Surface Plasmon Resonance Scattering and Absorption of anti-EGFR ...

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Letter
Surface Plasmon Resonance Scattering and Absorption
of anti-EGFR Antibody Conjugated Gold Nanoparticles
in Cancer Diagnostics:  Applications in Oral Cancer
Ivan H. El-Sayed, Xiaohua Huang, and Mostafa A. El-Sayed
Nano Lett., 2005, 5 (5), 829-834 • DOI: 10.1021/nl050074e
Downloaded from http://pubs.acs.org on February 8, 2009
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NANO
LETTERS
Surface Plasmon Resonance Scattering
2005
Vol. 5, No. 5
and Absorption of anti-EGFR Antibody
829-834
Conjugated Gold Nanoparticles in
Cancer Diagnostics: Applications in
Oral Cancer
Ivan H. El-Sayed,*,† Xiaohua Huang,‡ and Mostafa A. El-Sayed*,‡
Department of Otolaryngology-Head and Neck Surgery, ComprehensiVe Cancer
Center, UniVersity of California at San Francisco, San Francisco, California 94143,
and Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia
Institute of Technology, Atlanta, Georgia 30332
Received January 13, 2005
ABSTRACT
Gold nanoparticles with unique optical properties may be useful as biosensors in living whole cells. Using a simple and inexpensive technique,
we recorded surface plasmon resonance (SPR) scattering images and SPR absorption spectra from both colloidal gold nanoparticles and
from gold nanoparticles conjugated to monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodies after incubation in cell cultures
with a nonmalignant epithelial cell line (HaCaT) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3). Colloidal gold
nanoparticles are found in dispersed and aggregated forms within the cell cytoplasm and provide anatomic labeling information, but their
uptake is nonspecific for malignant cells. The anti-EGFR antibody conjugated nanoparticles specifically and homogeneously bind to the
surface of the cancer type cells with 600% greater affinity than to the noncancerous cells. This specific and homogeneous binding is found
to give a relatively sharper SPR absorption band with a red shifted maximum compared to that observed when added to the noncancerous
cells. These results suggest that SPR scattering imaging or SPR absorption spectroscopy generated from antibody conjugated gold nanoparticles
can be useful in molecular biosensor techniques for the diagnosis and investigation of oral epithelial living cancer cells in vivo and in vitro.
The increasing availability of nanostructures with highly
preparation, ready bioconjugation, and potential noncyto-
controlled optical properties in the nanometer size range has
toxicity.6 Immunogold nanoparticles conjugated to antibodies
created widespread interest in their use in biotechnological
have provided excellent detection qualities for cellular
systems for diagnostic application and biological imaging.1-2
labeling using electron microscopy.7
Cellular imaging utilizing microscope techniques provides
Gold nanoparticles have the ability to resonantly scatter
anatomic details of cells and tissue architecture important
visible and near-infrared light upon the excitation of their
for cancer diagnostics and research. Currently used optical
surface plasmon oscillation. The scattering light intensity is
probes include chemiluminescent, fluorimetric, and colori-
extremely sensitive to the size and aggregation state of the
metric techniques.3 Markers attached to antibodies provide
particles.8 Preliminary studies have reported their use as
specific information about the presence of specific molecules.
contrast agents for biomedical imaging using confocal
Quantum dots are widely used and studied for this application
scanning optical microscopy,9 multiphoton plasmon reso-
due to their unique size-dependent fluorescence properties.4,5
nance microscopy,10 optical coherence microscopy,11 and
But potential human toxicity and cytotoxicity of the semi-
third-harmonic microscopy.12 Gold nanoparticles have several
conductor material are two major problems for its in vitro
advantages for cellular imaging compared to other agents.
and in vivo application. Colloidal gold nanoparticles have
They scatter light intensely and they are much brighter than
become an alternative consideration due to their ease of
chemical fluorophores. They do not photobleach and they
can be easily detected in as low as 10-16 M concentration.13
Sokolov9 described the scattering of anti-EGFR/Au nano-
* Corresponding authors. Ivan El-Sayed: ielsayed@ohns.ucsf.edu. Tel:
415-353-2401, Fax:
415-353-2603; Mostafa El-Sayed:
mostafa.el-
particles for cervical cancer when stimulated with a laser at
sayed@chemistry.gatech.edu. Tel: 404-894-0292. Fax: 404-894-0294.
†
single wavelength. Irradiation with a laser will only scatter
University of California at San Francisco.
‡ Georgia Institute of Technology.
light that is close to the laser wavelength used. Gold
10.1021/nl050074e CCC: $30.25
© 2005 American Chemical Society
Published on Web 04/15/2005

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nanoparticles scatter light of many colors when illuminated
from a mercury lamp on top of the samples. A 100×/1.35
with white light at appropriate angles. The wavelength
oil Iris Ph3 objective (UPLANAPO) was used to collect only
distribution of the light in this case is determined by the shape
the scattered light from the samples. When the light beam
and size of the nanoparticles.14 This color scattering property
direction is optimized, the center illumination light beam does
offers the potential for labeling studies with a white light
not enter the light collection cone of the microscope
source, which has not yet been characterized for cellular
objective, and only the scattered light of the side beam by
imaging and detection.
the sample is collected. This presents an image of a bright
In the present work, we used a very simple and inexpen-
object in a dark background. The absorption spectra of gold
sive conventional microscope with proper rearrangement of
nanoparticles inside a single cell were measured using a
the illumination system and the light collection system to
SEE1100 microspectrometer under 20× magnification.
image cells that were incubated with colloidal gold or with
In the present work gold nanoparticles with the average
anti-epidermal growth factor receptor (anti-EGFR) antibody
sizes of 35 nm are chosen after experimental determination
conjugated gold nanoparticles. The optical properties of the
of the particle uptake efficiency, the cellular labeling
gold nanoparticles incubated in single living cancerous and
efficiency, and the light scattering intensities of the nano-
noncancerous cells are compared for different incubation
particles. Gold nanoparticles are introduced into cells by the
methods. It is found that the scattering images and the
endocytosis process during cell differentiation and prolifera-
absorption spectra recorded from anti-EGFR antibody con-
tion processes. Smaller nanoparticles cross the cytoplasmic
jugated gold nanoparticles incubated with cancerous and
membrane more easily, but their scattering light cross-section
noncancerous cells are very different and offer potential
is smaller than larger nanoparticles. They also give more
techniques for cancer diagnostics.
greenish scattered color which cannot be easily resolved from
Gold NPs were prepared by the citrate reduction of
the scattered green light from the cellular organelles. Larger
chloroauric acid.15 The nanoparticles have an absorption
nanoparticles have higher scattering cross-section but have
maximum at 529 nm, and TEM shows that the nanoparticles
smaller labeling efficiency, possibly due to steric hindrance.
have an average size of 35 nm. The anti-EGFR/gold
For this experiment, we also used 15 and 60 nm nano-
conjugates were prepared according to the method described
particles, but neither one is found to be more efficient than
by Sokolov.8 Briefly, the gold NPs were diluted in 20 mM
the 35 nm nanoparticles in either the amount of colloid
HEPES buffer (pH 7.4, Sigma) to a final concentration with
nanoparticles uptaken into cells or the labeling efficiency
optical density of 0.8 at 529 nm. 40 uL anti-EGFR mono-
for cancer cell detection when anti-EGFR antibodies are
clonal antibodies (host mouse; Sigma) was added to 960 uL
used.
of the same HEPES buffer to form 1 mL dilute solution.
Figure 1 shows the light scattering images of the HaCaT
Then 10 mL of the gold solution prepared above was mixed
noncancerous cells (left column), HOC cancerous cells
with the dilute antibody solution for 20 min. 0.5 mL of 1%
(middle column) and HSC cancerous cells (right column)
poly(ethylene glycol) (MW 20 000; Sigma) was added to
without nanoparticles. All the cells show dim greenish light
the mixture to prevent aggregation, and the solution was
(two images were shown for each type of cells for compari-
centrifuged at 6000 rpm for 30 min. Then the anti-EGFR/
son). This green light is due to autofluorescence and scattered
gold pellet was redispersed in PBS buffer (pH ) 7.4, Cellgro)
light from the cell organelles in cell cytoplasm and mem-
and stored at 4 °C.
brane. From this figure we can see that the three types of
One nonmalignant epithelial cell line HaCaT (human
cells have different structure characteristics. HOC cancer cells
keratinocytes) and two malignant epithelial cell lines HOC
are almost four times larger than HaCaT or HSC cells.
313 clone 8 and HSC 3 (human oral squamous cell
HaCaT and HSC cells show almost homogeneous diamond
carcinoma) were cultured on 18 mm diameter glass cover
shapes while HOC cells have other shapes for some cells.
slips in a 12-well tissue culture plate in DMEM plus 5%
When incubated in the presence of nanoparticles, the cells
FBS at 37 °C under 5% CO2. The cover slips were coated
grow at a normal rate and the nanoparticles are accumulated
with collagen type I (Roche) in advance for optimum cell
inside the cells. The incorporated gold nanoparticles scatter
growth. For the incubation of colloidal gold, nanoparticles
strong yellowish light and make individual cells easily
(∼ 0.3 nM) were added into the medium and the cells were
identifiable. Three images for each kind of cell are shown
grown for 48 h. The cells on the cover slips were then rinsed
to test reproducibility (Figure 2). Examination reveals that
with PBS buffer and fixed with 1.6% paraformaldehyde and
gold nanoparticles are predominantly accumulated inside the
sealed with another cover slip with a small amount of
cytoplasm of the cells. In most HaCaT noncancerous cells
glycerol. For the incubation of conjugated nanoparticles, the
(left column) the gold nanoparticles demonstrate a spotted
cells were grown on the cover slips for 48 h and then the
pattern inside the cytoplasm while the nanoparticles are
cell monolayer was immersed into the conjugated nanopar-
homogeneously distributed in the cytoplasm of HOC (middle
ticle solution for 40 min, rinsed with PBS buffer, fixed with
column) and HSC (right column) cancerous cells. The
paraformaldehyde, and sealed as above.
difference of the distribution of nanoparticles inside cells
The light scattering images were taken using an inverted
may reflect the difference of the cell differentiation and
Olympus IX70 microscope in which the illumination system
proliferation processes. The HSC specimens give the stron-
was removed and replaced by an illumination condenser (U-
gest scattering light due to the large amount of accumulated
DCW), which delivers a very narrow beam of white light
gold nanoparticles.
830
Nano Lett., Vol. 5, No. 5, 2005

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Figure 1. Light scattering images of HaCaT noncancerous cells (left column), HOC cancerous cells (middle column), and HSC cancerous
cells (right column) without gold nanoparticles. Two different images of each kind of cells were shown to test reproducibility. The weak
greenish scattered light from the cells shows large difference in the sizes and shapes of the three different types of cells. Scale bar: 10 µm
for all images.
Using micro-UV-visible spectroscopy, the absorption
The light scattering pattern of gold nanoparticles is
spectra of gold nanoparticles from single cells are obtained
significantly different when anti-EGFR antibodies were
shown in the bottom row of Figure 2. To statistically
conjugated to gold nanoparticles before incubation with the
characterize the surface plasmon absorption of the gold
cells (Figure 3). The HaCaT noncancerous cells are poorly
nanoparticles inside the cells, 25 cells of each kind are
labeled by the nanoparticles and the cells could not be
measured. The NPs inside all cells have a major peak around
identified individually (Figure 3, three images on the left
545 nm, characteristic of the surface plasmon absorption of
column). When the conjugates are incubated with HOC
the individual nanoparticles inside the cytoplasm of the cells
(Figure 3, three images on the middle column) and HSC
that are red shifted by 16 nm compared to the colloid
(Figure 3, three images on the right column) cancerous cells
nanoparticle suspension at 529 nm. This suggests that the
for the same amount of time, the nanoparticles are found on
nanoparticle surface has a different dielectric environment
the surface of the cells, especially on the cytoplasm
when present inside the cells. The broad absorption around
membranes for HSC cancer cells. This contrast difference
700 nm of the gold nanoparticles inside the cells is
is due to the specific binding of overexpressed EGFR on
characteristic of the aggregated gold nanoparticles. Aggrega-
the cancer cells with the anti-EGFR antibodies on the gold
tion of the nanoparticles is likely induced by the salts both
surface. The nanoparticles are also found on the HaCaT
in the growth medium and in the cytoplasm of the cells. The
noncancerous cells due to part of the specific binding, but
capping material could also be dissolved inside cells and thus
mostly due to the nonspecific interactions between the
leads to aggregation of the resulting metallic nanostructures.
antibodies and the proteins on the cell surface, and thus the
In HSC cells, the aggregates have the absorption maximum
nanoparticles are randomly distributed on the whole cells.
around 715 nm. In HaCaT cells, the size of these large
The nonspecific interaction between the anti-EGFR antibod-
aggregates is smaller as concluded from the shorter wave-
ies and the collagen matrix also exists, which is shown as
length surface plasmon absorption maximum. The absorption
the reddish scattering light of the gold nanoparticles on the
of the aggregates inside HOC is not as resolved due to the
collagen background.
shorter wavelength (679 nm), which is close to the absorption
When anti-EGFR antibodies are attached to the gold
maximum of the surface plasmon absorption of the individual
nanoparticles, all the absorption spectra on different cells
nanoparticles. The different sizes of the aggregates inside
become narrower and similar for each cell type. No absorp-
different kind of cells may reflect the difference in the cell
tion bands due to aggregation are observed. The nanoparticles
cytoplasm medium or differences in the intracellular process-
bound to HOC and HSC cancer cells have similar absorption
ing of the nanoparticles by the cells. The ability to resolve
maxima at around 545 nm, which is 9 nm red shifted
aggregates within cells by SPRA spectroscopy suggests that
compared to the isolated anti-EGFR/Au solutions at 536 nm.
different capping agents could be utilized to monitor intra-
This red shift is due to the specific binding of the anti-EGFR
cellular processes as aggregates are formed.
antibodies on the gold surface to EGFR on the cell surface.
Nano Lett., Vol. 5, No. 5, 2005
831

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Figure 2. Light scattering images and microabsorption spectra of HaCaT noncancerous cells (left column), HOC cancerous cells (middle
column), and HSC cancerous cells (right column) after incubation with unconjugated colloidal gold nanoparticles. Three different images
of each kind of cells are shown to test reproducibility. The images show that the particles are inside the cells in the cytoplasm region but
do not seem to adsorb strongly on the nuclei of the cells. The absorption spectra were measured for 25 different single cells of each kind.
They show that nanoparticles have an SPR absorption maximum around 548 nm, independent of the cell type. The broad long wavelength
tails in the absorption spectra suggest the presence of aggregates. It also shows that no specific difference is observed in either the scattering
images or the absorption spectra of the gold nanoparticles in the cancerous and the noncancerous cells. Scale bar: 10 µm for all images.
It also could be due to the interparticle interaction resulting
and 0.07 for HSC cells. One can conclude that the binding
from the arrangement of the conjugates on the cell surface
ability of the anti-EGFR antibody conjugated nanoparticles
in two dimensions. Such spectroscopic binding undoubtedly
to HOC and HSC cancerous cells is 600% and 700%,
changes the dielectric constant around the surface of the gold
respectively, over the HaCaT noncancerous cells. This is
nanoparticles. One can use the maximum at 545 nm to
undoubtedly due to the difference of the EGFR concentration
characterize the conjugated nanoparticles binding to the
on the surface of the cancer and noncancerous cells. Current
EGFR on the cell surface. For HaCaT noncanerous cells,
optical staining techniques do not have the ability to quantify
the particles with maximum at 545 nm are found to have a
nonspecific binding in this manner. Our results correlate well
maximum absorption of 0.01 (Figure 3) for the 25 cells
with previously published studies which qualitatively report
measured. The rest of the nanoparticles have their maximum
that most cancerous cells accumulate significantly higher
at 552 nm. This red shift indicates that these nanoparticles
amounts of EGFR during the carcinoma process.16
are nonspecifically bound. The maximum absorbance of the
In summary, cellular imaging with improved contrast due
conjugated particles to cancer cells is of 0.06 for HOC cells
to the strong resonant light scattering of gold nanoparticles
832
Nano Lett., Vol. 5, No. 5, 2005

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Figure 3. Light scattering images and microabsorption spectra of HaCaT noncancerous cells (left column), HOC cancerous cells (middle
column), and HSC cancerous cells (right column) after incubation with anti-EGFR antibody conjugated gold nanoparticles. Three different
images of each kind of cells are shown to test reproducibility. The absorption spectra were measured for 25 different single cells. The
figure shows clearly distinguished difference for the scattering images from the noncancerous cells (left column) and the cancerous cells
(right two columns). The conjugated nanoparticles bind specifically with high concentrations to the surface of the cancer cells (right two
columns). Conjugated nanoparticles did not show aggregation tendency (no long wavelength broad tail is observed). Scale bar: 10 µm for
all images.
incubated inside or on the surface of cells is obtained using
in Figure 3) with an absorption maximum at 545 nm. The
a very simple and inexpensive student microscope with
binding to noncancerous cells seems to be nonspecific and
proper rearrangement of the illumination system and the light
at random, with absorption maximum mostly around 552 nm.
collection system. The nonconjugated gold nanoparticles are
Thus both SPR scattering imaging and SPR absorption
accumulated inside cells and aggregation takes place (Figure
spectroscopy from anti-EGFR antibodies conjugated gold
2). The observed difference in the scattering of different types
nanoparticles are found to distinguish between cancerous and
of cells in this figure is mostly due to the difference in the
noncancerous cells. This makes either technique potentially
size of the cells (Figure 1) and not to the specific interaction
useful in cancer diagnostics.
of the nanoparticles with different cells. However, there is a
distinct difference in the distribution of anti-epidermal growth
Acknowledgment. We thank Prof. Paul Edmonds, Prof.
factor receptor antibody conjugated nanoparticles when
Mohan Srinivasarao, Prof. Rob Dickson, Dr. Lynn Peyser,
incubated with cancerous and noncancerous cells (Figure 3).
Mr. Sandeep Patel, and Mr. Jie Zheng at the Georgia Institute
Conjugated nanoparticles bind homogeneously and specif-
of Technology for assistance and use of their facilities. I.H.E.
ically to the surface of the cancer cells (two right columns
thanks Prof. Randall Kramer and Mr. Moon Lim at the Oral
Nano Lett., Vol. 5, No. 5, 2005
833

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Cancer Research Center at the University of California at
(8) Sokolov, K.; Aaron, J.; Hsu, B.; Nida, D.; Gillanwater, A.; Follen,
San Francisco for their aid, instruction, and use of their
M.; Macaulay, C.; Adler-Storthz, K.; Korgel, B.; Discour, M.;
Pasqualini, R.; Arap, W.; Lam, W.; Richartz-Kortum, R. Technol.
facilities, and Dr. Patrica Leake and Dr. Russell Snyder in
Cancer Res. Treatment 2003, 2(6), 491-504.
the Epstein Laboratory (UCSF) for the use of their facilitites.
(9) Sokolov, K.; Follen, M.; Aaron, J.; Pavlova, I.; Malpica, A.; Lotan,
The financial support of the Chemical Science, Geosciences
R.; Richartz-Kortum, R. Cancer Res. 2003, 63, 1999-2004.
(10) Yelin, D.; Oron, D.; Thiberge, S.; Moses, E.; Silberberg, Y. Opt.
and Bioscience Division of the Department of Energy (NO:
Express 2003, 11, 1385-1391.
DE-FG02-97 ER14799) is acknowledged.
(11) Raub, C. B.; Orwin, E. J.; Haskell, R. J. Biomech. Eng. 2003, 125,
1-6.
References
(12) Yelin, D.; Oron, D.; Korkotian, E.; Segal, M.; Silberberg, Y. Appl.
Phys. B 2002, 74 (Suppl.), S97-S101.
(1) Alivisatos, A. P. Nat. Biotech. 2004, 22, 47-52.
(13) Yguerabide, J.; Yguerabide, E. E. Anal. Biochem. 1998, 262, 137-
(2) Wickline, S. A.; Lanza, G. M. Circulation 2003, 107, 1092-1095.
156.
(3) Roda, A.; Pazzagli, M.; Kricka, L. J.; Stanley, P. E. Bioluminescence
(14) Yguerabide, J.; Yguerabide, E. E. Anal. Biochem. 1998, 262, 157-
and Chemiluminescence: PerspectiVes for the 21st Century; Wiley:
176.
Chichester, 1999.
(15) Hayat, M. A. Principles and techniques of electron microscopy:
(4) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P.
biological applications; Van Nostrand Reihold: New York, 1970.
Science 1998, 281, 2013-2015.
(16) Nicholson, R. J.; Gee, J. M.; Harper, M. E. Eur. J. Cancer 2001, 37
(5) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016-2018.
(Suppl. 4), S9-S15.
(6) West, J. L.; Halas, N. J. Cur. Opin. Biotech. 2002, 11, 215-217.
(7) Hayat, M. A. Colloidal gold: principles, methods and applications;
Academic Press: San Diego, 1989; Vol 1.
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