Dual functional AuNRs@MnMEIOs nanoclusters for magnetic
resonance imaging and photothermal therapy
Yao-Chen Chuang
a
, Chia-Jung Lin
a
,1
, Shih-Feng Lo
a
, Jei-Lin Wang
a
, Shey-Cherng Tzou
a
,
Shyng-Shiou Yuan
b
,**
, Yun-Ming Wang
a
,c
,*
aDepartment of Biological Science and Technology, Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, 75 Bo-Ai Street,
Hsinchu 30068, Taiwan
bTranslational Research Center, Cancer Center, and Department of Obstetrics and Gynecology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan cDepartment of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
a r t i c l e i n f o
Article history:
Received 24 January 2014 Accepted 12 February 2014 Available online 7 March 2014 Keywords:
MR imaging Gold nanorods
Manganese-doped iron oxide Theranosis
Photothermal
a b s t r a c t
A novel dual functional theranosis platform is developed based on manganese magnetism-engineered iron oxide (MnMEIO) and gold nanorods (AuNRs) to combine magnetic resonance (MR) imaging and photothermal therapy in one nanocluster. The platform showed improved T2-weighted MR imaging and
exhibited a near-infrared (NIR) induced temperature elevation due to the unique characteristics of AuNRs@MnMEIOs nanoclusters. The obtained dual functional spherical-shaped nanoclusters showed low cytotoxicity, and high cellular uptake efficiency. The AuNRs@MnMEIOs nanoclusters also demonstrated a 1.9 and 2.2 folds r2relaxivity value higher than those of monodispersed MnMEIO and Resovist. In
addition, in vivo MR imaging study found that the contrast enhancements weree 70.4 4.3% versus e 7.5 3.0% in Her-2/neu overexpression tumors as compared to the control tumors. More importantly, NIR laser irradiation to the tumor site resulted in outstanding photothermal therapeutic efficacy and without damage to the surrounding tissue. In additional, the prepared dual functional AuNRs@MnMEIOs display high stability and furthermore disperse even in the presence of external magnet, showing that AuNRs@MnMEIOs nanoclusters can be manipulated by an external magnetic field. Therefore, such nanoclusters combined MR imaging and photothermal therapeutic functionality can be developed as a promising nanosystem for effective cancer diagnosis and therapy.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, nanostructured materials have received great
atten-tion in biomedical imaging, diagnosis, and therapy
[1,2]
. Each of
these methods possesses characteristic strengths and weaknesses,
but none of them are capable of independently providing complete
structural and functional information. The accuracy of tumor
detection can be greatly improved by integrating the different
in-dividual imaging modalities. Therefore, various types of hybrid
nanoparticles that have been used for multimodal imaging, based
on the incorporation of several contrast agents into one single
system, will enable the development of multifunctional
nano-medical platforms for multimodal imaging
[3,4]
. Moreover, these
hybrid platforms could simultaneous diagnose and treat patients
by combining therapeutic and diagnostic capabilities into one
sin-gle agent. Thus the hybrid systems provide more speci
fic and
effi-cient ways for the treating diseases than single-modality
nanoparticle
[5]
. Among various nanomaterials, gold and
super-paramagnetic iron oxide (SPIO) nanoparticles are highly promising
for the development of
“theranosis” agents
[6
e9]
. Combining iron
oxide and gold in one multifunctional agent could enhance
diag-nosis and therapeutic heating in the targeting tumors due to
excellent magnetic properties (from iron oxide), and optical (from
gold). Over the past few years, many research efforts have been
focused on developing such dual functional nanomaterials
including heterodimer nanocompositions
[10,11]
and core
eshell
nanostructure
[12
e15]
. Among various forms of particle
geome-tries, gold nanomaterials, especially gold nanorods (AuNRs) can be
adjusted by tuning their morphology to exhibit distinctive optical
properties in near-infrared (NIR) region (700
e850 nm). Due to
* Corresponding author. Department of Biological Science and Technology, Institute of Molecular Medicine and Bioengineering, National Chiao Tung Univer-sity, 75 Bo-Ai Street, Hsinchu 30068, Taiwan. Tel.:þ886 3 5729287; fax: þ886 3 5729288.
** Corresponding author.
E-mail address:ymwang@mail.nctu.edu.tw(Y.-M. Wang).
1 Author for equal contribution forfirst author.
Contents lists available at
ScienceDirect
Biomaterials
j o u r n a l h o m e p a g e : w w w . e l s e v i e r. co m/ lo ca t e / b i o m a t e ri a l s
http://dx.doi.org/10.1016/j.biomaterials.2014.02.026
minimal attenuation by water and hemoglobin at these
wave-lengths, which absorb weakly in soft tissue and blood, AuNRs is
particularly interesting for the development of NIR absorption
photothermal therapy
[16
e18]
. Hence, a nanostructure that
com-bines MR image diagnosis and NIR photothermal ablation would
greatly increase the treatment ef
ficacy and simultaneously
mini-mize the damage to normal cells and tissues. However, the gold
coating on iron oxide nanoparticles display intense NIR absorbance
is dif
ficult to synthesize;
[19
e21]
On the other hand, controlling the
hydrodynamic size of nanocompositions between 10 nm and
100 nm to avoid elimination by reticuloendothelial system (RES) is
also a major challenge to design ideal platforms
[22,23]
.
Herein, we described a novel strategy of dual functional
nano-clusters based on manganese-doped magnetism-engineered iron
oxide (MnMEIO) and AuNRs which combined with optical and
magnetic properties useful in MR imaging and photothermal
ablation functions. The size of the dual functional nanocluster was
controlled below 100 nm to avoid elimination by clearance organs.
Furthermore, we studied the effect of nanocluster shape on cellular
uptake. The MnMEIO in our platform can be used as MR imaging
contrast agent and the photothermal ablation function of AuNRs
was also demonstrated in this work.
Scheme 1
depicted the novel
dual functional nanocluster. MnMEIO attracted AuNRs through a
well-understood chemistry of Au
eS bond
[24]
. Then AuNRs and
MnMEIOs nanoclusters (abbreviated as AuNRs@MnMEIOs) were
further stabilized with acryl-modi
fied poly(ethylene glycol)
(PEG-Ac) and conjugated with Herceptin for targeting to Her-2/neu
hu-man breast cancer cells (SKBR-3). In vitro and in vivo ef
ficacies of the
AuNRs@MnMEIOs-Herceptin
for
theranosis
were
evaluated
through confocal
fluorescent microscopic studies, MR imaging,
optical imaging and photothermal therapy studies.
2. Experimental section 2.1. Chemicals and materials
Osmium tetroxide (1%), iron (III) acetylacetonate (Fe(acac)3, 99.9%), manganese
(II) acetylacetonate (Mn(acac)2, 99%), oleic acid (90%), oleylamine (90%),
1,2-hexadecandiol (90%), benzyl ether (98%), poly(ethylene glycol) methyl ether (mPEG, average Mwy 2000), N-ethyl-N0-(3-dimethylaminopropyl) carbodiimide
(EDC), fluorescein isothiocyanate (FITC), sodium cacodylate trihydrate (98%),
tannic acid (99%), and glutaraldehyde (25%) were purchased from Sigma Aldrich (MO, USA). Acetyl chloride (96%), cetyl trimethylammonium bromide (CTAB) (98%), and (3-mercaptopropyl) trimethoxysilane (MPTES, 95%) were purchased from Alfa Aesar (MA, USA). (3-Aminopropyl) triethoxy silane (APTES, 98%) and N0 -hydrox-ysuccinimide (NHS, 97%) were purchased from Fluka (Buchs, Switzerland). Herceptin (Trastumab) was purchased from Roche (Basel, Switzerland). Molecular-porous membrane tubings (average Mw y 12e14 kDa and average Mw y
300 kDa) were purchased from Spectrum (TX, USA). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide and thiazole Blue were purchased from Merck (Dietikon, Switzerland). S162 Scientific Formvar/Carbon200-mesh copper (50-grid) was purchased from Agar Scientific (Stansted, UK). Matrigel was purchased from BD Bioscience (MA, USA). Spurr’s resin was purchased from Agar Scientific (Essex, UK). All solvents were purchased from Echo Chemical (Miaoli, Taiwan). Dulbecco‘s minimal essential medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (NY, USA).
2.2. Cell line and tumor model
SKBR-3, a human breast cancer cell line, and Colo-205, a human colorectal cancer cell line, were obtained from Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan). Cells were cultured in DMEM and supplemented with 10% FBS, 100 U mL1penicillin and 100mg mL1streptomycin at 37C in an
atmosphere of 5% CO2. Nu mice (4 weeks old) were obtained from the BioLASCO
(Yi-Lan, Taiwan). Animal experiments were performed in accordance with institute guidelines.
2.3. Characterization
Relaxation time values (T1 and T2) of MnMEIO samples were measured to
determine relaxivity r1and r2. All measurements were made using the relaxometers
(NMS-120 & MQ60 Minispec, Bruker, Ontario, Canada) operating at 20 and 60 MHz and 37.0 0.1C. Hydrodynamic size and zeta potential were analyzed by Zetasizer
(Nano ZS90, Malvern Instruments, UK). The compositions of AuNRs@MnMEIOs-PEG were obtained by an Energy-dispersive X-ray spectrometer (EDX, S-3000N, Hitachi, Tokyo, Japan) and inductively coupled plasma atomic emission spectroscopy (ICP-AES, Jobin-Yvon JY 38type III, Edison, New Jersey, USA). Magnetic properties of the MnMEIO-PEG and AuNRs@MnMEIOs-PEG were studied with superconducting quantum interference devise magnetometer (SQUID, VSM Model 7400, OH, USA) at fields ranging from 10 to 10 kOe and at 300 K. The average core size, size distri-bution, and morphology were examined using a transmission electron microscope (TEM, JEOL JEM-2000 EX II, Tokyo, Japan) at a voltage of 80 kV. The composite dispersion was drop-cast onto a 200-mesh copper grid and the grid was air-dried at room temperature before being loaded into the microscope.
2.3.1. Synthesis of methoxy poly(ethylene glycol) acrylate (mPEG-Ac)
Poly(ethylene glycol) methyl ether (mPEG, 40 g, 20 mmol) was dispersed in 200 mL of anhydrous dichloromethane (DCM) in a 250 mL round-bottomflask and cooled to 0 C using an ice-bath with stirring. Then, acetyl chloride (2.4 mL,
Scheme 1. Schematic illustration showed that AuNRs@MnMEIOs nanoclusters were prepared. The AuNRs@MnMEIOs nanoclusters were used as a T2constrast agent and
30 mmol) and triethanolamine (TEA) (4.2 mL, 30 mmol) were added and the mixture was stirred at room temperature for 48 h under nitrogen atmosphere. The reaction mixture wasfiltered using a filter funnel and the solvent was evaporated to obtain the crude product, which was further precipitated in hexane. Thefinal product was dried under vacuum for overnight. 1H NMR (300 MHz, CDCl3) d (ppm): 3.37 (s, 3H,
CH3eO), 3.63 (m, 104H, PEG chain protons), 4.30 (t, J ¼ 4.7 Hz, 2H, CeCH2eC]O),
acryl group: 5.56 (1H), 6.15 (1H), 6.37 (1H).
2.3.2. Synthesis of functionalized magnetic nanoparticles
MnMEIOs capped with oleic acid was synthesized by thermal decomposition under nitrogen atmosphere[25]. Briefly, Fe(acac)3(1.4 g, 4 mmol), Mn(acac)2(0.51 g,
2 mmol), 1,2-hexadecanediol (2.58 g, 10 mmol), oleic acid (1.7 g, 6 mmol), oleyl-amine (1.6 g, mmol), and benzyl ether (5 mL) were mixed and stirred under nitrogen atmosphere. The mixture was heated to 200C and maintained to reflux for 2 h. Then, it was heated to 300C and refluxed for 1 h. The reaction mixture was cooled to room temperature. Then, the black solution was precipitated with acetone and separated via centrifugation. After discarding the supernatant, the black product was dissolved in hexane. Centrifugation (3000 rpm, 10 min) was applied to remove the undispersed residue. The product, MnMEIOs, was then precipitated with acetone, centrifuged to remove the solvent, and redispersed into chloroform. The surface of the MnMEIOs was functionalized with organosilane molecules (APTES and MPTES) to generate an amine and thiol terminated surface[26]. APTES and MPTMS functionalization was performed by silane ligand exchange. Briefly, 0.5% (v/ v) APTES and 0.25% (v/v) MPTES were added to the MnMEIOs in toluene containing 0.01% (v/v) acetic acid causing precipitation of the particles. The mixture was son-icated for 6 h and then rinsed and redispersed with water to remove excess APTES and MPTES.
2.3.3. Synthesis of gold nanorods (AuNRs)
Gold nanorods were modified and synthesized using the previous procedures
[27,28]. Briefly, the formation of short AuNRs was produced by the addition 3.2 mL of 5 mMHAuCl4 and 72mL HCl (37 wt. % in water, 12.1M) to 15 mL of 0.2 mMCTAB
aqueous solution and stirred for 15 min at 700 rpm. The solution was then mixed with 2.56 mL of 10 mMascorbic acid and 16mL of 100 mMAgNO3. Next, 40mL of
10 mMNaBH4wasfinally added to the mixture to initiate growth to yield gold
nanorods of aspect ratio about 3. The formation of short nanorods was visually observed by the formation of a red-colored solution within 30 min. Complete short AuNRs was monitored by solution extinction measurements. Finally, excess CTAB was removed by centrifuging twice at 10,000 rpm, the supernatant was remove the solvent, and redispersed into deionized water.
The long AuNRs were synthesized using the binary surfactant. Briefly, the seed solution for AuNRs growth was prepared as follows: 5 mL of 0.5 mMHAuCl4was
mixed with 5 mL of 0.2MCTAB solution, then, 600mL of fresh 10 mMNaBH4was
injected to the Au(III)-CTAB solution under vigorous stirring at 1,200 rpm. The seed solution was aged at room temperature for 30 min before use. The growth solution containing 0.137 g of CTAB and 0.025 g of sodium oleate in 5 mL of deionized water was reacted at 30C and mixed with 19.2mL of 100 mMAgNO3solution. Then which
5 mL of 1 mMHAuCl4solution was added and stirred for 90 min and 72mL of HCl
(37 wt. % in water, 12.1M) was added to adjust the pH. After another 15 min of slow stirring at 400 rpm, 1.25 mL of 64 mMascorbic acid was added and the solution was vigorously stirred for 30 s. Finally, 16mL of seed solution was injected into the growth solution. The resultant mixture was stirred for 30 s and left undisturbed at 30C for
12 h for AuNR growth. The final products were isolated by centrifugation at 7,000 rpm for 30 min followed by removal of the supernatant.
2.3.4. Preparation of spherical-shaped and rod-shaped AuNRs@MnMEIOs-PEG-Herceptin
Using the reported extinction coefficients of the longitudinal plasmon peaks
[29], the molar concentration in nanoparticles of all AuNR solutions were prepared before used. To establish spherical-shaped AuNRs@MnMEIOs, 2 mL of functionalized-MnMEIOs was added into 4 mL of 10 nMCTAB-AuNRs solution (both short aspect ratio and long aspect ratio AuNRs) and shaken for 12 h at room tem-perature. Then, 300mL of 2% mPEG-Ac was added to the mixture and stirred for 3 h at room temperature. The spherical-shaped AuNRs@MnMEIOs-PEG was obtained and purified by dialysis to remove excess mPEG-Ac and CTAB. To establish rod-shaped AuNRs@MnMEIOs, the process was similar with spherical-shaped AuNRs@Mn-MEIOs, after removing excess mPEG-Ac and CTAB, 300mL of 2% NH2-PEG3500-SH was
added to the mixture and stirred for 3 h at room temperature to modify the surface AuNRs, the rod-shaped AuNRs@MnMEIOs-PEG was obtained and purified by dialysis to remove excess NH2-PEG3500-SH. To prepare dual functionalization
nano-compositions, AuNRs@MnMEIOs-PEG-Herceptin, three hundreds microliters of 1.6 mg mL1EDC and 1 mg mL1NHS were added into both spherical-shaped and rod-shaped AuNRs@MnMEIOs-PEG solution. Then, 2mL of 22 mg mL1Herceptin was added into the mixture and incubated for 90 min at room temperature. The Herceptin-modified AuNRs@MnMEIOs-PEG was isolated by centrifugation at 8,000 rpm for 30 min at 4C to remove the excess EDC, NHS, Herceptin and unbound MnMEIOs (i.e. spherical-shaped AuNRs@MnMEIOs-PEG-Herceptin and rod-shaped AuNRs@MnMEIOs-PEG-Herceptin, respectively). Thefinal products were kept at 4C in PBS before used.
2.4. Evaluation of intracellular distribution of AuNRs@MnMEIOs nanoclusters Human breast cancer cell line (SKBR-3) (1 106cells) were seeded onto round
glass coverslips placed in 6-well plates and cultured overnight. After cells attached, cells were incubated with 1 mM (based on Fe and Mn concentration) of AuNRs@MnMEIOs-FITC-PEG-Herceptin for 24 h at 37C. At the end of the incuba-tion period, the cells were washed with PBS for 2 times to remove any free AuNRs@MnMEIOs-FITC-PEG-Herceptin and thenfixed with 4% formaldehyde solu-tion at room temperature for 30 min. Coverslips were placed onto glass microscope slides, and the distribution of AuNRs@MnMEIOs-FITC-PEG-Herceptin was analyzed using a confocal laser scanning (CLS) imaging system (TCS-SP5-X AOBS, Leica, Germany) consisting of Olympus BX51 microscope (Olympus, Tokyo, Japan) and a 20 mW-output 488 nm argon ion laser.
2.5. Cell viability determination
SKBR-3 cells were seeded in a 24-well plate at a density of 1 105
cells per well and were allowed to attach for 24 h at 37C in a 5% CO2incubator before the
treatment. Cells were maintained at 37C for 24 h after treatment with CTAB-AuNRs
or AuNRs@MnMEIOs-PEG-Herceptin at different concentrations (625 pMe2 nM
based on AuNRs). Cell viability was then determined using the MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay. Viable cells are capable of metabolizing the MTT reagent while dead cells are not. The treated cells were incubated with the MTT reagent for 4 h. Then 500mL DMSO was added to each well and incubated for 10 min and the absorbance at 540 nm was read. Each con-centration was repeated in triplicate and the results expressed as percentages relative to the control.
2.6. Nanoclusters cellular uptake study
To compare the cellular uptake of efficiency of different morphologies of AuNRs@MnMEIOs-PEG nanoclusters (with and without Herceptin), both Prussian blue staining and TEM were used to analyze. For Prussian blue staining, SKBR-3 (approximately 1 106
cells) were seeded in T25flask. After cells attached, media was replaced with medium containing spherical-shaped or rod-shaped AuNRs@MnMEIOs-PEG-Herceptin (56mg mL1, based on the concentration of Fe and Mn). At the end of the incubation period, the cells were washed with PBS for 2 times to remove unbound AuNRs@MnMEIOs-PEG-Herceptin. Cells werefixed with 4% paraformaldehyde for 40 min. Then, cells were incubated with fresh prepared Perls’ reagent (4% potassium ferrocyanide/20% HCl, 1:1, v/v) for 30 min and subse-quently observed by an inverted optical microscope. The uptake efficiency of different morphologies was also monitored by TEM to further validate the cellular uptake in SKBR-3 cells. For this experiment, SKBR-3 cells (approximately 1 107cells per T75flask) were incubated with medium containing
spherical-shaped or rod-spherical-shaped AuNRs@MnMEIOs-PEG-Herceptin (56mg mL1, based on the concentration of Fe and Mn) for 4 h. Treated cells were centrifuged andfixed with paraformaldehyde (1.6%)-glutaraldehyde (2.5%) solution followed by OsO4
solution. Then, cells were dehydrated in a graded series of ethanol and acetone and embedded in Spurr resin. These cell-containing resin blocks were sectioned using an ultramicrotome and ultrathin sections (70e90 nm thickness) were transferred onto TEM grid.
2.7. Intracellular iron content measurement
For the intracellular iron content quantification, cells were incubated with AuNRs@MnMEIOs nanocluster (56mg mL1, based on the concentration of Fe and Mn) for 12 h. The cells were washed, collected, and counted. After 1500 g centri-fugation for 10 min, the cell pellets were resuspended in 100mL 12% HCl solution and incubated at 60C for 6 h. After incubation, the suspension was centrifuged at
12,000 g for 10 min, and the supernatants were collected for iron concentration quantification. FiftymL of 1% ammonium persulfate was added into 50mL sample solution to oxidize the ferrous ions to ferric ions. Finally, 100mL of 0.1Mpotassium thiocyanate was added to the solution and incubated for 5 min to form the red color iron-thiocyanate. The absorption was read by a microplate reader set at 490 nm.
2.8. In vitro and in vivo MR imaging studies
MR imaging was performed on a 7.0 T MR imaging system (Bruker, Ettlingen, Germany). For in vitro MR imaging studies, SKBR-3 and Colo-205 cells (1 106cells)
were incubated with AuNRs@MnMEIOsnanoclusters (56mg mL1 based on the concentration of Fe and Mn) at 37C for 24 h and washed three times in PBS. A T2
-weighted spin-echo sequence (TR/TE¼ 3000 ms/22 ms) was used for MR imaging. For in vivo MR imaging studies, cultured SKBR-3 and Colo-205 tumor cells (3 106cells/mouse) were injected in their right and left thigh regions to establish
the tumors bearing mice model. After the tumors developed up to a size of approximately 200 mm3, AuNRs@MnMEIOs-PEG-Herceptin (10 mg kg1 body
weight) were intravenous (i.v.) injected via the tail vein into the mice. The whole-body imaging of pentobarbital-anesthetized mice was performed with a 7.0 T MR imaging system. All samples were scanned by a fast gradient echo pulse sequence with the following parameters: TR/TE¼ 3000 ms/22 ms, matrix size ¼ 256 256,
FOV¼ 6 6 mm and data are represented as mean SD (n ¼ 5). The contrast enhancement (%) was calculated by the following equation(1):
Enhancementð%Þ ¼ SIpost SIpre
SIpre 100 (1)
where SIpostis the value of signal intensity in tumor cells treated with the contrast
agents, AuNRs@MnMEIOs-PEG-Herceptin, and SIpre is the value of signal intensity for tumor cells alone.
2.9. In vivo systematic toxicity
For in vivo systematic toxicity study, a total of 4 tumors bearing mice were allocated to 2 groups, a) control group (without AuNRs@MnMEIOs-PEG-Herceptin treatment, b) with AuNRs@MnMEIOs-PEG-Herceptin treatment (10 mg kg1). Af-ter AuNRs@MnMEIOs-PEG-Herceptin injection, were sacrificed by CO2asphyxiation.
A full necropsy was performed and the organs were harvested,fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological exaination using standard techniques. Examined tissues included: liver, spleen, lung, kidney, and heart.
2.10. Thermal response measurements
The average temperature elevation of AuNRs@MnMEIOs nanoclusters was measured by placing the AuNRs@MnMEIOs nanoclusters with different concentra-tions (100mL, 0.12 nMe1 nMbased on the concentration of AuNRs) in 96-well plates and irradiated using a 808 nm diode continuous wavelength (CW) laser source (Pretek, Taiwan) at a power density of 5 W cm2. A themometer (TM-924C) from Lutron (Taipei, Taiwan)fitted with a K-type thremocouple (not exposed to the laser beam) was immersed in the AuNRs@MnMEIOs solutions to record the temperature. For control experiment, same volume of water without the AuNRs@MnMEIOs so-lution was irradiated with CW laser and recorded its temperature.
2.11. Photothermal ablation study
Human breast cancer cell line SKBR-3 (1 104cells) were seeded in 96-well
plates and cultured overnight at 37C in a 5% CO2incubator before the treatment.
Afterward cells were incubated with AuNRs@MnMEIOs-PEG-Herceptin nano-clusters for 6 h in the cell culture medium. Cells were irradiated at 808 nm CW laser for 10 min with power intensity of 5 Wcm2. To evaluate cell viability after irradi-ation, the cells were then stained with Calcein AM (BD Bioscience, CA, USA) and Propidium Iodide (PI) (Calbiochem, NJ, USA). Then, the cells were observed using a laser scanning confocal imaging system. For in vivo experiments, after the mice were intravenous injected with 200mL of AuNRs@MnMEIOs-PEG-Herceptin (10 mg kg1 body weight) for 24 h, the tumor on each mouse was radiated by the 808 nm NIR laser at the power density of 5 W cm2for 10 min. Four groups of mice (untreated, laser only, Herceptin only, and AuNRs@MnMEIOs-PEG-Herceptinþ laser) with five mice per group were used as the control. The tumor sizes were measured by a caliper every four day and calculated by the following equation(2):
Volume¼ tumor lengthð Þ tumor widthð Þ2
=2 (2)
Relative tumor volumes were calculated as V/V0(V0was the tumor volume
when the treatment was initiated). Mice with tumor sizes exceeding 4,000 mm3
were euthanatized according to the animal protocol.
3. Results and discussion
3.1. Characterization of AuNRs@MnMEIOs nanoclusters
The monodispersed MnMEIOs and CTAB-AuNRs were
synthe-sized following the previously reported methods. The morphology
of MnMEIOs and CTAB-AuNRs can be clearly observed in
Fig. 1
. Two
hundred particles were counted and measured per sample. TEM
images showed that the monodispersed MnMEIOs have an overall
particle size of 8.2
1.3 nm and the CTAB-AuNRs with average
length and width 31.2
2.9 and 10.6 1.5 nm, respectively.
Sub-sequently, the AuNRs@MnMEIOs nanoclusters were prepared
through the conjugation of thiol-functionalized MnMEIOs with
AuNRs. A near sphere nanocluster was shown in
Fig. 1
C, the average
size of AuNRs@MnMEIOs nanoclusters is 53.2
6.9 nm. The
hy-drodynamic size and zeta potential were determined (
Table S1
).
The hydrodynamic size of MnMEIO and AuNRs had a relatively
narrow size distribution with a mean size of 15.2
2.5 nm
thiol-functionalized MnMEIO, CTAB-AuNRs, PEG-Ac and Herceptin
antibody, the hydrodynamic size of the nanoclusters increased to
64.2
6.3 nm. Furthermore, the zeta potential values of MnMEIO,
CTAB-AuNRs, and AuNRs@MnMEIOs nanoclusters were found to
be
þ29.4 4.3, þ46.3 1.5, and 6.3 0.5 mV, respectively. The
decrease in zeta potential can be attributed to CTAB removal from
the surface of AuNRs, and PEG masking of the surface charges
[30
e
32]
.
Figure S1
showed the UV/vis spectra of MnMEIO-PEG,
CTAB-AuNRs, and AuNRs@MnMEIOs-PEG. The CTAB-AuNRs has two
absorption bands at 520 nm (transverse band) and 800 nm
(lon-gitudinal band). After conjugating with MnMEIOs, the lon(lon-gitudinal
band was blue-shifted from 800 to 788 nm and the color of
AuNRs@MnMEIOs-PEG was near red-wine as shown in
Figure S1
A
inset. We speculated that slightly blue-shift of
AuNRs@MnMEIOs-PEG may result from incomplete protection in modi
fied process
[33,34]
. Compare with CTAB-AuNRs, the longitudinal band was
evidently blue-shifted and CTAB-AuNRs would aggregate during
CTAB removal from the surface of AuNRs (
Figure S1B
). Photograph
in
Figure
S2A
illustrated
excellent
colloidal
stability
of
AuNRs@MnMEIOs-PEG in PBS even in presence of an external
magnet, indicating that AuNRs@MnMEIOs-PEG can be manipulated
in the presence of an external magnetic
field. Compared with
others reports, most Au-SPIO complexes lose their colloidal
sta-bility in external magnet
[35
e37]
, and severe aggregations would
preclude their applications in vivo, because their circulation time is
very short and they can sometimes cause organ damage result from
capillary occlusion
[38]
. In this study, the high colloidal stability of
AuNRs@MnMEIOs-PEG could be attributed to steric stabilization
provided by AuNRs and PEG to avoid MnMEIOs interact to each
other and resulted in good colloidal stability
[39,40]
. Stability of
AuNRs@MnMEIOs-PEG dispersions to excess electrolytes (PBS)
exhibited a similar trend. The images shown in
Figure S2B
indicate
that PEG produces stable dispersions after 6 month of storage. The
normalized saturation magnetization of AuNRs@MnMEIOs-PEG at
300 K was 52 emu g
1(
Figure S2C
). To convert from magnetization
per total mass of particles to a basis of per mass of [Fe
þ Mn], the
mass ratio of Au/Fe/Mn was about 3:2:1, as determined by ICP-AES
and EDX. The magnetization of approached the value of
104 emu g
1 [FeþMn], which was similar to magnetization of
MnMEIO-PEG (
z105 emu g
1[Feþ Mn]), indicating no signi
ficant
interference from the AuNRs conjugation. None of the samples
showed hysteresis, indicating that both MnMEIO-PEG and
AuNRs@MnMEIOs
nanoclusters
retained
superparamagnetic
property.
3.2. Evaluation of intracellular distribution of AuNRs@MnMEIOs
nanoclusters
To con
firm the composition of AuNRs@MnMEIOs nanoclusters,
ICP-AES and EDX were performed. The spectrum in
Figure S3
con
firmed the presence of Fe, Mn, and Au atoms in the
nano-clusters. Moreover, confocal laser scanning (CLS) microscopy was
used to further investigate the distribution of both AuNRs and
MnMEIOs. To do this study, we labeled the
fluorescent dye, FITC on
the MnMEIOs of AuNRs@MnMEIOs-PEG-Herceptin and applied
intense laser to excite plasmon emission of AuNRs and induce the
fluorescence
[41,42]
to examine the intracellular distribution of the
AuNPs and MnMEIOs using CLS microscopy.
Fig. 2
showed the FITC,
AuNRs,
fluorescence images and their overlaps for SKBR-3 cells
incubated with AuNRs@MnMEIOs-FITC-PEG-Herceptin. The
intra-cellular staining results indicated that the green-
fluorescent spots
of FITC (located on MnMEIOs,
Fig. 2
A) and red-
fluorescent spots of
AuNRs (
Fig. 2
B) were generally merged and the yellow spots were
observed in cytoplasm (
Fig. 2
C). The line pro
files of the fluorescence
mapping determined by CLS microscopy (
Fig. 2
D) evidenced the
AuNRs and MnMEIO combined with each other. As shown in
Fig. 2. Fluorescence images of SKBR-3 cell after treated with AuNRs@MnMEIOs-PEG-FITC-Herceptin for 24 h. (A) Greenfluorescence showed the location of MnMEIOs; (B) red fluorescence showed the location of AuNRs; (C) physically overlaid image of panels (A) and (B); and (D) A cross sectional compositional (green: MnMEIOs and red: AuNRs) line profile of a single cell under CLS microscopy image. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Figure S4
, we also observed the co-localization for a
three-dimensional analysis. These results also indicated that MnMEIOs
and AuNRs combined with each other.
3.3. Cytotoxicity assessment
The AuNRs-based nano-platform has been realized and applied
in biomedical
field, however, AuNRs were reported to induce DNA
damage, mitochondria damage and elevate intracellular reactive
oxidative species (ROS) due to CTAB
[43,44]
. Regarding the
cyto-toxicity of AuNRs@MnMEIOs nanoclusters, the cell viability was
assessed as shown in
Fig. 3
. As expected, MTT cytotoxicity tests
revealed no obvious toxic effect of the
AuNRs@MnMEIOs-PEG-Herceptin was observed while concentration was below 2 n
M(based
on
AuNRs
concentration).
It
is
noteworthy
that
AuNRs@MnMEIOs nanoclusters evoked excellent biocompatibility
and
reasonable
noncytotoxicity.
We
speculate
that
strong
AuNRs@MnMEIO binding through MPTES would remove residual
CTAB from AuNRs surface, and reduce cytotoxicity of AuNRs to a
negligible level.
3.4. Effect of nanocluster shape on cellular uptake
Recent observations in biological systems suggest that particle
shape has been considered to play an important role in cellular
uptake
[45,46]
. To our knowledge, it is not clear whether the shape
of AuNRs@MnMEIOs nanoclusters have great impact on cellular
uptake. To address the effects of shape on cellular uptake,
spherical-shaped and rod-shaped
AuNRs@MnMEIOs-PEG-Hercep-tin were chosen in this study (
Figure S5
). The TEM images (
Fig. 4
)
was conducted to estimate nanoclusters internalization by cells. As
shown in
Fig. 4
, for a single cell, multiple vesicles containing
spherical-shaped AuNRs@MnMEIOs-PEG-Herceptin were readily
observed (
Fig. 4
B
eF). On the other hand, the TEM image of
rod-shaped AuNRs@MnMEIOs-PEG-Herceptin uptake showed no
uptake for the same conditions (
Fig. 4
A). According to visual
assessment,
the
cellular
uptake
of
AuNRs@MnMEIOs-PEG-Herceptin is highly dependent on the shape, in which the results
are consistent with cellular uptake of AuNRs
[45
e47]
. Typical
Prussian blue staining was performed to demonstrate the
nano-cluster internalization. As shown in
Figure S6A-B
, rod-shaped
AuNPs@MnMEIOs-PEG-Herceptin internalization were less than
that of spherical-shaped AuNRs@MnMEIOs-PEG-Herceptin ones. In
addition, Herceptin-modi
fied nanoclusters showed higher uptake
in comparison to PEG-coated nanoclusters (
Figure S5C-D
). The
intracellular iron contents for the four types of AuNRs@MnMEIOs
nanocluster in SKBR-3 was carried out as shown in
Figure S5E
.
SKBR-3 incubated with spherical-shaped
AuNPs@MnMEIOs-PEG-Herceptin resulted in a striking increase in iron contents by a factor
of 5 as compared to other three groups. We speculated that the
rod-shaped nanoclusters need larger contact area with the cell
mem-brane receptors than the spherical-shaped ones, and the lower
amounts of rod-shaped nanoclusters uptake was observed in
comparison to the spherical-shaped ones. By taking the overall
uptake nanoclusters, spherical-shaped nanoclusters was selected
for the following experiments.
3.5. In vitro MR imaging study
To assess the potential use of AuNRs@MnMEIOs nanoclusters as
an enhanced MR imaging contrast agent, the T
2relaxivity value (r
2)
of the AuNRs@MnMEIOs-PEG was determined in 20- and 60-MHz
relaxometers and compared with that of the MnMEIO-PEG. As
shown in
Table S2
, we summarized the relaxivity values of
MnMEIO-PEG, AuNRs@MnMEIOs-PEG and Resovist using 20-MHz
and 60-MHz relaxometer. The r
2relaxivity value of monodispered
MnMEIO-PEG was 191.2
2.3 m
M1s
1at 0.5 T. In contrast,
MnMEIOs decorated with AuNRs, AuNRs@MnMEIOs-PEG,
exhibi-ted a dramatically increased r
2value of 364.0
2.8 m
M1s
1.
Resovist, a clinically approved T
2-weighted MR imaging contrast
agent, has slightly lower relaxivity value than that of MnMEIO-PEG
and
signi
ficant
lower
relaxivity
values
than
that
of
AuNRs@MnMEIOs-PEG
[48]
. Signi
ficant improvement in the r
2relaxivity value could be attributed to the synergistic magnetic
effect of multiple MnMEIOs nanoparticles in one cluster
[49,50]
.
These results suggested that the AuNRs@MnMEIOs-PEG should
have a stronger magnetization than that of monodispered MnMEIO
and could serve as a more effective contrast agent for MR imaging
in clinical diagnosis. T
2-weighted images were performed using a
7.0-T MR imaging system. As shown in
Figure S7
the negative
contrast enhancements were 55.7
2.4%, 32.6 2.7%, 30.8 3.2%,
12.7
1.9%, and 6.2 1.2% by comparing their results to that of
MnMEIO-PEG. AuNRs@MnMEIOs-PEG exhibited better negative
contrast enhancements than MnMEIO-PEG at different
concentra-tions (12, 25, 50, 100, and 200
m
M).
To illustrate the utility of AuNRs@MnMEIOs-PEG-Herceptin for
biomedical imaging, we also evaluated cellular uptake of
AuNRs@MnMEIOs-PEG-Herceptin in two different cancer cells
(Colo-205 and SKBR-3). As shown in
Fig. 5
, T
2-weighted MR images
of Colo-205 cells treated with AuNRs@MnMEIOs-PEG-Herceptin
and SKBR-3 cells treated with AuNRs@MnMEIOs-PEG exhibited
minimal
contrast
enhancement.
The
MR
images
of
AuNRs@MnMEIOs-PEG-Herceptin treated SKBR-3 cells
(over-express HER-2) showed much more (negative) contrast
enhance-ments (
37.2 3.4%, 49.1 4.5%, 66.1 2.7%, 84.7 5.1%,
and
90.0 4.9% along with increasing contrast agent
concen-tration). Different contrast enhancements between SKBR-3 and
Colo-205 is most likely attributed to ligand-receptor-mediated
internalization of AuNRs@MnMEIOs-PEG-Herceptin by targeted
cells
[51]
.
3.6. In vivo MR imaging study
Furthermore, in vivo T
2-weighted MR images were observed on
nude mice bearing subcutaneous xenografts of SKBR-3 and
Colo-205
tumor
before
and
after
intravenously
injection
of
AuNRs@MnMEIOs-PEG-Herceptin (10 mg kg
1). As shown in
Fig. 6
,
it is clear that high negative contrast enhancement observed for
SKBR-3 tumor (
70.4 7.2%) indicated more accumulation of
contrast agents, and moderate or slight negative contrast
0
20
40
60
80
100
120
140
0.00
0.06
0.12
0.25
0.50
1.00
2.00
Cell viability
(%)
Concentration of AuNRs (nM)
CTAB-AuNRs AuNRs@MnMEIOs-PEG-HerceptinFig. 3. In vitro cytotoxicity of AuNRs@MnMEIOs-PEG-Herceptin and CTAB-AuNRs at different concentrations (62.5 pMe2 nMbased on AuNRs) against SKBR-3 cells assessed by MTT assay.
enhancement observed for Colo-205 tumor (
7.5 1.4%). By
comparing these results, MR images at 24 h post injection showed
signi
ficant negative contrast enhancement in SKBR-3 tumor.
In addition, the systematic toxicity of
the established
AuNRs@MnMEIOs-PEG-Herceptin nanoclusters was examined
in vivo in our experiment. Histological examinations of major
or-gans indicated no evident tissue damage was observed in these
major organs (
Figure S8
).
3.7. Targeted photothermal tumor treatment
AuNRs are excellent photothermal agents due to their strong
absorption and high light-to-thermal conversion ef
ficiency which
is about
five orders of magnitude higher than that of fluorescent
dye
[52]
. To demonstrate the photothermal conversion effect,
AuNRs@MnMEIOs nanoclusters were exposed under the 808 nm
laser radiation, which is close to the longitudinal band of
AuNRs@MnMEIOs
nanoclusters,
and
the
temperature
was
measured. As shown in
Figure S9
a remarkable increase
tempera-ture more than 30
C was observed at the aqueous solution
con-taining 1 n
Mof AuNRs under 5 W cm
2and 808 nm laser irradiation
for 10 min. As previous reports, after laser irradiation, the
longi-tudinal band of AuNRs markedly decrease even if grafted
macro-molecules on the AuNR surfaces (
Figure S10
)
[33,53]
. The blue shift
of absorption band would be attributed to the aggregation of the
AuNRs. Indeed, to evaluate the aggregate formation of the
AuNRs@MnMEIOs-PEG, we measured the absorption spectra of the
AuNRs@MnMEIOs-PEG after NIR laser irradiation and found
AuNRs@MnMEIOs-PEG showed negligible loss in their longitudinal
band after laser irradiation for 10 min (
Figure S10
). It is likely that
Fig. 4. TEM images of SKBR-3 cells incubated with (A) rod-shaped and (B) spherical-shaped AuNRs@MnMEIOs-PEG-Herceptin for 4 h (C), (D), (E), (F) represent enlarged views of SKBR-3 cells incubated with spherical-shaped AuNRs@MnMEIOs-PEG-Herceptin nanoclusters.
Fig. 5. A) Top: in vitro T2-weighted MR images of a) untreated SKBR-3 cells, b) SKBR-3 treated with AuNRs@MnMEIOs-PEG and c) Colo-205 treated with
AuNRs@MnMEIOs-PEG-Herceptin; B) bottom: in vitro T2-weighted MR images of SKBR-3 cells treated with AuNRs@MnMEIOs-PEG-Herceptin nanoclusters at different concentrations. All of signal
the AuNRs@MnMEIOs-PEG nanoclusters have superb thermal
sta-bility so that the AuNRs@MnMEIOs-PEG nanoclusters could be
particularly suited to long term thermal therapy. Recent studies
have reported the use of con
fined and thermally-stable materials
such as carbon and silica to trap AuNRs, thereby enabling highly
thermal stability
[54]
. We speculated that such limitations could be
overcome by embedding AuNRs with MnMEIOs nanoparticles to
protect from aggregation.
Effective internalization provides an opportunity to apply
se-lective destruction of cancer cells upon irradiation by 808 nm NIR
laser. To demonstrate the photothermal effect in vitro, the
SKBR-3 cells were treated with AuNRs@MnMEIOs-PEG-Herceptin and
laser irradiated at varying intensities. The localized cell
photo-thermal effect was further investigated as shown in
Fig. 7
.With the
treatment of AuNRs@MnMEIOs-PEG-Herceptin, a clear
demarca-tion line between dead (red) and live cell (green) regions can be
observed in presence of laser exposure at 5 W cm
2for 10 min. It is
demonstrated that AuNRs@MnMEIOs-PEG-Herceptin could kill
cells only through the photothermal effect induced by NIR laser
irradiation, while neither the AuNRs@MnMEIOs-PEG-Herceptin
itself nor the laser irradiation alone can kill SKBR-3 cells.
3.8. Photothermal therapy in vivo
For an in vivo photothermal therapy, nude mice subcutaneously
transplanted with SKBR-3 cells on their right thighs were used.
After the mouse was injected with
AuNRs@MnMEIOs-PEG-Herceptin for 24 h, tumor of each mouse in the treatment group
was exposed to an 808 nm laser at a power density of 5 W cm
2for
10 min and recorded with an IR camera in real time (
Figure S11
). As
shown in
Fig. 8
A, after mouse tumors were
AuNRs@MnMEIOs-PEG-Herceptin, followed by irradiation with the laser, the tumor growth
was signi
ficantly suppressed. In addition, a charring spot appears
on the tumor site. In this irradiation group, one mouse tumor was
completely eliminated on day 5 of irradiation. In contrast, the
tu-mors in the control animals injected with solely PBS or
AuNRs@MnMEIOs-PEG-Herceptin without irradiation or solely
laser grow markedly over time (
Fig. 8
B). Importantly, mice in the
four control groups had an average life span of 14
e26 days, while
mice
in
the
treated
group
(AuNRs@MnMEIOs-PEG-Herceptin
þ laser) were tumor-free after treatment and survived
over 40 days (
Fig. 8
C). Our results suggest that
AuNRs@MnMEIOs-PEG-Herceptin is a powerful agent for in vivo photothermal
ther-apy of cancer.
4. Conclusions
We have reported the successful strategy toward the ultimate
goal
of
cancer
diagnosis
and
therapy.
This
established
AuNRs@MnMEIOs nanoclusters features high stability,
monodis-persity with an average hydrodynamic diameter of approximately
60 nm, superparamagnetic property, low cytotoxicity, and highly
Fig. 7. Cells incubated with AuNRs@MnMEIOs-PEG-Herceptin and then irradiated by an 808 nm laser for 10 min at different power densities. Thefirst row shows laser alone while the second row AuNRs@MnMEIOs-PEG-Herceptin and 808 nm laser treatment. Each column shows cells treated at specific laser intensity: 0 W cm2((A), (F)), 1 W cm2((B), (G)),
2 W cm2((C), (H)), and 5 W cm2((d), (i)) Viable cells appear green from calcein AM staining while red areas of PIfluorescence are cells destroyed by photothermal irradiation. Scale bar 200mm for all images. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)
ef
ficient photothermal effect. The AuNRs@MnMEIOs nanoclusters
showed transversal relaxivity at least about 2 times higher than
commercial contrast agents. On the other hand, the
spherical-shaped nanoclusters were easily engulfed by cells and
demon-strated that the fabricated AuNRs@MnMEIOs nanocluster can kill
cancer cells effectively with exposure to NIR laser. In addition, these
dual functional nanoclusters have a great potential for in vitro and
in vivo MR imaging diagnosis and photothermal tumor therapy.
Since the AuNRs of these nanoclusters can provide heat while NIR
laser treatment, this technique will provide functional options for
achieving an advanced controlled-release system using AuNRs for
drug therapy responding to NIR stimuli.
Acknowledgments
This work was supported by grants NSC
102-227-M-009-004-MY3 and NSC 101-2923-M-009-002-MY3 from the National
Science Council of Taiwan and MOHW 103-TD-B-111-05 from
Ministry of Health and Welfare. This research was also
partic-ularly supported by
“Aim for the Top University Plan” of the
National Chiao Tung University and Ministry of Education. The
authors thank to Ms. C.-Y. Chien of Precious Instrument
Center (National Taiwan University) for the assistance in TEM
experiment and Ms. H.
eF. Chen for the assistance in MRI
experiment.
Fig. 8. In vivo photothermal therapy. (A) Representative photos of tumors with and without laser irradiation or AuNRs@MnMEIOs-PEG-Herceptin treatment; (B) Tumor growth curves in different groups of mice after treatment (n¼ 5) (where the arrows labeled). The tumor volumes were normalized to their initial sizes. Error bars were based on SD; (C) Survival curves of mice bearing SKBR-3 tumor after various treatments indicated.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://
dx.doi.org/10.1016/j.biomaterials.2014.02.026
.
References
[1] Katz E, Willner I. Integrated nanoparticle-biomolecule hybrid systems: synthe-sis, properties, and applications. Angew Chem Int Ed Engl 2004;43:6042e108. [2] Ferrari M. Cancer nanotechnology: opportunities and challenges. Nat Rev
Cancer 2005;5:161e71.
[3] Lal S, Clare SE, Halas NJ. Nanoshell-enabled photothermal cancer therapy: impending clinical impact. Acc Chem Res 2008;41:1842e51.
[4] Bao G, Mitragotri S, Tong S. Multifunctional nanoparticles for drug delivery and molecular imaging. Annu Rev Biomed Eng 2013;15:253e82.
[5] Gao J, Gu H, Xu B. Multifunctional magnetic nanoparticles: design, synthesis, and biomedical applications. Acc Chem Res 2009;42:1097e107.
[6] Lu W, Singh AK, Khan SA, Senapati D, Yu H, Ray PC. Gold nano-popcorn based targeted diagonosis, nanotherapy treatment and in-situ monitoring of pho-tothermal therapy response of prostate cancer cells using surface enhanced raman spectroscopy. J Am Chem Soc 2010;132:18103e14.
[7] Koo HM, Huh S, Sun IC, Yuk SH, Choi K, Kim K, et al. In vivo targeted delivery of nanoparticles for theranosis. Acc Chem Res 2011;44:1018e28.
[8] Xie J, Liu G, Eden HS, Ai H, Chen X. Surface-engineered magnetic nanoparticle platforms for cancer imaging and therapy. Acc Chem Res 2011;44:883e92. [9] Fan Z, Shelton M, Singh AK, Senapati D, Khan SAP, Ra C. Multifunctional
plasmonic shell magnetic core nanoparticles for targeted diagnostics, isola-tion, and photothermal destruction of tumor cells. ACS Nano 2012;6:1065e73. [10] Bao J, Chen W, Liu T, Zhu Y, Jin P, Wang L, et al. Bifunctional Au-Fe3O4
nanoparticles for protein separation. ACS Nano 2007;1:293e8.
[11] Xu C, Xie J, Ho D, Wang C, Kohler NE, Walsh G, et al. Au-Fe3O4dumbbell nanoparticles as dual-functional probes. Angew Chem Int Ed 2008;47:173e6. [12] Lyon JL, Fleming DA, Stone MB, Schiffer PM, Williams E. Synthesis of Fe oxide core/Au shell nanoparticles by iterative hydroxylamine seeding. Nano Lett 2004;4:719e23.
[13] Xu Z, Hou Y, Sun S. Magnetic core/shell Fe3O4/Au and Fe3O4/Au/Ag nano-particles with tunable plasmonic properties. J Am Chem Soc 2007;129:8698e9. [14] Wang L, Bai J, Li Y, Huang Y. Multifunctional nanoparticles displaying magnetization and near-IR absorption. Angew Chem Int Ed 2008;47: 2439e42.
[15] Ma LL, Feldman MD, Tam JM, Paranjape AS, Cheruku KK, Larson TA, et al. Small multifunctional nanoclusters (nanoroses) for targeted cellular imaging and therapy. ACS Nano 2009;3:2686e96.
[16] Hirsch LR, Stafford RJ, Bankson JA, Sershen SR, Rivera B, Price RE, et al. Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance. Proc Natl Acad Sci 2003;100:13549e54.
[17] Chen J, Wang D, Xi J, Au L, Siekkinen A, Warsen A, et al. Immuno gold nanocages with tailored optical properties for targeted photothermal destruction of cancer cells. Nano Lett 2007;7:1318e22.
[18] Madsen SJ, Baek SK, Makkouk AR, Krasieva T, Hirschberg H. Macrophages as cell-based delivery systems for nanoshells in photothermal therapy. Ann Biomed Eng 2012;40:507e15.
[19] Larson TA, Bankson J, Aaron J, Sokolov K. Hybrid plasmonic magnetic nano-particles as molecular specific agents for MRI/optical imaging and photo-thermal therapy of cancer cells. Nanotechnology 2007;18:325101e8. [20] Elsherbini AA, Saber M, Aggag M, El-Shahawy A, Shokier HA. Laser and
radiofrequency-induced hyperthermia treatment via gold-coated magnetic nanocomposites. Int J Nanomedicine 2011;6:2155e65.
[21] Narayanan S, Sathy BN, Mony U, Koyakutty M, Nair SV, Menon D. Biocom-patible magnetite/gold nanohybrid contrast agents via green chemistry for MRI and CT bioimaging. ACS Appl Mater Interfaces 2012;4:251e60. [22] Liu H, Chen D, Li L, Liu T, Tan L, Wu X, et al. Multifunctional gold nanoshells on
silica nanorattles: a platform for the combination of photothermal therapy and chemotherapy with low systemic toxicity. Angew Chem Int Ed 2011;50: 891e5.
[23] Melancon MP, Lu W, Zhong M, Zhou M, Liang G, Elliott AM, et al. Targeted multifunctional gold-based nanoshells for magnetic resonance-guided laser ablation of head and neck cancer. Biomaterials 2011;32:7600e8.
[24] Bardhan R, Lal S, Joshi A, Halas NJ. Theranostic nanoshells: from probe design to imaging and treatment of cancer. Acc Chem Res 2011;44:936e46. [25] Sun S, Zeng H, Robinson DB, Raoux S, Rice PM, Wang SX, et al. Monodisperse
MFe2O4(M¼ Fe, Co, Mn) nanoparticles. J Am Chem Soc 2004;126:273e9.
[26] Levin CS, Hofmann C, Ali TA, Kelly AT, Morosan E, Nordlander P, et al. Mag-netic plasmonic coreshell nanoparticles. ACS Nano 2009;3:13791388. [27] Si S, Leduc C, Delville MH, Lounis B. Short gold nanorod growth revisited: the
critical role of the bromide counterion. Chemphyschem. 2011;13:193e202.
[28] Ye X, Zheng C, Chen J, Gao Y, Murray CB. Using binary surfactant mixtures to simultaneously improve the dimensional tunability and monodispersity in the seeded growth of gold nanorods. Nano Lett 2013;13:765e71.
[29] Orendorff CJ, Murphy CJ. Quantitation of metal content in the silver-assisted growth of gold nanorods. J Phys Chem B 2006;110:3990e4.
[30] Wu SC, Lin KL, Wang TP, Tzou SC, Singh G, Chen MH, et al. Imaging specificity of MR-optical imaging agents following the masking of surface charge by poly(ethylene glycol). Biomaterials 2013;34:4118e27.
[31] Lipka J, Semmler-Behnke M, Sperling RA, Wenk A, Takenaka S, Schleh C, et al. Biodistribution of PEG-modified gold nanoparticles following intratracheal instillation and intravenous injection. Biomaterials 2010;31:6574e81. [32] Niidome T, Yamagata M, Okamoto Y, Akiyama Y, Takahashi H, Kawano T, et al.
PEG-modified gold nanorods with a stealth character for in vivo applications. J Control Release 2006;114:343e7.
[33] Yamashita S, Fukushima H, Niidome Y, Mori T, Katayama Y, Niidome T. Controlled-release system mediated by a retro Diels-Alder reaction induced by the photothermal effect of gold nanorods. Langmuir 2011;27:14621e6. [34] Kawamura G, Yang Y, Nogami M. Facile assembling of gold nanorods with
large aspect ratio and their surface-enhanced Raman scattering properties. Appl Phys Lett 2007;90:261908e10.
[35] Wang DW, Zhu XM, Lee SF, Chan HM, Li HW, Kong SK, et al. Folate-conjugated Fe3O4@SiO2@gold nanorods@mesoporous SiO2 hybrid nanomaterial: a theranostic agent for magnetic resonance imaging and photothermal therapy. J Mater Chem B 2013;1:2934e42.
[36] Ma M, Chen H, Chen Y, Wang X, Chen F, Cui X, et al. Au capped magnetic core/ mesoporous silica shell nanoparticles for combined photothermo-/chemo-therapy and multimodal imaging. Biomaterials 2012;33:989e98.
[37] Wang C, Chen J, Talavage T, Irudayaraj J. Gold nanorod/Fe3O4nanoparticle “nano-pearl-necklaces” for simultaneous targeting, dual-mode imaging, and photothermal ablation of cancer cells. Angew Chem Int Ed 2009;48:27592763. [38] Kievit FM, Zhang M. Surface engineering of iron oxide nanoparticles for
tar-geted cancer therapy. Acc Chem Res 2011;44:853e62.
[39] Liong M, Lu J, Kovochich M, Xia T, Ruehm SG, Nel AE, et al. Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery. ACS Nano 2008;2:889e96.
[40] Lee N, Choi Y, Lee Y, Park M, Moon WK, Choi SH, et al. Water-dispersible ferrimagnetic iron oxide nanocubes with extremely high r2 relaxivity for highly sensitive in vivo MRI of tumors. Nano Lett 2012;12:3127e31. [41] Goldys EM, Sobhan MA. Fluorescence of colloidal gold nanoparticles is
controlled by the surface adsorbate. Adv Funct Mater 2012;22:1906e13. [42] Eustis S, El-Sayed M. Aspect ratio dependence of the enhancedfluorescence
intensity of gold nanorods: experimental and simulation study. J Phys Chem B 2005;109:16350e6.
[43] Zhao F, Zhao Y, Liu Y, Chang X, Chen C, Zhao Y. Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials. Small 2011;7:1322e37. [44] Rayavarapu RG, Petersen W, Hartsuiker L, Chin P, Janssen H, van Leeuwen FW,
et al. In vitro toxicity studies of polymer-coated gold nanorods. Nanotech-nology 2010;21:145101e10.
[45] Chithrani BD, Chan WC. Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Lett 2007;7:1542e50.
[46] Chithrani BD, Ghazani AA, Chan WC. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 2006;4:662e8.
[47] Qiu Y, Liu Y, Wang L, Xu L, Bai R, Ji Y, et al. Surface chemistry and aspect ratio mediated cellular uptake of Au nanorods. Biomaterials 2010;31:7606e19. [48] Qin J, Laurent S, Jo YS, Roch A, Mikhaylova M, Bhujwalla ZM, et al. A
high-performance magnetic resonance imaging T2 contrast agent. Adv Mater 2007;19:1874e8.
[49] Yoon TJ, Lee H, Shao H, Hilderbrand SA, Weissleder R. Multicore assemblies potentiate magnetic properties of biomagnetic nanoparticles. Adv Mater 2011;23:4793e7.
[50] Lee JE, Lee N, Kim H, Kim J, Choi SH, Kim JH, et al. Uniform mesoporous dye-doped silica nanoparticles decorated with multiple magnetite nanocrystals for simultaneous enhanced magnetic resonance imaging,fluorescence imaging, and drug delivery. J Am Chem Soc 2010;132:552e7.
[51] Meier R, Henning TD, Boddington S, Tavri S, Arora S, Piontek G, et al. Breast cancers: MR imaging of folate-receptor expression with the folate-specific nanoparticle P1133. Radiology 2010;255:527e35.
[52] Yi DK, Sun IC, Ryu JH, Koo H, Park CW, Youn IC, et al. Matrix metalloproteinase sensitive gold nanorod for simultaneous bioimaging and photothermal ther-apy of cancer. Bioconjug Chem 2010;21:2173e7.
[53] Cheng L, Yang K, Li Y, Zeng X, Shao MS, Lee T, et al. Multifunctional nano-particles for upconversion luminescence/MR multimodal imaging and magnetically targeted photothermal therapy. Biomaterials 2012;33:2215e22. [54] Yang HW, Liu HL, Li ML, Hsi IW, Fan CT, Huang CY, et al. Magnetic gold-nanorod/PNIPAAmMA nanoparticles for dual magnetic resonance and pho-toacoustic imaging and targeted photothermal therapy. Biomaterials 2013;34: 5651e60.
