
The effects of NP injection and laser exposure on oocyte
viability were independently assessed based on average RMPs
and leak current measurements (Figure 2, D). Taken together,
these data demonstrate that oocyte RMPs are maintained and that
leak currents remain within tolerable limits following injection
with the NP vehicle and subsequent laser irradiation, suggesting
neither membrane integrity nor cell health are compromised by
the laser light or by the subsequent photothermal release of the
fluorescent dye from functionalized NPs. Visual inspection of
the cells revealed no major change in membrane shape or color
following photothermal release, which is consistent with a high
survival rate. Only one oocyte was determined to be dead after
the release event, and discoloration of the cell membrane around
the injection site suggests this was due to membrane degeneration because of microinjection (Figure 2, D, inset).
We then chose to study the performance of our delivery
vehicle in CHO cells as a model for its behavior in mammalian
tissues, 26 allowing us to assess cellular uptake of the decorated
NPs and their effects on tissue cell viability before and after laser
irradiation. The decorated NPs were introduced to CHO cells via
growth media containing FBS, facilitating adsorption of serum

913

Figure 5. Electopherograms showing the mixture of compounds resulting

from exposure of decorated Au NPs to pulsed laser irradiation ( = 532 nm,
10 Hz, 100 mW) (top) and a reference mixture composed of authentic
samples of fluorescein and the furan-functionalized derivative (bottom).
Peaks were assigned based on adding the authentic samples to the reaction
mixture (see Supporting Information for details).

proteins to the NPs for subsequent receptor-mediated

endocytosis. 7,9 In addition, a fluorescent Tfn derivative known
to localize within early endosomes was included in the modified
cell culture media to further elucidate the mechanism of NP
uptake. 27 After incubation, the cells were washed and the growth
media replaced with fresh, NP-free media to ensure that any
fluorescence observed upon sample irradiation could be
attributed to photothermal release from intracellular NPs. CHO
cells were then either kept in the dark or exposed to pulsed laser
radiation. The cells were then fixed with paraformaldehyde to
kill them (to limit progressive autolysis of cellular structures) and
to increase their mechanical strength and rigidity (by anchoring
cellular components to one another through a polymer network)
so that cell morphologies would be retained during later sample
processing and analysis.
Internalization of the NP vector and its ability to undergo
photothermal release in CHO cells were studied by comparing
confocal fluorescence microscope images of irradiated and
nonirradiated cells exposed to the decorated NPs. Nuclear
staining with blue, fluorescent DAPI reveals roughly equivalent
numbers of cells in the irradiated (Figure 3, bottom panel) and
nonirradiated samples (Figure 3, top panel). Red emission from
early endosomes stained with the Tfn derivative indicates
endocytotic activity in both groups during incubation with
decorated NPs. The appearance of green luminescence in CHO
cells following pulsed laser irradiation is consistent with
photothermal release of the modified fluorescein ligand. As
with the oocytes, the absence of fluorescein emission prior to
irradiation suggests that the fluorescein ligand is stable on the NP
surface under physiological conditions in the absence of light.
Very little colocalization of the endosomal marker and
fluorescein derivative following photothermal release was
observed. Emission from the Tfn conjugate is apparent in
discrete patches, whereas emission from the released fluorescein
derivative is throughout the cytosol, albeit it is punctate.

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Interpretation of these results as evidence of nonendocytotic

internalization of the NP vehicle would conflict with a previously
reported example of receptor-mediated endocytosis facilitated by
adsorption of serum proteins to Au NPs, although it cannot be
ruled out that a different entry mechanism was utilized by our
decorated NPs. 8 Alternatively, the possibility of rapid diffusion
of the released fluorescein derivative through the endosomal
membrane prior to fixation with paraformaldehyde would
reconcile our results with those reported in the literature.
The fluorescence microscopy data acquired from both groups
of CHO cells were quantified and compared in terms of voxel
intensities (Table 1). These results suggest that similar quantities
of the fluorescent Tfn derivative were endocytosed by the
irradiated cells and by the control group, supporting the
reasonable assumption that both populations internalized
comparable amounts of the NP vehicle. Additionally, the
average voxel intensity per cell attributable to emission from
the dissociated fluorescein derivative was much greater in
irradiated cells than in control groups. These data indicate
successful photothermal release of the fluorescent dye in tissuecultured cells irradiated under sterile conditions. Given the
amount of background fluorescein emission observed in
nonirradiated samples, it can be concluded that the functionalized NP vehicle is also reasonably stable in the cellular
environment and that very small quantities of the fluorescein
derivative are released in the absence of laser irradiation. Similar
levels of background fluorescence were observed from cells that
had been incubated with functionalized NPs for 4 hours prior to
fixation and confocal microscopic analyses.
The effects of NP uptake and pulsed laser irradiation on
CHO cell viability and proliferation were examined using an
assay that quantifies cellular physiological activity based on the
amount of a luminescent dye, resorufin, generated metabolically
from an administered precursor, the nonfluorescent dye
resazurin. As a positive control, CHO cells were separately
treated with the proteasome inhibitor Lactacystin. Results from
experiments performed 4 hours after laser exposure suggest
that uptake of the decorated Au NPs, laser irradiation or a
combination of both had little to no immediate effect on cell
viability. An increase in luminescence intensity observed among
all groups except those treated with lactacystin was observed
24 hours after laser irradiation, which indicates that neither the
presence of the Au NPs nor laser exposure had reduced the rates
of cell proliferation. In contrast, cells exposed to lactacystin
did not exhibit a corresponding increase in luminescence after
24 hours, which is consistent with cell-cycle inhibition and
affirms the validity of our AlamarBlue viability data.
The possibility that luminescence from the modified
fluorescein ligand might interfere with the results of the
AlamarBlue assay was addressed by emission measurements
performed on a solution of the decorated NPs using the
AlamarBlue filter set, before and after exposure to pulsed laser
irradiation. The slight increase in luminescence intensity that
was observed indicates a small amount of bleed-through.
However, the amount of interference is negligible in comparison with the increase in luminescence intensity observed in
cells treated with the NP vector following irradiation. This
finding confirms that the observed increase in fluorescence is not

due to free fluorescein adducts but to metabolized resazurin. It

also confirms that these procedures were not cytotoxic, nor did
they affect cell proliferation.
To identify what fluorescent compound or compounds are
released as a result of the photothermal reaction, samples of Au
NPs were irradiated (532 nm) and analyzed by capillary
electrophoresis, whereby the electrophoretic mobility of each
component in the mixture was characterized based on the time
it took to migrate past an excitation light source (Ar laser, 488
nm) and fluorescence detector positioned alongside the
capillary. The mixture was shown to consist of a major and a
minor product. The major product was identified as the furanfunctionalized fluorescein, which confirms that the increase in
fluorescence intensity accompanying pulsed laser irradiation of
the decorated Au NPs is due primarily to photothermolysis of
the oxabicycloheptene motif via the retro-Diels-Alder reaction.
The minor product was identified as free fluorescein, which is
presumably generated by hydrolysis of the furfuryl ester. These
results are consistent with previously reported data that show
minimal AuS bond breaking and release of thiols under
similar conditions. 28
In summary, we succeeded in preparing a NP-based drug
delivery vehicle and demonstrated its ability to elicit photothermal release within live oocytes and tissue cell cultures. Our
viability studies indicate high cell survival rates following
treatment with functionalized NPs and subsequent laser
irradiation, highlighting the possible extension of intracellular
photothermal phenomena to noncytotoxic applications, such as
gene therapy and stem cell differentiation. Our future work
will combine these design principles with established celltargeting methodologies utilizing antibodies to test our
system's ability to deliver targeted chemical agents having
therapeutic properties.

Appendix A. Supplementary data

Supplementary data to this article can be found online at
doi:10.1016/j.nano.2011.10.012.

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