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Biomaterials 33 (2012) 989e998

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Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

Au capped magnetic core/mesoporous silica shell nanoparticles for combinedphotothermo-/chemo-therapy and multimodal imaging

Ming Ma, Hangrong Chen*, Yu Chen, Xia Wang, Feng Chen, Xiangzhi Cui, Jianlin Shi*

State Key of Laboratory of High Performance Ceramic and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Science, Shanghai 200050, PR China

a r t i c l e i n f o

Article history:Received 22 September 2011Accepted 10 October 2011Available online 24 October 2011

Keywords:Mesoporous silicaDrug deliveryGold nanorodPhotothermal

* Corresponding authors. Tel.: þ86 21 52412712; faE-mail addresses: hrchen@mail.sic.ac.cn (H. Ch

(J. Shi).

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.10.017

a b s t r a c t

Uniform Au NRs-capped magnetic core/mesoporous silica shell nanoellipsoids (Au NRs-MMSNEs) wereprepared by coating a uniform layer of Au NRs on the outer surface of a magnetic core/mesoporous silicashell nanostructure, based on a two-step chemical self-assembly process. This multifunctional nano-composite integrate simultaneous chemotherapy, photo-thermotherapy, in vivo MR-, infrared thermaland optical imaging into one single system. The obtained multifunctional nanoellipsoids showed verylow cytotoxicity, and the cancer cell uptake and intracellular location of the nanoellipsoids wereobserved by confocal laser scanning microscopy and bio-TEM. Importantly, the prepared multifunctionalnanoellipsoids showed high doxorubicin loading capacity and pH value-responsive release mainly due tothe electrostatic interaction between DOXmolecules and mesoporous silica surface. Besides, a synergisticeffect of combined chemo- and photo-thermo therapy was found at moderate power intensity of NIRirradiation based on the DOX release and the photothermal effect of Au NRs.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The biomedical applications of inorganic nanoparticles, such asquantum-dots, gold and silica nanoparticles et al, have receivedextensive attentions due to their special characters at nano-scale.Among them, high chemical stability and drug loading capacityhave established mesoporous silica nanoparticles (MSNs) as a newdrug delivery carrier for potential biomedical applications [1e3].Especially, plenty of silanol groups in both interior and exteriorsurfaces ofMSNsmake it easy to combinewith various nanoparticlesthrough covalent and/or electrostatic self-assembly [4,5]. Forexample, Heyon et al. reported the fabrication of MSNs decoratedwith multiple magnetite nanocrystals, and built a platform formagnetic resonance imaging and drug delivery [4]. Our grouprecently synthesized magnetic mesoporous nanoellipsoids coatedwith fluorescent quantum dots through a polyelectrolyte mediatedself-assembly technique [3]. The ever-increasing interests have beenfocused on combining additional functional nanoparticles intoMSNsto integrate different therapeutic and diagnostic modes into onesingle system, which is of great potential in biomedical applications.

Gold nanoparticles (Au NPs) with particular shapes have alsoattracted great attentions for their large optical adsorption coeffi-cients in the near-infrared region, where the biological tissues are

x: þ86 21 52413122.en), jlshi@sunm.shcnc.ac.cn

All rights reserved.

transparent [6e12]. It has been reported that gold nanoparticlescould precisely accumulate in the tumor after a long period of bloodcirculation and produce heat cytotoxicity upon the remote NIR laserirradiation [13]. Among three differently shaped Au NPs, goldnanorods (Au NRs) have superior spectral bandwidth and largerextinction coefficient than other shaped Au NPs [14]. Unlike theformation of gold shells and gold cages by complicated gold coatingprocess, Au NRs are much easier to synthesize using a seed-mediated route without irreversible aggregation among thenanorods. Furthermore, PEG-stabilized Au NRs have longer circu-lation time and more tumor accumulation, thus Au NRs arebelieved to be more promising and therefore have attractedintensive attentions, compared to other types of Au NPs.

Current clinical therapy investigation has shown that a combi-nation of chemo- and thermotherapy could result in additionallyenhanced anti-cancer efficacy, because hyperthermia can promotedrug delivery into tumor [15], and increase the drug toxicity (e.g.,doxorubicin and cisplatin et al.) [16]. However, such a combinedtherapy is often confronted with side-effects and unsatisfactorytherapeutic efficacy because the thermotherapy and drugs do nottake effect simultaneously in the same tumorgenic regions. Theadvances in biomedical nanotechnology in recent years have pre-sented new approaches for current thermo-/chemo-therapy. Forexample, PLGA-Au half-shell [17] and mesoporous silica rattle-Aushell [10] have been used in the co-delivery of gold nanoparticlesand drugs into the targeted tumor, resulting in an apparentlyenhanced therapeutic efficacy compared to the treatments by each

M. Ma et al. / Biomaterials 33 (2012) 989e998990

single component. However, there still lacks suitable methods toconjugate Au NRs on the exterior surface of MSNs for the combinedtherapy, because the shape and size of Au NRs do notmatchwith thespherical morphology of MSNs of about 100 nm in diameter. Thismis-match between the elongated Au NRs andMSNs spheres wouldlead to very limited and unstable connecting of Au NRs on theexterior surface of spherical MSNs. To solve the problems, here wepropose that the equally elongated MSNs, e.g., ellipsoidal MSNs ofrelatively large aspect ratio, which has been reported to be moreeasily internalized by cells [18], should be well connected with AuNRs of similar aspect ratio by chemical bonding.

Herein, well-dispersed Au NRs-capped magnetic core/meso-porous silica shell nanoellipsoids (Au NRs-MMSNEs) have beenfabricated, based on a two step chemical self-assemblymethod. Thenumerous Au NRs are homogenously covalently conjugated/distributed on the exterior surface of magnetic mesoporous silicananoellipsoids (MMSNEs). This multifunctional nanocomposite isdesigned to integrate chemotherapy, photo-thermotherapy, in vivoMRI, and infrared thermal imaging simultaneously into one singlesystem. Into this system, anti-cancer drugs could be highly andefficiently loaded into themesopores of themesoporous silica shell,and release in a pH-responsive way in acid environment. Cellproliferation experiments and in vivo thermal imaging are used todemonstrate the feasibility of using these nanocomposites inphotothermal treatment of cancer cells. Moreover, a synergistic

Fig. 1. (a) Schematic microscopic structure of Au NRs-MMSNEs: Numbers of Au NRs being cacovalent bonding; (bef) TEM micrographs of (b) Fe2O3@SiO2@mSiO2, (c, d, e) Au NRs-MMSNon the surface of a gold nanorod.

effect of combined chemo- and photo-thermo therapy was ex-pected to decrease both the dosage-limiting toxicity of the anti-cancer drugs and tissue damage by over-heating.

2. Materials and methods

2.1. Materials

Tetraethyl orthosilicate (TEOS), ethanol, ammonia solution, urea, NaH2PO4 andNaBH4 were obtained from Sinopharm Chemical Reagent Co.; Octadecyltrimethox-ysilane (C18TMS) was purchased from Tokyo Chemical Industry Co., Ltd.;Fe(ClO4)3$6H2O, HAuCl4, AgNO3, hexadecyltrimethylammonium bromide (CTAB,�99%), phosphate-buffered saline (PBS), MES solution (1M)，L(þ)-ascorbic acid,4,7,10,13,16,19,22,25,32,35,38,41,44,47,50,53-hexadecaoxa-28,29-dithiahexapentaco-ntanedioic acid (PEG acid disulfide), O-[(N-succinimidyl)succinyl-aminoethyl]-O0-methylpolyethylene glycol 2000 (mPEG2000eNHS), and O-[2-(3-mercaptopropionyl-amino)ethyl]-O0-methylpolyethylene glycol 5000 (mPEG5000eHS) were purchasedfrom SigmaeAldrich; 3-aminopropyltriethoxysiliane was purchased from AcrosOrganics, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) were purchased from Fluka; Doxorubicin (DOX) wasprovided by Beijing HuaFeng United Technology Co., Ltd. PBS solution (10 mmol/L, pH7.2e7.4) was purchased from Beijing Zoman Biotechnology Co., Ltd. All aqueous solu-tions were prepared in deionized water.

2.2. Synthesis of NH2-modified Fe3O4@SiO2@mSiO2 ellipsoids

Uniform300nmsized Fe3O4@SiO2@mSiO2 nanoellipsoidswere synthesized usingthe previously reported method [19,20]. Firstly, 200 nm sized the hematite spindleswere prepared by aging a solution containing 11.6 g Fe(ClO4)3$6H2O, 1.5 g urea, and

pped on the outer surface of a magnetite core/mesoporous silica shell nanoellipsoid viaEs and (f) the designed structure model. The arrow in the Figure c shows the PEG layer

M. Ma et al. / Biomaterials 33 (2012) 989e998 991

0.16 gNaH2PO4 in 250mLdeionizedwater at 100 �C for 24h. For transitional SiO2 layercoating, 60 mg Fe2O3 spindles was well suspended in a mixed solution of 142.8 mLethanol, 20 mL H2O and 6.28 mL ammonia solution. Then 0.50 mL TEOS dissolved in4 mL ethanol was added by an injecting pump at a speed of 4 mL/h under vigorousstirring. For mesoporous silica coating, 0.6 mL TEOS and 0.4 mL C18TMS mixtures in4 mL ethanol were added dropwise using the same injection method. The resultantFe2O3@SiO2@mSiO2 precipitate was collected by centrifugation and washed withdeionizedwater and ethanol several times. Then, the productwere dried at 100 �C andcalcined at 550 �C for 6 h. Finally, the reduction was carried out by the thermaltreatment of Fe2O3@SiO2@mSiO2 inmixed H2 and Ar (volume ratio 5:95) gas at 410 �Cfor 5 h. For amine functionalization, Fe3O4@SiO2@mSiO2 (20 mg) was dispersed in20 mL ethanol, to this 0.025mL of 3-aminopropyltriethoxysilianewas added, and themixture were refluxed overnight. After centrifugation with ethanol, NH2 modifiedFe3O4@SiO2@mSiO2 was resuspended in water.

2.3. Synthesis of COOH-modified Au NRs

Au NRs with an aspect ratio ofw4.5 were synthesized through a seed-mediatedapproach as previously reported [6e8]. Briefly, the 3 nm sized gold seed particleswere prepared by adding 0.6 mL of an ice-cold solution of 10 mM NaBH4 into 10 mLaqueous solution consisting of 0.1 M CTAB solution and 0.25 mM HAuCl4. Thebrownish-yellow seed solutionwas formed and kept at 25 �C for synthesis of Au NRs.A growth solution was prepared in the following order: 100 mL of 0.1 M CTABsolution, 3 mL of 0.025 M chloroauric acid, 0.52 mL of 50 mM silver nitrate solution,and 1.5 mL of 80mM ascorbic acid. Finally, 0.3 mL of seed solutionwas added and theentire solution was left overnight at 28 �C. The excess CTAB was removed bycentrifugation (three times at 13000 rpm, 15 min each). To 5 mL of Au NRs solution5 mL of 0.5 mg/mL aqueous mPEG5000eHS solution was added, and the mixture wassonicated for 30 s and left to proceed for 2 h. The excess mPEG5000eHS moleculeswere removed by centrifugation (two times at 13000 rpm, 15 min each), then thePEGylated Au NRswere resuspended in the 5mL of deionizedwater. To functionalize

Fig. 1. (cont

the Au NRs with carboxylic groups, 5 mg of PEG acid disulfide was added, and themixturewas sonicated at 30 �C for 1 h. The excess PEG acid disulfidewas removed bycentrifugation (two times at 13,000 rpm, 15 min each).

2.4. Synthesis of Au NRs-MMSNEs

The COOH-modified Au NRs were suspended in 5mL 0.1 M MES solution (pH 5.0).To 5 mL of this were added 2 mg N-(3-dimethylaminopropyl)-N0-ethylcarbodiimidehydrochloride (EDC) and 2 mg N-hydroxysuccinimide (NHS), and the mixture wasgently stirred at room temperature for 30min. Excess activation agents were removedby washing with cold water and centrifuging at 13,000 rpm and 4 �C for 20 min. Theactivated Au NRs solution was suspended in 5 mL H2O, to this was added dropwise2 mL of aqueous NH2-modified Fe3O4@SiO2@mSiO2 (2 mg/mL), and stirring proceedfor 2 h at room temperature. The productwas purified by centrifuging at 6000 rpm for10min, and suspended in 5mL of ethanol. Finally, the resulting composite was stirredat room temperature for 3 h, followed by adding 2 mg of mPEG2000eNHS dissolved in1 mL ethanol to react with the residual amino- groups on the particle surface. Theproductswerepurifiedbycentrifugation, and suspended in 0.1 M PBS solution (pH7.4).The Au, Si and Fe element contents in Au NRs-MMSNEs dispersed in the deionizedwaterwere determined by inductively coupled plasma atomic emission spectrometry(ICP-AES; Vista AX). The molar ratio of Au:Si:Fe was calculated to be 6:9:1.

2.5. Nanoparticles characterization

TEM micrographs were obtained on a JEMe2010 electron microscope with anaccelerating voltage of 200 kV. The UV adsorption spectrums of different nano-composties dispersed in deionized water were collected on a Shimadzu UV-3101PCUVevis absorption spectrophotometer. T2-MR imaging of as-prepared samples insolution were tested on a 3.0 T clinical MRI instrument (GE Signa 3.0 T). N2

adsorptionedesorption isotherms at 77 K were measured on a Micrometitics Tristar3000 system. The pore size distributions were calculated from desorption branches

inued).

Fig. 2. The UVevis absorption spectra of (1) Fe3O4@SiO2@mSiO2, (2) Au NRs dispersedin CTAB solution, and (3) Au NRs-MMSNEs.

M. Ma et al. / Biomaterials 33 (2012) 989e998992

of isotherms by the Barrett-Joyner-Halenda (BJH) method. Pore volume and specificsurface area were calculated by using Barrett-Joyner-Halenda (BJH) and Langmuirmethods, respectively.

2.6. Drug loading and release

2.6.1. Drug loadingA fitting straight line was obtained with UVevis absorption intensity at 485 nm

as the ordinate and DOX concentration in PBS solution as the abscissa, according tolinear relation in the concentration range from 0 to 0.5 mg/mL. The DOX-Au NRs-MMSNEs were obtained by mixing 5.0 mg of Au NRs-MMSNEs with 3 mg DOX (in6 mL PBS solution) at the room temperature in the dark, and the mixture was thenmagnetically stirred for 24 h. The supernatant DOX solutions were collected bycentrifugation, and the concentration of unloaded DOX was measured through the

Fig. 3. (a) T2 phantom images of Au NRs-MMSNEs at different Fe concentrations; (b) Relaxaa mouse before and after intratumor injection of Au NRs-MMSNEs; (d) Photographs of Auattraction.

above fitting line, and the drug loading content could be easily obtained. DOX drugloading amount was measured using the below equation:

Loading amount (%) ¼ 100 � (total DOX used-DOX in supernatant)/(total Au NRs-MMSNEs used þ total DOX used-DOX in supernatant).

2.6.2. In vitro drug release of DOX4 mg of DOX-loaded Au NRs-MMSNEs were transferred into a dialysis bag (cut

off molecular weight 8000 g mol�1), then the bag was put into a tube containing20mL PBS solution. The pH value of PBS solution could be adjusted by adding 1 MHClsolution. The tubes were placed into a shaking table with 140 rpm at 37 �C. At timeintervals, 3.0 mL solution was collected and measured using UVevis absorption,then back to the tube.

2.7. Treatment effectiveness evaluation

2.7.1. Cell cultureHuman breast cancer MCF-7 cells were chosen as a cancer cell model to evaluate

the toxicity of Au NRs-MMSNEs and the therapeutic effect of combination therapy incomparison with every single one. MCF-7 cells were cultivated in Dulbecco’smodified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum, 100units/mL penicillin and 100 mg/mL streptomycin in a humidified incubator at 37 �C,5% CO2.

2.7.2. Cell viability and proliferationIn vitro cytotoxicities of Au NRs-MMSNEs against breast cancer MCF-7 cells were

evaluated based on the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) assays. Cancer cells were seeded in 96-well plate at a density of 2 � 104

cells in 100 mL DMEM for 8 h. The mediumwith different concentrations of Au NRs-MMSNEs (0e400 mg/mL) were added into the 96-well plate, and the cells werecultured for 24 h. After extraction of medium in wells, MTT with a concentration of0.7 mg/mL in 100 mL DMEMwere added, and the cells continued to be incubated for4 h. Then DMSO of 100 mL/well was added to replace the medium, and theabsorption strength of every well at 490 nm was recorded by using a microplatereader (Bio-Tekn Elx 800).

2.7.3. Photo-thermal treatmentCancer cells were seeded in 96-well plate at a density of 2 � 104 cells in 100 mL

DMEM for 8 h. The DMEM medium with different concentrations of Au NRs-MMSNEs (0, 40, 60 and 80 mg/mL) were added into the 96-well plate, and the

tion rate 1/T2 of Au NRs-MMSNEs as a function of Fe concentration; (c) In vivo MRI ofNRs-MMSNEs dispersed in water before (left) and after (right) an external magnetic

Fig. 4. (a) UVevis absorption spectra of initial 0.5 mg/mL of DOX PBS solution andsupernatant DOX solution after loading into Au NRs-MMSNEs. (b) In vitro releaseprofiles of Au NRs-MMSNEs-DOX using dialysis membrane against PBS solution at pH7.4 and 5.5.

M. Ma et al. / Biomaterials 33 (2012) 989e998 993

cells were subsequently cultured for 4 h. The photothermal treatment was per-formed using a 808 nm high power multimode pump laser (Shanghai Connet FiberOptics Company). The beam diameter was 5 mm and the power density of lasersource was fixed at 2.0 W cm�2. The cells were exposed under the 808 nm laser for5 min, and then back incubated for another 20 h. The cell viability was measuredusing MTT assay and the necrotic cell death immediately after irradiation weredetected by propidium iodide (KeyGen) staining method according to the manu-facturer’s instruction.

2.8. Bio-TEM and confocal fluorescence microscopy images of cells uptake

The MCF-7 cells were incubated in 10 mL of DMEM at a density of 2 � 104 cellsfor 4 h, and then 10 mL of DMEM with 20 mg/mL Au NRs-MMSNEs were added toreplace the initial medium. The cells were cultured for 4 h at 37 �C, 5% CO2, and thecells were collected by centrifugation at 1500 rpm for 5 min. After removingsupernatant solution, the cells were fixed by adding 2.5% glutaraldehyde solution.The fixed sample was treated with phosphate buffer solution and dehydrated withgraded ethanol and propylene oxide. The sample were embedded in EPSM812 andpolymerized in the oven at 37 �C for 12 h. Ultrathin sections were cut using a dia-tome diamond knife on a Leica UC6 ultramicrotome and observed by JEM-1230electron microscopy. Besides, the localizations of DOX were directly observed bythe Olympus Confocal Microscope (FluoView� FV1000).

2.9. In vivo MR and infrared thermal imaging

Animal procedures were in agreement with the guidelines of the InstitutionalAnimal Care and Use Committee. Walker 256 cells (5�106 cell/site) were implantedsubcutaneously into SD mice. The anesthetized mouse was intratumoral-injectedwith 0.5 mL PBS solution containing 1.4 mg/mL Au NRs- MMSNEs. After 0.5 h, theMR imaging was measured on a clinical 3.0 T MRI instrument (GE Signa 3.0T) by thesequences as follows: field of view FOV ¼ 12 cm2, thickness ¼ 4 mm,spacing ¼ 0.5 mm, NEX ¼ 1, TR ¼ 4000 ms, TE ¼ 13, 26, 39, 52. TE ¼ 13, 26, 39, 52.In vivo infrared thermal imaging was performed using the same optical sourcementioned above. The anesthetized mouse was intratumoral-injected with 0.5 mLAu NRs-MMSNEs PBS solution (1.4 mg/mL), then the tumor were irradiated atdifferent laser power inputs. The distance between the beam head and the surface oftumor was about 2 cm. Temperature distribution of the tumor sites were detectedusing a thermal camera (FLIR T390) for different time periods.

3. Results and discussion

3.1. Materials synthesis, structure and cytotoxicity

The MMSNEs were synthesized following the previously re-ported method [19,20]. The double-shelled core/shell nano-structure Fe2O3@SiO2@mSiO2 was fabricated and followed byhydrogen reduction to transform Fe2O3 core to Fe3O4. The core/shellstructure can be clearly seen in Fig. 1b. The morphology ofFe3O4@SiO2@mSiO2 is ellipsoidal as inherited from the initialspindle hematite template. TEM image shows that the core/shellnanostructure is uniform with the overall particle size ofca.300 � 180 nm, and hematite core size of ca.200 � 80 nm. Thesurface of mesoporous silica was reacted with 3-aminopropyltriethoxysilane (APS) for amino functionalization.The FT-IR spectra (Figure S1) shows the appearance of NeH band(1460 cm�1), CeH band (2900 cm�1), and the decrease of SieOHband (960 cm�1), which demonstrated that the surface of meso-porous silica have been partially functionalized with amine group.After post-modification with APS, the surface zeta potential ofFe3O4@SiO2@mSiO2 changed from (�43.63 � 0.72) mV to(�11.1 � 0.75) mV.

Au NRs of ca.40 � 15 nm in length � diameter were synthesizedthrough the seed-mediated approach as previously reported[21e23]. To functionalize Au NRs with carboxyl groups, PEG aciddisulfide (Fig. 1a) molecules were used in order to enhance thebiocompatibility and higher solubility in aqueous solution. Thesurfactant CTAB molecules play an important role in stabilizing AuNRs [6e8], which form a bilayer on the surface of gold surface.However, CTAB are severely toxic to cells [24]. To remove all theCTAB molecules, the gold naonorods were firstly mixed withmPEG5000eHS (long chain PEG molecule), used as a temporary

steric barrier to avoid the irreversible aggregation among Au NRsduring severe replacement reaction [25], followed by the robustreplacement of the residue CTAB using plenty of PEG acid disulfidemolecules.Wemeasured the surface electric potentials of Au NRs ineach step of Au NRs-CTAB (þ41.4 mV), Au NRs-PEG5000 (�14.4 mV)and Au NRs-PEG-COOH (�24.9 mV), respectively, and the lowestsurface electric potentials of Au NRs-PEG-COOH showed that PEGdisulfide molecules were successfully bonded onto the surface ofAu NRs (Table S1). The negligible amount of Br element in resultingAu NRs-PEG-COOH solution demonstrated the completely removalof toxic CTAB molecules by this route (Table S2). After reaction,a dense PEG molecule layer (w1 nm in thickness) formed on thesurface of Au NRs, as seen in Fig. 2c. The Au NRs could be welldispersed in acidic, alkaline and salt solutions for more than 4months.

Au NRs were activated by the N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide(NHS) in MES buffer solution, followed by self-assembly on theexterior surface of Fe3O4@SiO2@mSiO2 by direct chemical reactionbetween terminal succinimidyl ester on Au NRs and amino groupson silica surface. Each nanoellipsoid contains multiple Au NRs, andthe final molar ratio for Au:Si:Fe is 6:9:1 by inductively coupled

M. Ma et al. / Biomaterials 33 (2012) 989e998994

plasma atomic emission spectrometry (ICP-AES). No free goldnanorods were detected in the supernatant liquid after centrifu-gation at 6000 rpm, showing effective conjugation of nanorods onthe Fe3O4@SiO2@mSiO2. In the end, the residue amine groups onthe exterior surface of Au NRs-MMSNEs reacted withmPEG2000eNHS to minimize the aggregations and prevent non-specific protein adsorption during the blood circulation. Au NRs-MMSNEs in aqueous media had a mean diameter of 386.6 nmwith a unimodal size distribution, as measured by DLS (Figure S2).As expected, Au NRs-MMSNEs are non-toxic for MCF-7 cells ata concentration up to 100 mg/mL by MTT assay (Figure S3), asa result of completely removing the CTAB bilayers on the nanorods.This reaction provides a facile method to conjugate highly disper-sive Au NRs on the exterior surface of Fe3O4@SiO2@mSiO2, thusinhibiting the decrease of longitudinal plasmon resonanceabsorption in the NIR region which is caused by the irreversibleaggregations among the Au NRs. The UVevis absorption spectra(Fig. 2) of Au NRs-MMSNEs display a maximum absorption peak at790 nm, with a slightly broadened adsorption peak in comparisonwith initial free Au NRs dispersed in CTAB solution, due to thevariation of local dielectric field around the Au NRs.

3.2. In vitro and in vivo magnetic resonance imaging and celloptical imaging

The magnetic property of Au NRs-MMSNEs is shown in Fig. 3dand Fig. S4. Such a type of PEG coated magnetic core/mesoporoussilica shell drug delivery system showed highly efficiency forcarrying anti-cancer drug to the designated organs using an

Fig. 5. (a) Selected bio-TEM images of the Au NRs-MMSNEs’ uptake by MCF-7 cells, showing(a-1), then the formation of endosomes containing the aggregations of Au NRs-MMSNEsenlarged image in Figure a-1 shows the Au NRs are still capping MMSNEs in the cytoplasm. (MMSNEs-DOX for 4 h: blue fluorescence shows the nuclei stained with DAPI (b-1) and red fl

(b-2).

external magnetic field, which was recently reported by our group[26]. Meanwhile, Au NRs-MMSNEs could also be used as a T2-weighted MRI contrast agent. The incremental signal intensity isindicated by the enhanced darkness at increased Fe concentrationfrom 0 to 0.032 mM (Fig. 3a). A high T2 relaxivity coefficient (r2)value of 393.8 mM

�1s�1 was measured on a clinical 3.0 T MRIinstrument for Au NRs-MMSNEs (Fig. 3b). In addition, the tumorcarried in mouse was also clearly visible by in vivo MR imaging asdemonstrated in Fig. 3c. The tumor exhibits an apparently dark-ened area at the injection site compared to the control, in 0.5 h afterintratumoral injection of Au NRs-MMSNEs, showing the potentialof Au NRs-MMSNEs for in vitro MRI labeling use.

Au NRs can be used for cell optical imaging based on its highlight scattering rate, and the present Au NRs-MMSNEs also showeda clear dark-field optical imaging capability for MCF-7 cells asillustrated in Figure S5.

3.3. DOX loading and pH-responsive release

The DOX drug molecules can be readily loaded into the meso-pore network in the mesoporous silica shell of Au NRs-MMSNEs bysimply mixing them in the phosphate-buffered saline (PBS) solu-tion of DOX, as evidenced by the significant UVevis absorptiondecrease of a 0.5 mg/mL PBS solution of DOX (Fig. 4a), thanks to thelarge surface area of the mesoporous silica (Figure S6). The Au NRs-MMSNEs could encapsulate a high amount of DOX up to 30 wt%.The release rate of DOX from Au NRs-MMSNEs using dialysismembrane against PBS solution at pH 7.4 and 5.5 (Fig. 4b) wasevaluated to simulate neutral environment of blood circulation and

the cell membrane invagination of Au NRs-MMSNEs before crossing into the cytoplasm(a-2), subsequently the Au NRs-MMSNEs’ escape from the endsomes (a-3). The insetb) Confocal fluorescent microscopic images of MCF-7 cells after incubated with Au NRs-uorescence shows the location of DOX (b-2); and (b-3) is the superposition of (b-1) and

M. Ma et al. / Biomaterials 33 (2012) 989e998 995

acidic one in cellular endosomes, respectively. The DOX-loaded AuNRs-MMSNEswere almost inert in PBS at pH 7.4, only less than 5wt% of DOX was released in 20 h. However, when the pH value wasdropped to 5.5, DOX was quickly released to w40 wt% in 2 h, andw50 wt% in 6 h. This pH-responsive controlled-release property

Fig. 6. (a) Temperature of DMEM medium versus exposure time for MCF-7 cells treated witviabilities for MCF-7 cells treated with different concentrations of Au NRs-MMSNEs after inclaser irradiation are plotted as the control. (c) Representative confocal fluorescent microscirradiation for 5 min, necrotic MCF-7 cells were instantaneously stained by propidium iodi

can be attributed to the dissociation of electrostatic interactionbetween positively charged DOX molecules and negatively chargedsilanols of Au NRs-MMSNEs, due to the protonation of silanols atthe decreased pH value [27,28]. This property can effectivelyprevent the drug leakage during the blood circulation but increase

h different concentrations of Au NRs-MMSNEs under 2 Wcm�2 NIR irradiation. (b) Cellubation for 24 h, after the laser exposure for 5 min. The untreated MCF-7 cells but withopic images of MCF-7 treated with different concentrations of Au NRs-MMSNEs afterde (PI) (the scale bars are 50 mm).

M. Ma et al. / Biomaterials 33 (2012) 989e998996

the delivery efficacy in cancer cells, which is muchmore acidic thannormal cells and therefore such a pH-responsive drug releasefeature will be of great significance in the treatment of tumortissues [29].

3.4. Cell uptake

The endocytosis process and the distribution of Au NRs-MMSNEs in cytoplasm could be observed by bio-TEM imaging(Fig. 5a). Firstly, part of Au NRs-MMSNEs could be found near thecell membrane, inducing the cell membrane invagination beforethe Au NRs-MMSNEs cross the membrane into the cytoplasm, asmarked by the circle in Figs. 5a-1. Then, the Au NRs-MMSNEs arephagocytized by the cell and trapped into the intracellular endo-somes (Figs. 5a-2), and finally, escape from the endosomes into thecytoplasm (Figs. 5a-3). The enlarged image shows the maintainedmorphology of nanocomposites, indicative of the firm covalentbonding between gold rods and silica nanoellipsoids.

Fig. 4b shows confocal microscopy images of breast cancerMCF-7cells incubatedwithAuNRs-MMSNEs-DOX for 4 h, and then speciallymarked with DAPI for nuclei staining with blue fluorescence(Figs. 5b-1). Redfluorescence comes fromDOX (Figs. 5b-2), indicativeof the location of Au NRs-MMSNEs-DOX. The red purple signals inFigs. 5b-3,which is the superposition of the red and blue, suggest that

Fig. 7. Infrared thermal imaging under the photothermal heating by 808 nm laser irradiatio(b) 2 Wcm�2 irradiations, and (c) PBS solution-injected tumor under 2 Wcm�2 irradiation.

DOX, which is released quickly from the drug carriers Au NRs-MMSNEs in the acidic environment of cytoplasm, largely enteredinto the nucleolus. It has beenknown that the intracellular locationofDOX is within the nucleus, and delivery of DOX into cancer cells andaccumulation in the nucleus can enhance its anti-tumor activity [30].In short, all the analysis of bio-TEM, confocal fluorescence and dark-field images (Figure S5) confirms the highly efficient uptake of AuNRs-MMSNEs and Au NRs-MMSNEs-DOX by MCF-7 cells.

3.5. In vitro and in vivo photothermal effect for infrared thermalimaging and thermo- anti-cancer therapy

To demonstrate the high photothermal conversion effect of AuNRs-MMSNEs, the MCF-7 cells incubated with a certain concen-tration of Au NRs-MMSNEs were exposed under the 808 nm laserradiation (Figure S7), which is very close to the longitudinal plas-mon resonance wavelength of Au NRs-MMSNEs, and the temper-ature increases were measured. It can be clearly seen from theFig. 6a that the medium containing higher amount of Au NRs-MMSNEs generated more significant temperature increases uponexcitation. For example, the sample of 160 mg/mL was heated up to55 �C in only 2 min. On the contrary, no apparent temperaturechange was detected for the blank medium without Au NRs-MMSNEs, because of the little light absorption by the solution.

n for different time periods in Au NRs-MMSNEs-injected tumor under (a) 1 Wcm�2 and

Fig. 8. A comparison of inhibition rates forMCF-7 cells treated by Au NRs-MMSNEs-NIR(purple), Au NRs-MMSNEs-DOX (red) and Au NRs-MMSNEs-DOX-NIR (green). For pho-tothermal treatment, the media were under 808 nm laser irradiation for 5 min atdifferent power intensities, corresponding to themaximum temperature increases to39,42 and 45 �C. The additive therapeutic efficacies (blue) of combined treatment at eachtemperature were calculated by using the relation of Tadditive ¼ 100-(fAu NRs-MMSNEs-

DOX� fAu NRs-MMSNEs-NIR)� 100, here f is the cell viability of each treatment. Each data barrepresents an average of five parallels. (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)

M. Ma et al. / Biomaterials 33 (2012) 989e998 997

Therefore, an infrared thermal imaging function of tumor can bemade by detecting the temperature changes induced by theinfrared irradiation using Au NRs-MMSNEs.

The cell damage was examined using cell proliferation assay tocheck the long-term effect of laser irradiation on the cells (Fig. 6b),which were stained by propidium iodide (PI) to detect the necroticcell death immediately after irradiation (Fig. 6c). Comparatively thelaser irradiation alone caused no cell damage for a control groupwhere no Au NRs-MMSNEs were added by cell proliferation assay,and the darkness of confocal fluorescence image shows that all thecells have survived after the laser exposure [31]. With the increaseof the concentration of Au NRs-MMSNEs, the cell viability distinc-tively decreased after incubation for 24 h. When the concentrationof Au NRs-MMSNEs was 40 mg/mL, several red dots appeared after5 min exposure, which represented the death cells caused bylocalized hyperthermia for bearing relatively higher accumulationof Au NRs-MMSNEs in the cells. Nevertheless, most cells were stillalive because of the limited temperature rise in the case. However,with the further increase of Au NRs-MMSNEs concentrations, e.g.,80 mg/mL, the higher temperature (up to 50 �C) was reached whichcaused significant thermal cell death instantaneously, and morethan 70%MCF-7 cells were killed in 24 h due to the high heat stresswhich destroyed the integrity of cell membrane and enzymes forthe synthesis of DNA [32].

After confirming the in vitro photothermal effect of Au NRs-MMSNEs, the in vivo heating efficacy was further investigatedthrough intratumoral injection of PBS solution dispersed with AuNRs-MMSNEs (500 mL, 1.4 mg/mL). At 0.5 h post-injection when AuNRs-MMSNEs had spread over almost the whole tumor, this AuNRs-MMSNEs-treated tumor was exposed to a 808 nm laser beamwith a focal diameter of 5 mm (Figure S8). At a power intensity of1 Wcm�2, the temperature in exposed region increased by up to3 �C, as compared to the body temperature of about 37 �C (Fig. 7a).When the power intensity reached 2 Wcm�2 (Fig. 7b), the focalregion was heated up to a maximum of 55 �C in only 1 min ofexposure time, such a photothermal effect could induce thermalcell death and cause irreversible tumor damage. Subsequently, thered circle (the area above 50 �C) broadened with time, due to thegradually thermal accumulation/diffusion in/from the exposedregion, and the stable temperature distribution was reached inabout 5 min. For comparison, a control sample of the Au NRs-MMSNEs-free tumor is shown in Fig. 7c, no apparent grads-distribution and variation of temperature can be found. Thisdemonstrates that Au NRs-MMSNEs are efficient agent for thepotential in vivo photothermal treatment.

3.6. Synergistic photothermo- and chemo anti-cancer therapeuticeffect

To compare the in vitro cytotoxicity of combined therapy witheach single one, the inhibition rates of DOX-loaded Au NRs-MMSNEs (named as Au NRs-MMSNEs-DOX) without 808 nmlaser irradiation, as well as Au NRs-MMSNEs and DOX-loaded AuNRs-MMSNEs (named as Au NRs-MMSNEs-NIR and AuNRs-MMSNEs-DOX-NIR, respectively) under 808 nm laser irradia-tion were measured by using cell proliferation assay (Fig. 8). Thethree groups have an equivalent concentration of Au NRs-MMSNEsparticles (60 mg/mL), and the DOX concentration in drug treatmentwas 25 mg/mL. After MCF-7 cells were incubated with AuNRs-MMSNEs-DOX for 24 h, the inhibition rate of single chemo-therapy was about 40%. For Au NRs-MMSNEs-NIR and AuNRs-MMSNEs-DOX-NIR, we performed laser exposure experimentsafter incubation for 4 h, the cell mediumwas heated to about 39, 42and 45 �C, by controlling the power intensity of irradiation at1.4, 1.8 and 2.2 Wcm�2, respectively. Here we use the additive

therapeutic efficacy (Tadd.), obtained from a relation ofTadd. ¼ 100�(fchemo � fthermo) � 100 (where f is the cell viability ofeach treatment), as a reference to evaluate the interaction potencybetween two treatment [33,34]. In the case of 1.4 Wcm�2 powerintensity input (39 �C of the cell medium), the measured inhibitionrate of the combined treatment was 54%, which was higher thanthe calculated Tadd. value (43%) according to above formula, indic-ative of the significant synergistic anti-cancer therapeutic effect ofAu NRs-MMSNEs-DOX-NIR. It can be clearly seen from Fig. 8, theenhanced therapeutic efficacy by Au NRs-MMSNEs-DOX-NIR isobvious in between 39 and 42 �C. When the medium temperaturewas up to 45 �C, the inhibition rate by the combined therapyshowed no increase compared to single photothermal treatment,and moreover, such a high temperature would generate side effectto healthy tissues around tumor. From the synergistic effectbetween the chemotherapy and thermotherapy of the drug-loadedAu NRs-MMSNEs-DOX-NIR, we could lower the dosage of DOX bysimply heating the tumor up to a moderate temperature, in thisway both the dosage-limiting toxicity of the drug and tissuedamage by superheating can be effectively prevented.

4. Conclusion

In summary, we have synthesized the Au NRs-MMSNEs asa multifunctional platform to combine the chemotherapy, photo-thermotherapy, MR imaging, dark-field optical and infraredthermal imaging into one system simultaneously. The resulting AuNRs-MMSNEs, having the negative surface charge, could release thepositively charged DOX in the acidic intracellular environment ofcancer cells in a pH-responsive way. Cell experiments and in vivothermal effect indicated the feasibility of using the Au NRs-MMSNEs in photothermal treatment under near-infrared irradia-tion. Importantly, a synergistic effect in killing cancer cells wasfound by the combined photothermo-/chemo- therapy undermoderate heating to 39e42 �C. Such a multifunctional drugdelivery system shows apparent advantages to cancer therapy overconventional individual themo- or thermo-therapeutic system.

M. Ma et al. / Biomaterials 33 (2012) 989e998998

Acknowledgement

We greatly acknowledge financial supports from the NationalNatural Science Foundation of China (Grant No. 50823007,50972154, 51072212), National Basic Research Program of China(973 Program, Grant No. 2011CB707905). We greatly acknowledgeShengjian Zhang for MRI observation.

Appendix. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biomaterials.2011.10.017.

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