Biomaterials 32 (2011) 598e609

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Water-dispersible multifunctional hybrid nanogels for combined curcumin

and photothermal therapy
Weitai Wu a, Jing Shen a, Probal Banerjee a, b, Shuiqin Zhou a, *
a
b

Department of Chemistry, College of Staten Island, and The Graduate Center, The City University of New York, Staten Island, NY 10314, USA
CSI/IBR Center for Developmental Neuroscience, College of Staten Island, and The Graduate Center, The City University of New York, Staten Island, NY 10314, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 11 August 2010
Accepted 31 August 2010
Available online 8 October 2010

We design a class of water-dispersible hybrid nanogels for intracellular delivery of hydrophobic curcumin. The core-shell structured hybrid nanogels were synthesized by coating the Ag/Au bimetallic
nanoparticles (NPs) with a hydrophobic polystyrene (PS) gel layer as inner shell, and a subsequent thin
hydrophilic nonlinear poly(ethylene glycol) (PEG)-based gel layer as outer shell. The uniqueness of these
hybrid nanogels lies in the integration of the functional building blocks for combined curcumin and
photothermal therapy to signicantly improve the therapeutic efcacy. The Ag/Au core NPs cannot only
emit strong uorescence for imaging and monitoring at the cellular level, but also exhibit strong
absorption in the near-infrared (NIR) region for photothermal conversion. While the inner PS gel layer is
introduced to provide strong hydrophobic interactions with curcumin for high drug loading yields, the
external nontoxic and thermo-responsive PEG analog gel layer is designed to trigger the release of the
pre-loaded curcumin either by variation of surrounding temperature or exogenous irradiation with NIR
light. Such designed multifunctional hybrid nanogels are well suited for in vivo studies and clinical trials,
thereby likely to bring this promising natural medicine of curcumin to the forefront of therapeutic agents
for cancers and other diseases.
2010 Elsevier Ltd. All rights reserved.

Keywords:
Curcumin
Poly(ethylene oxide)
Gold
Drug delivery
Chemotherapy
Photothermal therapy

1. Introduction
Traditional medicine is known to be fertile ground for the source
of modern medicines [1,2]. Curcumin, a yellow compound presented in spice turmeric (Curcuma longa) that belongs to the ginger
(Zingiberaceae) family, has antioxidant, antibacterial, antifungal,
antiviral, anti-inammatory, antiproliferative, and pro-apoptotic
effects [2e5]. Chemopreventive and growth inhibitory activities of
curcumin against many tumor cell lines have been reported [3,7],
including cervical, prostate, breast, bladder, and colon cancers
[8e12]. Our recent in vivo study reveals that curcumin can block
brain tumor formation [13]. How a single agent could exhibit all
these medical effects is an enigma under intensive scrutiny [5].
However, one of the most important limitations with curcumin is
the hydrophobic nature of the molecule and consequently its poor
bioavailability [2]. Even doses as high as 8 g of curcumin per day
administered to human subjects only result in an average peak
serum concentration of about 1.77 mM of curcumin [14]. The rapid
metabolism of curcumin in vivo is another possible reason for the
observation of very low plasma levels of the compound [15].
* Corresponding author. Tel.: 1 718 718 3897.
E-mail address: shuiqin.zhou@csi.cuny.edu (S. Zhou).
0142-9612/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2010.08.112

Numerous approaches have been undertaken to enhance the

bioavailability. Some of the possible ways to overcome these
problems involve the use of adjuvants such as piperine that interfere with glucuronidation [16,17]. The structural analogs of curcumin have also been developed by ingenious organic synthesis
[17e20]. Liposomes [21e23], micelles [24,25], phospholipid
complexes [26,27], and other nanoparticle (NP)-based technologies
[28e31] are other promising formulations, which appear to provide
increased circulation time, improved permeability, and resistance
to metabolic processes.
On the other hand, a key attribute of drug delivery systems is
their ability to regulate the drug release to improve therapeutic
efcacy and reduce side effects [32]. Two approaches can be used to
regulate the release of therapeutic payload from the carrier:
endogenous and exogenous activation [33e39]. The endogenous
activation strategies exploit specic physiochemical characteristics
of the pathological microenvironment, e.g. local temperature
increase (by 2e5
C) and/or pH decrease by 1e2.5 pH units (acidosis)
that are found in many pathological processes in various tissues and
organs such as cancer and cystic brosis [33,40,41], providing biologically controlled release. The exogenous activation provides
a complementary approach, employing orthogonal external stimuli
to affect drug release. Recently, monolayer functionalized gold (Au)

W. Wu et al. / Biomaterials 32 (2011) 598e609

NPs in specic size and shape have been explored to provide photoand photothermally regulated drug release by using near-infrared
(NIR) irradiation [35e39]. For example, Xia et al. [37] developed
a platform based on Au nanocages covered with a thermo-sensitive
copolymer monolayer of poly(N-isopropylacrylamide-co-acrylamide). The Au nanocage can convert the absorbed NIR light to heat
to induce the collapse of the copolymer chains, thus provide a photothermally controlled drug release. Yoo et al. [38,39] deposited an
Au nanolayer onto half of the surface of the drug-loaded poly(lactide-co-glycolic acid) (PLGA) NPs to provide additional photothermally controlled drug release. In comparison with the
chemotherapy (doxorubicin loaded in PLGA NPs) or photothermal
treatment alone, the combined chemoephotothermal treatment
demonstrated a synergistic effect with a higher therapeutic efcacy.
However, few studies have been reported to trigger the delivery of
curcumin for treatments of cancer or other diseases. Thomass group
[29] reported an ultrasound-mediated release of curcumin from
microemulsions stabilized with phospholipids and a surfactant
Tween 80. Vemula et al.[30] employed enzymatic action of tumorassociated proteases to disassemble supramolecular hydrogels to
control the release of encapsulated acetaminophen and curcumin.
Despite of these demonstrated progresses, to regulate the release
and enhance the therapeutic efcacy of curcumin continues to be
highlighted as a major challenge.
In this text, we develop a class of core-shell structured hybrid
nanogels to integrate high curcumin loading yields, combinable
curcumin and photothermal therapy, and cellular imaging ability
into a single nano-object. As shown in Fig. 1, the hybrid nanogels are
composed of an Ag/Au bimetallic NP as core and a hydrophobicehydrophilic double-layer gel as shell. The small-sized Ag/
Au NP core (12  5 nm) is designed to provide both strong uorescence for cellular imaging and photothermal conversion ability.
The inner thick hydrophobic polystyrene (PS) chain networks are
designed to effectively load the chemotherapeutic curcumin
molecules via hydrophobic interactions. Poly(ethylene glycol)
(PEG) chains are known to be efcient steric protectors for various
particles, such as micelles, liposomes, nanoparticles, and nanocapsules in biological media [42]. The outer thin gel layer composed
of nonlinear PEG chain networks will not only increase the stability
of the hybrid nanogel dispersion in aqueous media, but also provide
a thermo-sensitive volume phase transition to trigger the drug

Fig. 1. Schematic illustration of curcumin-loaded Ag/Au@PS-PEG core-shell hybrid

nanogels with a small Ag/Au bimetallic NP (12  5 nm) core embedded in the PS-PEG
double-layer gel shell. The hydrophobic curcumin drug is encapsulated within the
inner PS gel layer, which is coated by an outer PEG-based gel layer to offer both
stability in aqueous media and temperature sensitivity. Such designed nanoparticlebased drug delivery approach has the potential to render the hydrophobic curcumin
dispersible in aqueous media, thus circumventing the pitfalls of poor solubility. Since
the Ag/Au bimetallic NP is NIR resonant, the curcumin release can be triggered by the
temperature-responsive swellingedeswelling transition of the outer PEG gel layer
under the local temperature increase of the target pathological zones or the heat
generated upon NIR irradiation.

599

release either endogenously by local temperature increase in the

pathological zone or exogenously by heat converted from the NIR
irradiation. We expect that such rationally designed waterdispersible sub-100 nm hybrid nanogels as the curcumin delivery
carrier should provide long circulation time [42,43], good cell
penetration ability, and high therapeutic efcacy from the
combined chemoephotothermal treatment.
2. Materials and methods
2.1. Materials
All chemicals were purchased from Aldrich. 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA, 95%), oligo(ethylene glycol)methyl ether methacrylate
(Mn 300 g/mol, MEO5MA) and poly(ethylene glycol) dimethacrylate (PEGDMA,
Mn z 550 g/mol, crosslinker) were puried with neutral Al2O3. Curcumin was
puried with anhydrous ethanol. Anhydrous ethanol, AgNO3, sodium citrate
(Na3C6H5O7$H2O), NaBH4, HAuCl4$3H2O, L-ascorbic acid, 0.1 N HCl standard solution,
styrene (St), divinylbenzene (DVB), 2,20 -azobis(2-methylpropionamidine) dihydrochloride (AAPH), and sodium dodecyl sulfate (SDS) were used as received without
further purication. The water used in all experiments was of Millipore Milli-Q
grade.
2.2. Synthesis of Ag/Au@PS-PEG hybrid nanogels
2.2.1. Synthesis of Ag NPs
Citrate-stabilized Ag NPs were rst prepared by dropwise addition of fresh
NaBH4 solution (10.6 mM, 2.5 mL) to an aqueous solution of AgNO3 (0.1 mM, 200 mL)
in the presence of sodium citrate (0.1 mM) under vigorous stirring. The resultant
solution was stirred for 1 h and aged for 7 days at ambient condition before use. The
long aging time is necessary for completely degrading the reducing agent of NaBH4.
SDS-stabilized Ag NPs were obtained by adding 3.6  104 mol SDS into 200 mL
aqueous solution of citrate-stabilized Ag NPs. The mixture was stirred and aged for
10 h under N2 atmosphere.
2.2.2. Synthesis of Ag@PS-PEG hybrid nanogels
The hybrid nanogels with double-layer gel shell were prepared according to the
recipes listed in Table 1. In a 250 mL round-bottom ask equipped with a stirrer, a N2
gas inlet, and a condenser, 200 mL as-prepared aqueous solution of SDS-stabilized
Ag NPs was heated to 30
C, followed by addition of St and DVB under stirring. After
30 min, the temperature was raised to 70
C and the polymerization was initiated by
adding AAPH. The polymerization was allowed to proceed for 5 h. The solution was
centrifuged three times at 20,000 rpm (30 min, Thermo Electron Co. SORVALL RC-6
PLUS superspeed centrifuge) with the supernatant discarded and the precipitate
redispersed in 200 mL deionized water. The resultant Ag@PS nanogels were used as
nuclei for subsequent precipitation polymerization to add the copolymer of p
(MEO2MA-co-MEO5MA) gel shell. The outer shell precursors of MEO2MA and
MEO5MA comonomer mixture in 1:2 M ratio, PEGDMA crosslinker, and SDS
(3.6  104 mol) were dissolved into the 200 mL puried Ag@PS nanogel dispersion.
The mixture was heated to 70
C under a N2 purge. After 1 h, AAPH initiator was
added to start the polymerization. The synthesis was allowed to proceed to total 8 h.
The resulted Ag@PS-PEG hybrid nanogels were puried with centrifugation/redispersion in water for three cycles, followed by 3 days of dialysis (Spectra/Por
molecularporous membrane tubing, cutoff 12,000e14,000) against very frequently
changed water at room temperature (e 22
C).
2.2.3. Synthesis of Ag/Au@PS-PEG hybrid nanogels
Au/Ag bimetallic NPs inside the nanogels were prepared using galvanic
replacement reaction [44,45]. 200 mL water suspension of the Ag@PS-PEG hybrid
nanogels was stirred in an ice water bath for 30 min, a solution of HAuCl4 (1.0 wt%,
50 mL) was then added dropwisely to the vial. The immediate color change revealed
the galvanic replacement reaction between Ag and Au(III). The solution was stirred
for another 20 min until the color was stable. After that, a solution of L-ascorbic acid
(100 mM, 400 mL) was added dropwisely to allow the seed-mediated growth of Au
nanoclusters. The reaction was continued for 15 min The nal hybrid nanogels with
Au/Ag bimetallic NP in the core, coded as Ag/Au@PS-PEG, were puried by centrifugation, decantation, and dialysis against water ate22
C, and then stored in a fridge
(e10
C) for further studies.
2.3. Incorporation of hybrid nanogels into mouse melanoma cells B16F10
Round glass coverslips were placed in wells of a 24-well plate and treated with
0.1% poly-L-lysine in 100 mM phosphate buffered saline (PBS) for 40 min. Following
the treatment, the solution was aspirated and the wells were washed with PBS 3
times each. Next, B16F10 cells (2  104 cell/well) were plated on the glass coverslips
at 80% conuence in DMEM containing 10% FBS and 1% penicillinestreptomycin.
After 24 h, 500 mL of different hybrid nanogels (3.0 mg/mL) in serum-free DMEM was

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W. Wu et al. / Biomaterials 32 (2011) 598e609

Table 1
Recipes used to prepared hybrid nanogels.
Hybrid Nanogels
e
DSG-1
e
DSG-2
a
b

St (mmol)
2.78
2.26
1.74
1.39

DVB (mmol)
0.22
0.18
0.14
0.11

AAPHa (mmol)
0.04
0.03
0.02
0.02

MEO2MA (mmol)
1.02
0.51
0.51
0.51

MEO5MA (mmol)
2.00
1.00
1.00
1.00

PEGDMA (mmol)
3.02
1.51
1.51
1.51






2

10
102
102
102

AAPHb (mmol)
0.04
0.03
0.02
0.02

Used for initiating the polymerization of St and DVB.

Used for initiating the polymerization of MEO2MA, MEO5MA and PEGDMA.

respectively added to the marked wells. In a control well, 500 mL of serum-free

DMEM was added. The plate was incubated at 37
C for 2 h. The medium was then
aspirated and fresh serum-free DMEM was added to each well. Finally, the coverslips
with cells were removed from the wells and mounted onto slides for confocal
microscopy study.
2.4. Drug loading and release
Curcumin was loaded to the hybrid nanogels by complexation method. 5 mL
hybrid nanogel dispersion, with the pH adjusted to 4.0 by using 0.1 N HCl, was
stirred in an ice water bath for 30 min. 2 mL fresh curcumin solution of 1 mg/mL in
anhydrous ethanol was then added dropwisely to the vial. The immediate slight
cloudy revealed the complexation of the curcumin molecules with the polymer
chains in hybrid nanogels. After stirring overnight, the suspension was centrifuged
at 5000 rpm for 30 min at 22
C. To remove free curcumin, the precipitate was
redispersed in 5 mL HCl solution of pH 4.0, and further puried by repeated
centrifugation and washing for at least six times. All the upper clear solutions were
collected, and the concentration of free curcumin was determined by UVevis
spectrometry at 435 nm. To conrm the loading amount, the emission intensity at
566 nm with the excitation wavelength of 420 nm was also recorded on the upper
clear solutions. The amount of loaded curcumin in the hybrid nanogels was calculated from the decrease in curcumin concentration, based on the linear calibration
curve with R2 > 0.99 measured using the curcumin solutions with known concentrations under the same condition. The loading content is expressed as the mass of
loaded drug per unit weight of dried hybrid nanogels.
The in vitro release test of curcumin from the hybrid nanogels was evaluated by
the dialysis method. Taking account of the solubility of free curcumin in acidic
solution, the curcumin-loaded hybrid nanogel dispersion was diluted to 0.15 mg/mL
for the release experiments. A dialysis bag lled with 1 mL diluted curcumin-loaded
hybrid nanogels (pH 4.0) was immersed in 50 mL 0.005 M buffer solutions of
pH 6.15 but at different temperatures. The release experiments at physiological
temperature of 37
C were also performed with 5 min NIR (1.5 W/cm2) irradiation at
a certain time interval. The released curcumin outside of the dialysis bag was
sampled at dened time period and assayed by UVevis spectrometry at 435 nm.
Cumulative release is expressed as the total percentage of drug released through the
dialysis membrane over time.
2.5. Cell viability evaluation of in vitro chemo, photothermal, and
chemoephotothermal treatments
The cytotoxicity test was carried out based on the method as described in our
previous paper [13]. Briey, B16F10 cells (6  103 cell/well) were cultured in DMEM
containing 10% FBS and 1% penicillinestreptomycin in a 96-well plate, and exposed
to free curcumin solution, ethanol, drug-free hybrid nanogels, and curcumin-loaded
hybrid nanogels. To cover the high concentrations, the nanogels were concentrated
and adjusted to an appropriate concentration in DMEM right before feeding into the
well. For photothermal or chemoephotothermal combined treatments, the cells
were irradiated with 1.5 W/cm2 NIR light for 5 min, whereas for chemotherapy
alone, the cells were not exposed to NIR light. The plate was incubated at 37
C for
24 h. The medium was then aspirated, and these wells were washed three times
using fresh serum-free DMEM. After that, 25 mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyltetrazolium bromide (MTT) solution (5 mg/mL in PBS) was added to the
wells. After incubation for 2 h, the solution was aspirated and 100 mL of the lysis
buffer (20% SDS, 50% dimethylformamide) was added to each well, the plate was
sealed and incubated overnight at 37
C with gentle mixing. Three portions of the
solution obtained from each well were transferred to three respective wells of a 96well plate. Cell viability was measured using a microplate reader at 570 nm. Positive
controls contained no drug or nanogels, and negative controls contained MTT.
Parallel wells (in triplicate) also contained only medium (no cells) and hybrid
nanogels with the same concentrations. For conrmation, the cells were also stained
with 10 mM Hoechst and cell viability was estimated with a Nikon uorescence
microscope tted with a Spot digital camera, as described in detail previously [46].
2.6. Characterization
The UVevis absorption spectra were obtained on a Thermo Electron Co. Helios

b UVevis Spectrometer. The PL spectra were respectively obtained on a JOBIN YVON

Co. FluoroMax-3 Spectrouorometer equipped with a Hamamatsu R928P photomultiplier tube, calibrated photodiode for excitation reference correction from 200
to 980 nm, and an integration time of 1 s. To conrm all emissions, the PL spectra
were also recorded on a VARIAN CARY Eclipse Fluorescence spectrophotometer
equipped with R928 photomultiplier tubes and self-optimized light lters. The pH
values were measured on a METTLER TOLEDO SevenEasy pH meter. The transmission electron microscopy (TEM) images were taken on a FEI TECNAI transmission
electron microscope at an accelerating voltage of 120 kV. Approximately 10 mL of the
diluted hybrid nanogel dispersion was air-dried on a carbon-coated copper grid for
the TEM measurements. The B16F10 cells incorporated with hybrid nanogels were
imaged using a confocal laser scanning microscopy (LEICA TCS SP2 AOBS) equipped with an HC PL APO CS 20  0.7 DRY len. A UV (405 nm) light was used as the
light source. Dynamic light scattering (DLS) was performed on a standard laser light
scattering spectrometer (BI-200SM) equipped with a BI-9000 AT digital time
correlator (Brookhaven Instruments, Inc.) to measure the hydrodynamic radius (Rh)
distributions. A HeeNe laser (35 mW, 633 nm) was used as the light source. All
hybrid nanogel dispersions were passed through Millipore Millex-HV lters with
a pore size of 0.80 mm to remove dust before the DLS measurements.

3. Results and discussion

3.1. Synthesis and structure of Ag/Au@PS-PEG hybrid nanogels
Our strategy to prepare the hybrid nanogels with hydrophobicehydrophilic double-layer polymer gel as shell and Ag/Au
bimetallic NP as core involves the rst synthesis of Ag NPs, followed
by immobilization of double-layer gel shell on the Ag NPs templates
and a moderate growth of Au on the surface of the encapsulated Ag
NPs. We have previously employed cetyltrimethylammonium
bromide capped Ag NPs (36  3 nm) to fabricate core-shell hybrid
nanogels with poly(N-isopropylacrylamide-co-acrylic acid) as gel
shells [47]. Herein, the much smaller Ag NPs (12  5 nm) capped
with surfactant SDS were used as nucleation centers for the freeradical precipitation polymerization of monomers to form a thick
PS gel shell. At concentrations of SDS approaching its critical
micelle concentration, the Ag/SDS interface is fully occupied by SDS
molecules, giving rise to a surface coverage of 3.3  106 mol/m2
(corresponding to an occupied molecular interfacial area of
w40e50 2) [48]. To avoid or minimize the formation of free
polymer nanogel spheres, we optimized the concentrations of Ag
NPs, SDS, and styrene monomers for the one-step encapsulation of
Ag NPs within the PS gel. These hydrophobic particles can serve as
nuclei for further polymerization to add the second layer of
nonlinear PEG-based gel, which consists of a copolymer of
MEO2MA and MEO5MA. The DLS results indicate that the growth of
the nonlinear PEG gel layer onto the Ag@PS nanogels increased the
average hydrodynamic size, but maintained the nearly monodispersed size distribution of hybrid nanogels (Supplementary
Fig. S1). The UVevis spectra also demonstrate a successful addition of the polymer gel shell onto the Ag NPs. After the coating of
PS-PEG double-layer gel shell, the absorption peak of the Ag NPs
red shifted from 396 to 422 nm (Fig. 2).
The encapsulated Ag NPs were further engineered to increase
the optical resonances of the hybrid nanogels in the NIR range for
successful photothermal conversion of high efcacy. The Ag/
Au@PS-PEG hybrid nanogels were thus prepared through the
titration of the template Ag@PS-PEG hybrid nanogels with the

AuCl
4 solution. The addition of AuCl4 induced an immediate

W. Wu et al. / Biomaterials 32 (2011) 598e609

601

Fig. 2. Typical UVeVis-NIR absorption spectra of Ag NPs, Ag@PS-PEG hybrid nanogels,

and Ag/Au@PS-PEG hybrid nanogels.

galvanic replacement reaction with Ag NPs, leading to the formation of Ag/Au bimetallic NPs in the interior of the hybrid nanogels.
The feeding ratio of Au(III) to Ag(0) is controlled very low
(Au:Ag z 1:16 mol/mol), so even a complete galvanic reaction
would not result in a complete replacement of the Ag atoms with
Au atoms. Briey, AuCl
4 oxidizes the sacricial Ag template to AgCl,
which is highly soluble at the reaction temperature, as estimated
from its solubility (1.92  103 g/L at 25
C). The standard reduction
potential of the AuCl
4 /Au pair (0.99 V) is higher than that of the
AgCl/Ag pair (0.22 V). The electrons generated in the oxidation
process migrate to the surface of the Ag NPs, where they reduce the
AuCl
4 to Au atoms. Because Au and Ag solids share the same facecentered cubic structure with closely matched lattice constants
(4.0786 and 4.0862 , respectively), the Au atoms are able to
epitaxially nucleate and grow on the surface of the Ag particles
[44,45]. Thus, the surface plasmon absorption change of the Ag/
Au@PS-PEG hybrid nanogels is expected due to the surface modication of the Ag NPs in the Ag@PS-PEG hybrid nanogels (Fig. 2),
whereas the shape and size of the metal core NPs did not show
signicant change as revealed by TEM image.
Fig. 3 shows the TEM images of the Ag/Au@PS-PEG hybrid
nanogels. While the markedly high electron density of noble metal
enables the direct visualization of the Ag/Au bimetallic NP core
within the polymer hydrogel, the clear boundary between the inner
and outer gel layer make the double-layer gel shell structure easily
distinguished. The clear double-layer shell structure is associated
with the fact that the hydrophobic PS-coated Ag NPs can hinder the
relatively hydrophilic nonlinear PEG chains of the outer shell from
interpenetrating into the inner shell area [49]. Importantly, the
thickness of both the inner and outer gel layer in the shell can be
well tuned by simply adjusting the feeding ratio of the monomers
to the nuclei NPs in the synthesis (Table 1), thus the overall size of
the hybrid nanogels can be controlled to be small. It should be
noted that the size of colloidal drug carriers is very important to
their fate in blood circulation since the recognition by the reticuloendothelial system is known to be the principal reason for the
removal of many colloidal drug carriers from the blood compartment. The sub-200 nm size is desirable for colloidal drug carrier
particles to extend their blood circulation time [42,43]. Herein, we
selected two Ag/Au@PS-PEG hybrid nanogels, coded as DSG-1
(Fig. 3B) and DSG-2 (Fig. 3D), respectively, with hydrodynamic
diameter well below 100 nm under the physiological condition to

Fig. 3. TEM images of Ag/Au@PS-PEG hybrid nanogels with different gel shell thickness, corresponding to the samples shown in Table 1 with the feeding of
St 2.78 mmol (A), 2.26 mmol (B), 1.74 mmol (C), and 1.39 mmol (D).

serve as the drug carrier for delivery of curcumin. The size of DSG-1
and DSG-2 hybrid nanogels is small enough to reasonably assume
that they have high extravasation efciency regardless of the cell
type and conditions.
3.2. Temperature-induced volume phase transition
Fig. 4 shows the temperature-dependent Rh (A) and the corresponding size distribution at 6
C and 50
C (B), respectively, of the
Ag/Au@PS-PEG hybrid nanogels dispersed in PBS of pH 6.15
measured at a scattering angle of q 60
. It is clear that the increase
in temperature-induced shrinkage of the hybrid nanogels. These
hybrid nanogels are nearly monodisperse regardless of their
swelling/shrinking states. The hydrophobic PS chains will not
undergo conformational and chemical changes in water when
giving external stimuli of temperature change. The observed
temperature-induced volume phase transitions of the hybrid
nanogels should be contributed from the outer gel layer containing
short oligo(ethylene glycol) side chains. The formation of hydrogenbonds between the ether oxygens of the side PEG chains and the
hydrogens of water is believed to be one of the key factors responsible for the unusual water solubility of this type of nonlinear PEG
polymers. This conformationally favorable effect in water is counterbalanced by the hydrophobicity of the apolar backbone [49,50].
Fig. 4A indicates that the proper feeding ratio of 1:2 between the two
macromonomers of MEO2MA (n 2) and MEO5MA (n 5) has led to
a volume phase transition region across the physiologically important temperature range. Linear plots of Rh versus temperature were
obtained over 37e42
C, which are typical abnormal temperature
range found in many pathological zones [33,40,41]. The hybrid
nanogels still did not reach a fully collapsed state at the experimental temperature limit of 50
C, which means that the partially
swollen PEG-based outer gel layer is still hydrophilic at temperatures below 50
C. This hydrophilic surface gel layer provides
excellent stability for the Ag/Au@PS-PEG hybrid nanogels. No sediment was observed after setting for a few weeks even at 42
C. More
importantly, while the thick inner PS gel layer can provide hydrophobic region for the storage of hydrophobic curcumin drugs, the

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W. Wu et al. / Biomaterials 32 (2011) 598e609

Fig. 4. (A) Temperature dependence of the average Rh values of the hybrid nanogels DSG-1 and DSG-2 in PBS of pH 7.38 at a scattering angle q 60
. (B) Size distributions of the
hybrid nanogels at different temperatures.

thermo-sensitive outer PEG-based gel layer can be utilized to

regulate the transport of the drug molecules from the inner gel layer
to the surrounding medium by temperature stimuli.
3.3. Photoluminescent properties and tumor cell imaging
Fig. 5 shows that strong uorescence can be readily detected
from the Ag/Au@PS-PEG hybrid nanogels. The frequency difference
between the emission peaks is independent of the excitation
wavelength. A comparison of the PL spectrum of the Ag@PS-PEG
hybrid nanogels and the Ag/Au@PS-PEG hybrid nanogels indicates
that the PL emissions were associated with the silver (Fig. 5 and
Supplementary Fig. S2). Considering the very low feeding ratio of Au
(III) to Ag (0), the surface galvanic replacement of Ag NPs with Au

nanoclusters should not signicantly affect the PL properties of the

silver [44]. The uorescence of noble metal NPs originates from the
radiative recombination of sp-band electrons and d-band holes,
which could be enhanced by 4e6 orders of magnitude due to the
surface plasmons of nanocrystals or rough metal surfaces [51].
Scaiano et al. believed that the emissions of Ag NPs are due to small
Ag cluster, predominantly Ag2 supported by the readily detectable
NPs [52]. In our hybrid nanogels, the Ag/Au bimetallic NPs are coated
with a thick inner PS gel layer and a subsequent external thin PEGbased gel layer. Although the outer PEG gel layer demonstrated
a signicant temperature-responsive volume phase transition
(Fig. 4), the temperature-induced PL property change of the hybrid
nanogels is negligible (Fig. 5). We speculate that the temperature
insensitive hydrophobic PS gel layer directly capping on the Ag/Au

W. Wu et al. / Biomaterials 32 (2011) 598e609

603

Fig. 5. Typical PL proles of the hybrid nanogels DSG-1 (A) and DSG-2 (B) recorded at low (6
C) and high temperature (50
C). All measurements were made in PBS of pH 7.38.
Excitation wavelength 334 nm.

bimetallic core protected the surface electronic structure of Ag

crystals, thus caused a little change of the local optical electric eld
at the metal surface. The slight variation in the PL intensity is
possibly due to the change in the refractive index of the gel shell
when the outer PEG-based gel layer swells and shrinks [47].
After conrming the strong uorescence, mouse melanoma cells
B16F10 were incubated with the Ag/Au@PS-PEG hybrid nanogels to
evaluate the cellular imaging function. It is clear that the hybrid
nanogels successfully penetrated into the B16F10 cells (Fig. 6,
Supplementary Fig. S3 and S4). Irradiated by the laser of 405 nm,
the Ag/Au bimetallic NPs embedded in the PS-PEG nanogels
produced a bright uorescence, which retained nearly the same PL
intensity even after 20 min irradiation. No signicant autouorescence was observed under similar conditions. The hybrid
nanogels were located in the cytoplasm and appear not to exist in
the karyons. As the complexity of molecular interactions governing

endocytosis are revealed, the mechanisms of endocytosis should be

viewed in a broader context than simple vesicular trafcking [53]. It
has been reported that small-sized (<100 nm) PEG-phosphatidylethanolamine micelles can penetrate deeply into poorly permeable
tumors and consequently increase accumulation in tumors via the
enhanced permeability and retention effect [43]. The surface
structure-regulated cell membrane penetration by monolayerprotected Au NPs [54] and gel-coated Ag NPs [47] has also been
reported. For our Ag/Au@PS-PEG nanogels, the endocytosis process
might be associated with the small size and specic surface properties. Nevertheless, it is obvious that these hybrid nanogels serving
as uorescence-labeling agents do not give dark regions in the cells
and the areas where the hybrid nanogels did not permeate are
clear. These results reveal that the hybrid nanogels designed in this
work can act as intracellular drug delivery systems and thus track
or monitor at the cellular level for advance therapy.

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W. Wu et al. / Biomaterials 32 (2011) 598e609

Fig. 6. Scanning confocal images of B16F10 cells incubated with hybrid nanogels of (A) DSG-1 and (B) DSG-2, respectively. Excitation laser wavelength 405 nm.

3.4. Curcumin loading and release properties

After demonstrating the tumor cell internalization and imaging
ability of the Ag/Au@PS-PEG hybrid nanogels, we next quantied the
loading yield and release kinetics of curcumin from the hybrid
nanogels at different temperatures. Curcumin is poorly soluble in
water at acidic or neutral pH with the macroscopic undissolved akes
visible in the solution (Fig. 7A). At pH above neutral value, the
dissociation of the compound increases the solubility, but
the compound undergoes rapid hydrolytic degradation [2,3,55]. The
half-life time for the hydrolytic degradation of curcumin in aqueous
solution containing 10% organic solvent has previously been determined to be 4.2  103 h, 15 h and 3.5  102 h at pH z 6, 7 and 8,
respectively [55]. To prevent any signicant degradation, the curcumin was loaded into the hybrid nanogels at pH 4.0. The hydrophobic aromatic groups in PS gel layer can form strong hydrophobic
associations with the aromatic phenols in curcumin molecules to
form intermolecular complexes, resulting in a high drug loading
efciency. The aqueous dispersion of the curcumin-loaded hybrid
nanogels is a homogeneous dispersed formulation with its hue
derived from the natural color of curcumin (Fig. 7B). The thicker the
inner PS gel layer, the higher the loading capacity of the hybrid
nanogels. A curcumin loading capacity of 24.5 wt% and 11.2 wt% was
determined for DSG-1 and DSG-2, respectively. The hybrid nanogels
with the same thickness of PEG gel layer but a thinner PS gel layer
(shown in Fig. 3C) exhibited a lower curcumin loading capability of
16.4 wt% as compared to DSG-1. Calculating from the approximate
concentrations of the hybrid nanogels in aqueous dispersion, the
loading capabilities are equivalent to 982.8 and 974.8 mM curcumin in
water for DSG-1 and DSG-2, respectively. Clearly, the overall curcumin concentration loaded in the water dispersed hybrid nanogels is
much greater than the solubility of free curcumin in water at acidic or
physiological pH (20.1 mM) [3,56]. It should be noted that the
current loading procedure has not been optimized to reach
a maximum loading yield as the hybrid nanogels only occupied less
than 10% of the total suspension volume. It is possible to cover even

higher dose by concentrating the hybrid nanogels through centrifugation. Importantly, the highly curcumin-loaded hybrid nanogels still
maintain excellent stability in aqueous media. No sediment was
observed for the ve-time concentrated curcumin-loaded hybrid
nanogels after setting for more than a month, due to the high
hydrophilicity of the outer PEG-based gel layer.
Fig. 7C shows the release kinetics of curcumin from the DSG-1 and
DSG-2 hybrid nanogels. To avoid the evident degradation of curcumin from the long time exposure in water, the in vitro release test was
made in a PBS of pH 6.15. A blank release experiment of free curcumin solution (containing 5% ethanol) with an equivalent amount of
drug to that trapped in the hybrid nanogels DSG-1, carried out under
the same conditions at 41
C, indicates that the dialysis membrane
played negligible role in the release of curcumin. The much slower
release rate of curcumin from the hybrid nanogels than from the free
curcumin solution demonstrates a sustained release of the curcumin
from the hybrid nanogels. The release kinetics of curcumin from the
hybrid nanogels were also temperature dependent. The increase in
temperature could signicantly speed up the release of curcumin
from both the DSG-1 and DSG-2 hybrid nanogels. For example, only
12.8% curcumin was released from DSG-1 at 22
C after 72 h. When
the temperature of releasing medium was increased to 37
C, 39
C,
and 41
C, the amount of curcumin released from the DSG-1 reached
34.6%, 54.9%, and 78.6%, correspondingly, during the same time
period of 72 h. The observed temperature dependency of curcumin
release should be associated with the thermo-sensitive outer PEGbased gel layer. The increase in temperature induces a gradual
shrinking and thus a thinner thickness of the outer PEG-based gel
layer (Fig. 4A), which reduces the restricted diffusion path length of
the curcumin from the inner PS gel layer to the medium outside the
hybrid nanogel particles. This hypothesis is supported by the effect of
the thickness of PEG-based outer gel layer on the release kinetics of
curcumin from the hybrid nanogels. The hybrid nanogels coated with
the same thickness of PEG-based gel layer (Fig. 3C) as DSG-1
demonstrated nearly the same curcumin release rate as DSG-1
within the experimental errors (Supplementary Fig. S5). For the

W. Wu et al. / Biomaterials 32 (2011) 598e609

605

Fig. 7. (A) Free curcumin is poorly soluble in aqueous media with macroscopic akes oating in the bottle. (B) In contrast, the nanogel-encapsulation renders curcumin completely
dispersible in aqueous media. (C) Releasing proles of curcumin from the hybrid nanogels DSG-1 (solid symbols) and DSG-2 (open symbols) at different temperatures. In the blank
(>), 1 mL diluted solution of free curcumin (containing 5% ethanol) with an equivalent amount of drug to that trapped in DSG-1 was performed at 41
C. All experiments were
carried out in 50 mL PBS (0.005 M) of pH 6.15.

hybrid nanogels DSG-2 possessing a thinner PEG-based gel layer than

the DSG-1, a quicker release rate was determined at all the investigated temperatures in comparison with the DSG-1 sample. It should
be mentioned that the increase in temperature may increase the
solubility of curcumin. It has been reported the solubility of curcumin
could be enhanced by heating the solution (1 or 5 mg/mL in water) to
boiling temperature for 10 min [56], but 98.5% of the curcumin was
still insoluble after the heating process. In our hybrid nanogels, the
temperature-dependent solubility change of curcumin is unlikely to
play a notable role in the temperature-trigged release, since the
concentration of curcumin-loaded hybrid nanogels used in the
releasing experiments has been controlled to have a total curcumin
amount well below the reported solubility. In addition, the solubility
change of curcumin in a temperature range of 37e42
C could be very

small. Nevertheless, the temperature-regulated drug release could be

exploited for intelligent drug delivery applications through environmental stimuli [32]. The abnormal temperature rise in pathological zones (e.g. by 1e5
C) could facilitate active drug release from
the Ag/Au@PS-PEG hybrid nanogels.
3.5. Curcumin release upon NIR irradiation
In addition to the endogenous activation strategy to regulate the
curcumin release via the temperature rise in the pathological
microenvironment, a highly orthogonal external NIR irradiation
can provide a spatioetemporal control of payload release. Fig. 8A
shows the release kinetics of curcumin from the DSG-1 and DSG-2
hybrid nanogels immersed in a buffer solution of pH 6.15 at 37
C

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W. Wu et al. / Biomaterials 32 (2011) 598e609

and irradiated by 1.5 W/cm2 NIR light for 5 min at 0, 5, 16, and 40 h,
respectively. An initial exposure to the NIR light led to a burst
release, followed by a gradual variation. Such a signicant release
within a brief period is undoubtfully attributed to the intensive
local heat, produced by the efcient photothermal conversion of
the Ag/Au NP core via electroneelectron and electronephonon
relaxations on the time scale of picoseconds [39,57,58]. When the
NIR light was turned off, heating immediately ceased and the drop
in temperature brought the drug release back to its regular rates.
The NIR-accelerated curcumin release from the Ag/Au@PS-PEG
hybrid nanogels may involve both the temperature-induced
shrinking of the PEG-based gel layer, producing a decreased diffusion path length for curcumin, and the increased mobility of

curcumin molecules at the elevated temperatures. We can safely

ignore the NIR absorption of water (KAu 318 W/
mK >> Kwater 0.6 W/mK) [59] and the dielectric PS-PEG gel shells.
Hence, the NIR-induced temperature increase should be mainly
localized at the surface of the Ag/Au NP core. The heat will then
dissipate into the surrounding medium. A quantitative description
of the temperature increase around a sphere placed in an innite
medium under steady state conditions is given by [59e62]

DTD R3 A=3DK

(1)

where D is the distance from the particle center, R is the particle

radius, K is the thermal conductivity of the surrounding medium,
and A is the heating rate per unit volume and proportional to the

Fig. 8. (A) Releasing proles of curcumin from the DSG-1 (-) and DSG-2 (C) hybrid nanogels immersed in buffer solutions at a constant temperature of 37
C, irradiated with
1.5 W/cm2 NIR for 5 min at cumulative time of 0, 5, 16, and 40 h. (B) Plots of accelerated release versus NIR exposure time. The samples were initially irradiated with 1.5 W/cm2 NIR
and harvested 3 h after irradiation.

W. Wu et al. / Biomaterials 32 (2011) 598e609

607

power density of the radiation at the particle location, respectively.

For a metal particle of a given radius, equation (1) can be simplied
as

DTD CP=D

(2)

where C is a constant including all the physical parameters

mentioned above, and P is the power of the radiation. At the same
power P of irradiation, the temperature rise DT decreases inversely
as the distance from the center of metal particle increases, which
affects the diffusion behavior of the curcumin molecules at
different regions inside the hybrid nanogels. The inverse relationship between DT and D has been experimentally demonstrated by
using photothermal imaging. For Au NPs of 5 nm, the photothermal
conversion at the heating power upto 20 MW/cm2 gives a temperature rise of about 15 K at the surface of the particle, which
decreases to 3 K at a distance of 13 nm from the center [57]. Oddershedes group mapped out the entire temperature prole
surrounding individual irradiated Au NPs of 80e200 nm by using
molecular partitioning and exploiting the phase dependent
quantum yield of uorophores in lipid bilayers [60]. For a certain
region near the Ag/Au NP core with a xed distance D, the
temperature rise DT in principle should linearly proportional to the
power P of incident NIR light applied on the particle. Fig. 8B shows
a plot of accelerated release of curcumin against the duration of NIR
exposure to the samples initially irradiated at 1.5 W/cm2 and harvested 3 h after irradiation. The accelerated release is calculated by
subtracting the cumulative release in the absence of NIR from that
in the presence of NIR irradiation. In all cases, the NIR-induced
temperature rise DT is far less than 1
C in the bulky dispersions.
Samples irradiated with NIR light less than 1.0 min exhibited little
or no accelerated release of curcumin. At some threshold of irradiation time between 1.0 and 1.5 min, the release becomes significantly greater than the corresponding cases without NIR
irradiation. The accelerated release of curcumin increases linearly
with the exposure time when the irradiation time is above 2 min.
Such a tunable NIR-trigged acceleration in drug release should
remarkably enhance the ability of the Ag/Au@PS-PEG hybrid
nanogels for specic drug delivery to the tumor site. Depending on
the stage and type of cancer, this NIR-accelerated drug release can
provide high dosage at a shorten treatment time if necessary.
Alternatively, by keeping the low NIR power, the delivered dosage
to tumor site can be minimized to give basal dosage for daily care.
3.6. Cell viability evaluation of in vitro chemo, photothermal, and
chemoephotothermal treatments
Having demonstrated the curcumin delivery ability of the hybrid
nanogels, we examined the in vitro cellular cytotoxicity of the drugfree and curcumin-loaded hybrid nanogels (Fig. 9). The results
indicate that the drug-free hybrid nanogels have negligible cytotoxicity against B16F10 cells after 24 h incubation in concentrations
up to 301.2 mg/mL and 448.5 mg/mL for DSG-1 and DSG-2 (equivalent
to drug-loaded hybrid nanogels containing 200 mM curcumin),
respectively. In contrast, the cell viability decreased to about 22%
and 18% for the curcumin-loaded DSG-1 and DSG-2, respectively, at
the same concentrations. The cytotoxicity of the curcumin-loaded
Ag/Au@PS-PEG hybrid nanogels should be attributed to the cellular
uptake of these particles and the sustained release of curcumin from
these hybrid nanogels. These results indicate that the curcuminloaded hybrid nanogels can maintain the high anticancer activity of
curcumin. Previously, we have found that the solubilized curcumin
in PBS containing 3% dimethyl sulfoxide can result in a marked
decrease in the viability of cancer cells, offering a safe therapeutic
strategy for treating brain tumors [13]. The cell viability of free

Fig. 9. Comparison of cell viability following chemo, photothermal, combined chemoephotothermal treatments with hybrid nanogels DSG-1 (A) and DSG-2 (B). (C) The
results of B16F10 cells treated with free curcumin solutions are included as a control.

curcumin solution at concentrations of 50, 100, and 200 mM in PBS

containing ethanol (Fig. 9C) was 30, 26, and 5%, respectively.
Considering that less than 30% of the loaded curcumin was released
in 24 h at 37
C (Fig. 7), the slightly lower cytotoxicity of the curcumin-loaded hybrid nanogels than the free curcumin solutions
(equivalent to loaded curcumin) is understandable.
To investigate the efcacy of combined chemoephotothermal
treatment, the B16F10 cells were treated with drug-free and

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W. Wu et al. / Biomaterials 32 (2011) 598e609

curcumin-loaded hybrid nanogels for 24 h, respectively, but adding

5 min initial NIR irradiation. Fig. 9 indicates that the B16F10 cells
treated with the combined NIR irradiation and curcumin-loaded
hybrid nanogels exhibited more cellular damage than those treated
with either curcumin-loaded hybrid nanogels (chemotherapy) or NIR
irradiation (photothermal therapy) alone. Cells irradiated with NIR
only under the same conditions exhibited no signicant response.
Cells treated with free curcumin solution at equivalent concentration
as those loaded in the hybrid nanogels exhibited a slight decrease
(3%) in cell viability. Our results indicate that the cytotoxicity of
B16F10 cells in response to the treatment of combined NIR irradiation
with the curcumin-loaded Ag/Au@PS-PEG hybrid nanogels is about
4-fold higher than that treated with free curcumin solution at the
equivalent concentration of curcumin. Although the photobiological
effects have been reported in photokilling of bacteria [6], we cannot
conclude weather the curcumin would be photobiological active for
cancer treatment because very limited information exists in either
literature or the clinical use. Nevertheless, the NIR-accelerated release
property of the curcumin-loaded hybrid nanogels should provide
another important and maybe essential scenario for such a remarkable enhancement in the therapeutic efcacy. Fig. 10 shows
a comparison of the therapeutic efcacies from combined chemoephotothermal treatment (calculated by subtracting the cell viability
from 100%) with the additive therapeutic efcacies of chemo- and
photothermal treatments, which were estimated using the relation of
Tadditive 100  (fchemo  fphotothermal)  100, where f is the fraction of
surviving cells after each treatment [62]. The therapeutic efcacy of
combined chemoephotothermal therapy with the curcumin-loaded

hybrid nanogels as drug carriers was higher than the additive therapeutic efcacy of chemo- and photothermal therapy alone. When ttest is used to compare the efcacy of combined treatment with the
efcacies of chemo- and photothermal treatments and their additive
value, all p-values are lower than 0.05, indicating a signicant
difference. Therefore, the synergistic effect of chemoephotothermal
treatment is clearly demonstrated on the curcumin-loaded Ag/
Au@PS-PEG hybrid nanogels.
4. Conclusion
Multifunctional water-dispersible Ag/Au@PS-PEG hybrid nanogels could be successfully synthesized through the precipitation
polymerization coating of the Ag/Au NPs with a hydrophobicehydrophilic double-layer gel shell. The hybrid nanogels
cannot only overcome cellular barriers to enter the intracellular
region and light up the B16F10 cells, but also serve as highly efcient
drug carriers for thermo-/photothermal-regulated curcumin
delivery. The inner hydrophobic PS gel layer can provide high
loading capabilities for hydrophobic curcumin. The thermo-sensitive outer PEG-based gel layer can provide good biocompatibility,
excellent stability in aqueous media, and thermally regulated drug
release. The curcumin-loaded into the hybrid nanogels can be
released more rapidly by either the temperature increase of the local
microenvironments (endogenous activation) or the heat generated
by NIR irradiation (exogenous activation). While the drug-free
hybrid nanogels are nontoxic to cells, the curcumin-loaded hybrid
nanogels exhibit potent cytotoxicity against B16F10 cells by
combined chemoephotothermal treatment, providing higher
therapeutic efcacy in comparison with the chemo-and photothermal treatment alone or their additive efcacy. The enhanced
therapeutic efcacy of curcumin-loaded hybrid nanogels is likely to
bring this natural product to the forefront of therapeutic agents for
the treatment of human diseases. Small, stable, and nontoxic hybrid
nanogels, designed by integration of functional building blocks, will
be highly benecial to both diagnostic and therapeutic purposes.
Acknowledgement
We gratefully acknowledge the nancial support from the US
Agency for International Development under the USePakistan
Science and Technology Cooperative Program (PGA-P280422) and
the National Science Foundation (CHE 0316078). We thank Phyllis
Langone at the CSI/IBR Center for Developmental Neuroscience for
the help with cell culture.
Appendix
Figures with essential color discrimination. All gures in this
article, are difcult to interpret in black and white. The full color
images can be found in the online version, at doi:10.1016/j.
biomaterials.2010.08.0112.
Appendix. Supplementary information
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biomaterials.2010.08.0112.

Fig. 10. Therapeutic efcacies of hybrid nanogels DSG-1 (A) and DSG-2 (B) were
calculated by subtracting cell viability from 100%. Additive therapeutic efcacies of
chemo-and photothermal treatments were estimated using the relation of
Tadditive 100  (fchemo  fphotothermal)  100, where f is the fraction of surviving cells
after each treatment. When the efcacy of combined treatment is compared with the
efcacies of chemo- and photothermal treatments and their additive value using t-test,
all p-values are lower than 0.05.

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