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Volume 19 | Number 16 | 2009

www.rsc.org/materials Volume 19 | Number 16 | 28 April 2009 | Pages 2261-2440
Published on 27 February 2009 on http://pubs.rsc.org | doi:10.1039/B816209C

Journal of Materials Chemistry

Image reproduced by permission of Guangshe Li
Downloaded on 13/05/2013 18:24:20.

Showcasing research from the Guangshe Li group, State As featured in:

Key Laboratory of Structural Chemistry, Fujian Institute
of Research on the Structure of Matter (FJIRSM), Chinese
Academy of Sciences, Fuzhou, P. R. China.
www.rsc.org/materials Volume 19 | Number 16 | 28 April 2009 | Pages 2261-2440

Title: Generation of tunable wavelength lights in core-shell CaWO4

microspheres via co-doping with Na+ and Ln3+ (Ln=Tb, Sm, Dy, Eu)

Under a single-wavelength excitation, tunable multicolour lights were

achieved in the visible region for self-assembled CaWO4 microspheres ISSN 0959-9428

PAPER
Chulhee Kim et al.
Cyclodextrin-covered gold
FEATURE ARTICLE
Alberto Credi et al.

via co-doping rare earth Ln3+ and alkali metal Na+. These findings
nanoparticles for targeted delivery of Artificial molecular shuttles: from
an anti-cancer drug concepts to devices 0959-9428(2009)19:16;1-T

could simplify the white light generation and relevant technologies.

Author Artwork: Yiguo Su, Liping Li and
Guangshe Li, J. Mater. Chem., 2009, 19,
2316–2322

www.rsc.org/materials
Pages 2261-2440

ISSN 0959-9428

Registered Charity
Registered Charity Number
Number 207890
207890 PAPER
Chulhee Kim et al. FEATURE ARTICLE
Cyclodextrin-covered gold Alberto Credi et al.
nanoparticles for targeted delivery of Artificial molecular shuttles: from
an anti-cancer drug concepts to devices 0959-9428(2009)19:16;1-T
 PAPER www.rsc.org/materials | Journal of Materials Chemistry

Cyclodextrin-covered gold nanoparticles for targeted delivery

of an anti-cancer drug†
Chiyoung Park,‡a Hyewon Youn,‡b Hana Kim,a Taiho Noh,a Yeon Hee Kook,a Eun Tax Oh,a Heon Joo Park*b
and Chulhee Kim*a
Received 16th September 2008, Accepted 12th January 2009
First published as an Advance Article on the web 27th February 2009
DOI: 10.1039/b816209c

We report on the therapeutic ability of a novel cyclodextrin-covered gold nanoparticle (AuNP) carrier
for noncovalent encapsulation of an anti-cancer drug. The surface of the AuNPs was functionalized
Published on 27 February 2009 on http://pubs.rsc.org | doi:10.1039/B816209C

with cyclodextrin as a drug pocket, anti-epidermal growth factor receptor (anti-EGFR) antibody as
a targeting moiety, and poly(ethyleneglycol) (PEG) as an anti-fouling shell. b-Lapachone, an anti-
cancer drug, was efficiently encapsulated into the hydrophobic cavity of cyclodextrin on the surface of
the AuNP carriers (AuNP-1). The glutathione-mediated release of b-lapachone from the surface of
AuNP-1 was demonstrated by an experiment with MCF-7 (low glutathione concentration) and A549
cells (high glutathione concentration). We also show that the introduction of an anti-EGFR antibody
onto the AuNP carriers (AuNP-2) increased the intracellular uptake of AuNP carriers as compared
with AuNP-1, which does not contain a targeting ligand. In the in vitro cytotoxicity study, AuNP-2 with
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b-lapachone exhibited a higher apoptosis effect than that caused by AuNP-1 with b-lapachone. This
work suggests that AuNPs covered with cyclodextrin and tumor-targeting ligands may find useful
applications for the development of nanoparticles with therapeutic and diagnostic modalities.

1. Introduction drugs to anchor them to the surface, inorganic nanoparticles

There has been considerable interest in nanomaterials for
medical applications, and various systems have been developed
for delivery vectors and diagnostic markers.1–4 Recently, the
combination of organic building blocks with anti-cancer drugs
and inorganic nanocrystals has been considered as a novel
approach in cancer therapy and diagnosis.4a–c Following the first
report of the unique ability of various inorganic nanomaterials
for diagnosis, the potential capability of gold nanoparticles
(AuNPs) for computed tomography imaging was recently
revealed.4d–f Furthermore, gold nanoparticles (AuNPs) have
recently been a focus of increased interest for their therapeutic
potential as drug-delivery carriers due to their attractive char-
acteristics such as size, robust stability, and biocompatibility.5
Recent work from several laboratories have shown that
AuNPs functionalized with drug molecules have a substantially
expanded performance as efficient therapeutic vehicles.6–10 In
these systems, drug molecules can be released from the surface of
AuNP by desorption through ligand-exchange processes or the
protonation of thiol groups at low pH.6–10 However, since most
methods for the integration of drug molecules with inorganic
nanoparticles reported to date require chemical modification of
have a limitation as therapeutic vehicles. Therefore, the use of
inorganic nanoparticles such as AuNPs as therapeutic vehicles
requires the development of a reliable approach that enables
noncovalent encapsulation of drug molecules without sacrificing
the unique advantages of inorganic nanoparticles.
Here we report the therapeutic ability of a novel AuNP carrier
system composed of a poly(ethylene glycol) (PEG) shell,
per-6-thio-b-cyclodextrin (SH-CD) as a drug pocket, and an
anti-epidermal growth factor receptor antibody (anti-EGFR
antibody) as a targeting ligand. PEG derivatives are biocom-
patible polymers and can protect nanocarrier and payload from
enzymatic degradation.11 Cyclodextrins (CDs) have been used as
drug-delivery agents because of their ability to protect drugs
from physical, chemical, and enzymatic degradation and also
to solubilize hydrophobic drugs.12 Therefore, SH-CDs on the
surface of AuNPs can provide sites to accommodate hydro-
phobic molecules. b-Lapachone, which can be encapsulated into
the cavity of b-CD (association constant: Kc ¼ (1.23
0.01) 
103 M1),13 was adopted as a chemotherapeutic drug, because it
exhibits superior activity against various cancer cells in breast,
lung, and prostate tissue.14

2. Results and discussion

a
Department of Polymer Science and Engineering, Inha University,
Incheon, 402-751, Korea. E-mail: chk@inha.ac.kr; Fax: +82-32-865- 2.1 Synthesis and characterization of AuNP carriers
5178; Tel: +82-32-860-7487
b
Department of Microbiology, Center for Advanced Medical Education by For the preparation of AuNP carriers functionalised with CD
BK21 Project, College of Medicine, Inha University, Incheon, 400-712, and PEG (AuNP-1), an aqueous solution of AuNPs (average
Korea. E-mail: park001@inha.ac.kr diameter 27 nm) stabilized with citrate (AuNP-0) was stirred
† Electronic supplementary information (ESI) available: Synthesis of
AuNPs, FT-IR spectra, TGA, dot blot analysis, DLS, fluorescence for 12 h with SH-CD and a-methoxy-u-mercapto-poly(ethylene
analysis and ESI-MS results. See DOI: 10.1039/b816209c glycol) (mPEG-SH, MW 2000 Da) (Scheme 1).15,16† AuNP-1.5,
‡ These authors contributed equally to this work. which contains succinimidyl-functionalized PEG and CD, was

2310 | J. Mater. Chem., 2009, 19, 2310–2315 This journal is ª The Royal Society of Chemistry 2009

Published on 27 February 2009 on http://pubs.rsc.org | doi:10.1039/B816209C
Scheme 1 Schematic illustration of the functionalization of AuNP
carriers with b-lapachone, using: i) SH-CD and mPEG-SH for AuNP-1
(RhoCD and mPEG-SH for RhoCD-AuNP-1); ii) SH-CD, mPEG-SH,
and NHS-PEG-SH for AuNP-1.5 (RhoCD, mPEG-SH, NHS-PEG-SH
for RhoCD-AuNP-1.5) and iii) anti-EGFR.
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prepared by the reaction of AuNP-0 with SH-CD, mPEG-SH,
and a-succinimidyl propionate-u-lipoic acid-poly(ethylene
glycol) (NHS-PEG-SH).† Both AuNP-1 and AuNP-1.5 were
purified by ultracentrifugation and washing with water.
The FT-IR spectra of AuNP-1 and AuNP-1.5 showed
absorption bands at 3430 cm1 (OH stretching H-bonded),
2920–2855 cm1 (CH2 stretching), 1632 cm1 (OH bending),
and 1041 cm1 (COC stretching), indicating that the surface of
AuNP-0 was functionalized with SH-CDs and PEG ligands17
(Fig. S1†). The thermogravimetric analysis (TGA) showed that
the organic content in AuNP-1 and AuNP-1.5 was 4.47 wt%
and 4.49 wt%, respectively (Fig. S2). Based on the results from
the TGA and 1H NMR studies, it was estimated that one
AuNP-1 carrier was covered with 800 SH-CDs and 2200
mPEG-SHs.
In the case of AuNP-1.5, the approximate number of SH-CDs,
mPEG-SH, and NHS-PEG-SH per particle was 800, 1800, and
180, respectively.†
For the introduction of a ligand able to target cancer cells that
overexpress EGFR (such as colon, lung, prostate, and breast
cancer cells), AuNP-1.5 were incubated with the anti-EGFR
solution (HEPES pH 7.4) for 12 h, and then the mixture (con-
taining AuNP-2) was purified by ultracentrifugation and washing
with water. The conjugation of anti-EGFR with the AuNP-1.5
was detected using the Dot blot method (Fig. S3†). The FT-IR
spectrum of AuNP-2 also confirmed the presence of anti-EGFR,
which exhibited absorption bands in the amide I (1651 cm1) and
amide II (1552 cm1) regions (Fig. S1). The amount of anti-
EGFR conjugated to the AuNP carriers, quantified using
a Micro BCA assay, was 15 mg mL1 of AuNP-2 (2.72 anti-
EGFRs per one AuNP-2). The aqueous solution of AuNP-
0 exhibited a surface plasmon resonance at 527 nm (3527nm ¼ 2.4
 109 M1 cm1). The absorption maxima of AuNP-1 and AuNP-
1.5 were shifted to 541 nm. The absorption maximum of the
AuNP-2 solution was further red-shifted to 544 nm (Fig. 1a).
AuNP-1.5 and AuNP-2 exhibited excellent colloidal stability in
This journal is ª The Royal Society of Chemistry 2009
water and various pH conditions (Fig. S4). Au carriers loaded
with b-lapachone (AuNP-1/lap and AuNP-2/lap) respectively
were prepared as shown in Scheme 1. To obtain Au nano-
carriers with b-lapachone, an excess amount of b-lapachone to
SH-CDs on Au nanocarriers was added to Au nanocarrier
solutions and sonicated for 3 min. AuNP-1/lap and AuNP-2/lap
were purified with filtration and ultracentrifugation respectively.
The relative absorbances of AuNP-1/lap and AuNP-2/lap over
24 h were constant, which indicated that no precipitation or
flocculation of the nanocarriers occurred in aqueous solution
(Fig. 1b). The average hydrodynamic diameters of AuNP-0,
AuNP-1, AuNP-2, AuNP-1/lap, and AuNP-2/lap measured by
dynamic light scattering (DLS) were 28.9 nm, 40.4 nm, 46.7 nm,
42.6 nm, and 47.0 nm, respectively. The increase of hydrody-
namic diameters in AuNP-1 and AuNP-2 revealed the formation
of a monolayer of PEG ligands and SH-CDs on the surface of
AuNP-0. The CONTIN analysis of the autocorrelation func-
tions for AuNP-0, AuNP-1/lap and AuNP-2/lap showed a mon-
omodal distribution, which indicates that no aggregation
occurred between Au nanocarriers after functionalization or
encapsulation of b-lapachone (Fig. 1c). The slope of the angular
dependence of the apparent diffusion coefficient (Dapp) was
zero, and the size of the aggregates was constant for the
investigated range of angle, which confirmed the spherical shape
of the carriers (Fig. 1d and S5).18 The transmission electron
microscopy (TEM) analysis revealed that the average diameters
of the Au core in AuNP-0, AuNP-1, AuNP-1.5, AuNP-2, AuNP-
1/lap, and AuNP-2/lap were 27.2
3 nm, 27.1
3 nm, 27.1
3
nm, 27.4
2 nm, 27.0
3 nm, and 27.6
3 nm, respectively
(Fig. 2 and S6). These results confirmed that there is no
significant transformation or deformation of AuNPs after the
surface functionalization.
The potential usefulness of AuNPs as a therapeutic carrier of
anti-cancer drugs was investigated as follows. First, the release
characteristics of functional molecules from the AuNP surface in
Fig. 1 (a) Absorption spectra of AuNP carriers. (b) Time dependence of
relative absorbance at 541 nm for AuNP-1/lap and AuNP-2/lap. (c) DLS
size distribution of AuNPs at a scattering angle of 90 . (d) Angular
dependence of apparent diffusion coefficient (Dapp) of AuNP-2/lap
(T ¼ 25  C).
J. Mater. Chem., 2009, 19, 2310–2315 | 2311
Published on 27 February 2009 on http://pubs.rsc.org | doi:10.1039/B816209C

Fig. 2 TEM images of (a) AuNP-0, (b) AuNP-1, (c) AuNP-1.5, (d)
AuNP-2, (e) AuNP-1/lap, and (f) AuNP-2/lap.
the intracellular environment were investigated. The release of
SH-CD from Au surfaces can be induced by ligand exchange
with glutathione in the intracellular matrix of cancer cells.7,8 To
trace the release of SH-CD from AuNP, rhodamine B was
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Fig. 3 The effect of anti-EGFR antibody and glutathione on the cellular
uptake and intracellular release in different cell lines. Time courses of
RhoCD fluorescence intensity from CLSM images in MCF-7 cell (low
glutathione) and A549 cell (high glutathione) incubated with RhoCD-
AuNP-1 or RhoCD-AuNP-2.
intracellular concentration of glutathione increased the RhoCD

release from the internalized RhoCD-AuNP-2.

conjugated with SH-CD (RhoCD)† and then introduced on the
surface of AuNP-1 to prepare RhoCD-AuNP-1. The fluorescence
of rhodamine B is quenched on the surface of Au nanoparticles 2.3 Intracellular uptake of AuNP
(Fig. S7). However, the fluorescence of rhodamine B can be
Fig. 4a shows the CLSM images of A549 cells incubated with
detected when RhoCD is removed from the Au surface. There-
RhoCD-AuNP-1 or RhoCD-AuNP-2 for 1 h. The stronger
fore, we can monitor the release behavior of SH-CD from the Au
RhoCD fluorescence in the cells incubated with RhoCD-AuNP-2,
surface during the intracellular migration of AuNP carriers.†
as compared with the cells incubated with RhoCD-AuNP-1,
demonstrated that anti-EGFR antibody effectively augmented
the internalization of AuNPs. The transmission electron
2.2 Glutathione-mediated release charateristics microscopy study (Fig. 4b) also demonstrated that the intracel-
The fluorescence intensity of the RhoCD moiety increased when lular uptake of AuNPs was greater in the A549 cells incubated
RhoCD-AuNP-1 was incubated in HEPES buffer at pH 7.4 with AuNP-2 compared with the cells incubated with AuNP-1. It
containing 10 mM glutathione, compared with RhoCD-AuNP-2 appeared that the increase in the internalization of AuNP-2
maintained in the buffer without glutathione. This result indi- mediated by anti-EGFR antibody increased the intracellular
cates that the release of RhoCD from the surface of RhoCD- uptake of AuNPs.
AuNP-2 was promoted by the presence of glutathione (Fig. S7†).
The importance of the role of glutathione in the intracellular
release of RhoCD from RhoCD-AuNP1 was further demon-
strated in the following experiment. We determined the intra-
cellular release of RhoCD from RhoCD-AuNP-1 in the MCF-7
cells containing a low concentration of glutathione and A549
cells containing a high concentration of glutathione (Fig. S8).
Based on the changes in the fluorescence intensity of the RhoCD
moiety from the confocal laser scanning microscopy (CLSM)
image, it could be concluded that RhoCD release from RhoCD-
AuNP-1 was faster in the A549 cells compared to the MCF-7
cells (Fig. 3). We then investigated how the presence of anti-
EGFR antibody on AuNP surface influences the intracellular
RhoCD content using RhoCD-AuNP-2. Note that the EGFR
expression on the surface of A549 cells has been reported to be
high.19 The results shown in Fig. 3 clearly indicate that the
concentration of RhoCD released from RhoCD-AuNP-2 was
greater, and increased more rapidly, in A549 cells than in MCF-7 Fig. 4 (a) Enhanced intracellular release: CLSM images of A549 cells
cells. Taken together, it is concluded that, in A549 cells, the incubated with Rho-AuNP-1 or Rho-AuNP-2 for 1 h. (b) Enhanced
relatively high level of EGFR expression on the cell surface intracellular uptake of AuNP carriers: TEM images of A549 cells incu-
augmented the internalization of RhoCD-AuNP-2, and the high bated with AuNP-1 or AuNP-2 for 4 h (scale bars: 10 mm).

2312 | J. Mater. Chem., 2009, 19, 2310–2315 This journal is ª The Royal Society of Chemistry 2009

Published on 27 February 2009 on http://pubs.rsc.org | doi:10.1039/B816209C
Fig. 5 (a) The effect of glutathione concentration on the release of
b-lapachone from AuNP-1/lap in HEPES buffer solution (pH 7.4). (b)
The percent apoptosis of A549 cells as determined by flow cytometry
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after incubation with free b-lapachone, AuNP-1, AuNP-1/lap, AuNP-2,
or AuNP-2/lap. Means of 5 experiments with duplicated cultures
S.E.
are shown.
2.4 The release of b-lapachone from AuNP carriers
The release of b-lapachone from AuNP-1/lap in the HEPES
buffer solution containing either 10 mM or no glutathione is
shown in Fig. 5a. The percent release of b-lapachone from
AuNP-1/lap was significantly greater in the presence of gluta-
thione. The mass analysis showed that both SH-CD and
b-lapachone were present in the buffer solution containing
10 mM glutathione, but not in the buffer solution without
glutathione (Fig. S9). It was evident that glutathione induced the
release of SH-CD from RhoCD-AuNP-1, followed by release of
b-lapachone from the cavity of CD. These results indicated that
the intracellular glutathione concentration significantly influ-
ences the release of b-lapachone from AuNP/lap in the cells.
2.5 In vitro cytotoxicity study of AuNP carriers with
b-lapachone
The effectiveness of 15 mM b-lapachone, AuNP-1/lap, and
AuNP-2/lap at inducing apoptosis in A549 cells was investigated
using flow cytometry (Fig. 5b). The percent of apoptosis caused
by AuNP-2/lap was slightly greater than that caused by free
b-lapachone after 4 h or 8 h treatment. Importantly, the
apoptosis caused by AuNP-2/lap was slightly greater than that
caused by AuNP-1/lap, indicating that the presence of anti-
EGFR antibody on the surface of AuNP increased the cellular
uptake of AuNP/lap (Fig. 4).
In the above in vitro cytotoxicity study, cells were exposed to
15 mM b-lapachone in the form of free b-lapachone, AuNP-1/lap,
and AuNP-2/lap for identical lengths of time. It has recently been
reported that AuNPs are cleared from blood circulation with
a half-life of 2–6 h after intravenous injection.20 Therefore, it may
be expected that the prolonged circulation of AuNP/lap would
This journal is ª The Royal Society of Chemistry 2009
result in the exposure of tumor cells to a higher concentration of
b-lapachone for a longer period, as compared with the situation
when free b-lapachone is administered. Furthermore, the pres-
ence of anti-EGFR would cause preferential accumulation of
AuNPs in tumor cells relative to normal cells.
3. Conclusions
In summary, we have developed a new type of Au nanocarrier
which is covered with SH-CDs, PEG, and a targeting antibody,
and evaluated its capability as a carrier vehicle for a hydrophobic
anti-cancer drug. It was found that AuNPs are a potentially
useful carrier of the anti-cancer agent b-lapachone, which can be
loaded in the cavity of CD on the surface of AuNPs. Since
a variety of hydrophobic drugs can be easily encapsulated into
the hydrophobic pocket of CDs, the multifunctional AuNP
carriers described here can serve as a versatile nanoplatform for
the delivery of other anti-cancer drugs as well. The use of AuNPs
as a drug carrier has the potential to prolong the blood circula-
tion time of the drug, thereby increasing the accumulation of the
drug in target tumors. Our results also indicated, as illustrated in
Fig. 6, that attaching anti-EGFR to AuNPs increased the cellular
uptake of drug-carrying AuNPs. The release of a cytotoxic
drug from the surface of AuNPs is greatly influenced by the
glutathione concentration in the cells. It was concluded that
AuNPs covered with SH-CD and a tumor-targeting ligand are
potentially useful carriers of hydrophobic anti-cancer drugs.
Furthermore, this approach may open up novel possibilities in
the development of the next generation of diagnostic and ther-
apeutic modalities based on inorganic nanoparticles.
4. Experimental
4.1 Materials and equipment
HAuCl4, trisodium citrate, b-cyclodextrin, iodine, NaOH,
N-hydroxysuccinimide, 1,3-diisopropylcarbodiimide, 4-(dime-
thylamino)pyridine, lipoic acid, mouse monoclonal anti-
epidermal growth factor receptor (anti-EGFR), and KHSO4
from Aldrich were used as received. Methoxy-poly(ethylene
glycol sulfhydryl) (mPEG-SH, MW 2000 Da) from SunBio and
a-carboxy u-hydroxy terminated poly(ethylene glycol) from
Polymer Source were used as received. All solvents were purified
by a literature procedure.21 1H and 13C NMR spectra were
Fig. 6 Schematic illustration for the enhanced cellular uptake of AuNPs
by anti-EGFR antibody and glutathione-mediated intracellular release of
AuNP-2 carriers.
J. Mater. Chem., 2009, 19, 2310–2315 | 2313
 recorded on a Varian UNITY INOVA 400 at 400 and 100 MHz
respectively. FT-IR spectra were obtained using VERTEX 80V
vacuum FT-IR spectrometer. UV–vis absorption spectra were
obtained using a Hewlett-Packard 8453A spectrophotometer.
All the fluorescence measurements were performed using
a Shimadzu RF-5301PC spectrofluorometer.
4.2 Cell lines and culture conditions
A549 human lung cancer cells and MCF human breast cancer
cells were used. The cells were cultured in 25 cm2 plastic tissue
culture flasks with Dulbecco’s modified Eagle’s medium
(DMEM, Hyclone Laboratories Inc., Logan, Utah) supple-
mented with 10% fetal bovine serum (FBS, Hyclone Laborato-
Published on 27 February 2009 on http://pubs.rsc.org | doi:10.1039/B816209C
ries Inc.) and 1% penicillin/streptomycin (Hyclone Laboratories
Inc.) in a humidified 5% CO2/95% air incubator at 37  C. The
cells in exponential growth phase were treated with b-lapachone,
AuNP-1, Au-NP-1/lap, AuNP-2 or Au-NP-2/lap.
4.3 Flow cytometric analysis
Control and drug-treated A549 cells were harvested using
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trypsin. The cells were then fixed with 70% ethanol and main-
tained at 20  C overnight, washed with PBS, and resuspended
in 1 mL PBS containing 30 units of DNase free RNase. After
incubation with propidium iodide (PI; Sigma-Aldrich, Inc., 5 mg
in 100 mL) in the dark at 37  C for 30 min, the cells were subjected
to apoptosis study with a FACSCalibur
flow cytometer
(Becton Dickinson Bioscience, San Jose, CA). A total of 10 000
cells were counted for each sample.
4.4 Confocal laser scanning microscopy
A549 cells were grown in eight-well chamber slides (or 24-well
coverslips) and treated with 15 mM free b-lapachone, AuNP-1,
Au-NP-1/lap, AuNP-2 or Au-NP-2/lap at 37  C for 0–24 h. Cells
were washed with PBS, fixed using 4% (v/v) paraformaldehyde
(Sigma–Aldrich) at 4  C for 30 min, and permeabilized with
0.25% (w/v) Triton X-100 (Sigma–Aldrich) at room temperature
for 2 min. After several washing with PBS, the nuclei were
stained with DAPI solution containing ProLong Gold antifade
reagent (Invitrogen, Eugene, OR). The glass slides were covered
with cover glasses, and the cells were examined with a fluores-
cence microscope.
4.5 Live cell imaging
For live cell time-lapse imaging, cells were plated on Lab-Tek II
coverglass bottom dishes and analyses were performed with
a Nikon TE200E inverted device 8 h after the addition of AuNPs
into the cultures. Single-plane confocal picture sequences were
taken every 15 min for confocal stacks and used for simultaneous
two-color-wide field imaging.
4.6 Intracellular glutathione concentration
The GSH concentration was measured using a glutathione assay
kit from Sigma (St. Louis, MO) following the manufacturer’s
instructions. Briefly, cells were seeded in a six-well plate and
either left untreated or treated with drugs before assaying for
2314 | J. Mater. Chem., 2009, 19, 2310–2315
GSH. Cell lysate was treated with 5% 5-sulfosalicylic acid and
centrifuged to remove protein precipitate. The supernatant was
then treated with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB).
GSH reduced DTNB to generate TNB, and the total TNB
formed was measured by absorption at 412 nm in a Beckman
DU640 spectrophotometer (Beckman Coulter, Fullerton, CA).
Acknowledgements
This work was supported by grants from the National R&D
Program for Cancer Control, Ministry of Health & Welfare,
Republic of Korea (0620340-1), the National Nuclear Tech-
nology Program (2007) from KOSEF, and the Korea Health
21 R&D Project (A062254).
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