Nanoscale

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aState Key Laboratory for Modication of

College of Materials Science and Engineerin

E-mail: hu.junqing@dhu.edu.cnbCollege of Chemistry and Chemical Engine

Science, Shanghai, ChinacDepartment of Orthopaedics, Shanghai Fi

University, 100 Haining Road, Hongkou DisdCenter of Super-Diamond and Advanced Fil

Materials Science, City University of Hong KeCollege of Chemistry, Chemical Engineering

Shanghai, China

† Electronic supplementary informa10.1039/c3nr06160d

‡ These authors contributed equally.

Cite this: Nanoscale, 2014, 6, 4361

Received 20th November 2013Accepted 5th February 2014

DOI: 10.1039/c3nr06160d

www.rsc.org/nanoscale

This journal is © The Royal Society of C

Cu2�xSe@mSiO2–PEG core–shell nanoparticles: alow-toxic and efficient difunctional nanoplatformfor chemo-photothermal therapy under nearinfrared light radiation with a safe power density†

Xijian Liu,‡ab Qian Wang,‡ac Chun Li,a Rujia Zou,ad Bo Li,a Guosheng Song,a

Kaibing Xu,a Yun Zhenge and Junqing Hu*a

A low-toxic difunctional nanoplatform integrating both photothermal therapy and chemotherapy for killing

cancer cells using Cu2�xSe@mSiO2–PEG core–shell nanoparticles is reported. Silica coating and further

PEG modification improve the hydrophilicity and biocompatibility of copper selenide nanoparticles. As-

prepared Cu2�xSe@mSiO2–PEG nanoparticles not only display strong near infrared (NIR) region

absorption and good photothermal effect, but also exhibit excellent biocompatibility. The mesoporous

silica shell is provided as the carrier for loading the anticancer drug, doxorubicin (DOX). Moreover, the

release of DOX from Cu2�xSe@mSiO2–PEG core–shell nanoparticles can be triggered by pH and NIR

light, resulting in a synergistic effect for killing cancer cells. Importantly, the combination of

photothermal therapy and chemotherapy driven by NIR radiation with safe power density significantly

improves the therapeutic efficacy, and demonstrates better therapeutic effects for cancer treatment than

individual therapy.

1 Introduction

Photothermal therapy (PTT) that uses optical absorbing agentsto “cook” cancer cells under light irradiation without damagingsurrounding healthy tissue has attracted signicant attention inrecent years as a promising alternative or supplement to tradi-tional cancer therapies.1–3 Ideal PTT agents should exhibitstrong absorbance in the near-infrared (NIR) region (l ¼ 700–1300 nm), which is a therapeutic window due to high opticaltransparency of biological tissues,4 and could efficiently transferthe absorbed NIR optical energy into heat. Furthermore, thePTT agents should have good biocompatibility.1,2 Currently, aseries of NIR-light-absorbing nanomaterials have shownpotential in PTT cancer treatment, such as gold nanorods,5,6

Chemical Fibers and Polymer Materials,

g, Donghua University, Shanghai, China.

ering, Shanghai University of Engineering

rst People's Hospital, Shanghai Jiaotong

trict, Shanghai, China

ms (COSDAF), Department of Physics and

ong, Hong Kong, China

and Biotechnology, Donghua University,

tion (ESI) available. See DOI:

hemistry 2014

gold shells,7,8 gold cages,9–12 palladium nanosheets,13,14 carbonnanotubes,15,16 graphene,17,18 polypyrrole,1,2 indocyanine dye,19

W18O49 nanowires,20 copper sulde,21–24 and copper selenidenanoparticles.25 However, one of the major limitations of usingPTT for clinical cancer treatment is the relatively high treatmentpower density of laser for the aim of obtaining sufficient heatingin cancer cell killing, which probably hurts normal tissues.26 Forclinical applications, the energy input of laser should be safe toavoid damage to healthy tissues and skin, which is always aprimary concern for PTT. If PTT agents can integrate photo-thermal therapy and other treatment modalities into a singlenanoplatform for cancer destruction, the treatment powerdensity of laser will be effectively reduced, and skin and normaltissues can be avoided fromharm. Numerous results showed thatthe combination of photothermal therapy and chemotherapyhad better effects on destroying cancer cells than chemotherapyor photothermal therapy alone due to the synergistic effect.For instance, Au@SiO2,27,28 Cu9S5@SiO2,29 hollow mesoporoussilica@Pd (HMSS-NH2@Pd),30 Pd@Ag@sSiO2@mSiO2-DihBen,31

graphene@mSiO2 (GSPI),32 DOX/ICG@lipid-polymer (DINPs)33

and Fe3O4@PPy34 nanocomposites have been fabricated andshowed remarkable cancer-cell killing efficiency. Therefore, thedevelopment of such novel multifunctional treatment systemscombining photothermal therapy and chemotherapy with a safeand efficient therapeutic effect is vital.

Recently, copper selenide has been supposed to be a prom-ising candidate as a photothermal agent due to high

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photothermal conversion efficiency and ease of synthesis.24,35

However, copper selenide is hydrophobic and less biocompat-ible when synthesized via hot injection. Though copper selenidewas coated by a polymer, a suspicious long-term toxicity invitro25 still limited its applications in in vivo therapy. Therefore,new methods of surface modication should be developed tofurther reduce the toxicity of copper selenide as PTT agents.Mesoporous silica nanoparticles (MSNs), which have shownexcellent stability, non-cytotoxicity, tunable porosity and facilemodication,30,36 appeal researchers to use them as a nano-carrier for multifunctional drug delivery in a drug controllablerelease area. Thus mesoporous silica coating is a very efficientway to reduce toxicity of photothermal materials, and to be usedas an anticancer drug carrier. So the combination of chemo-therapy and photothermal therapy in a platform will be ach-ieved by mesoporous silica coating.

In this work, we designed and prepared Cu2�xSe@mSiO2–PEGcore–shell nanoparticles by combining the hot injection reactionand the sol–gel reaction, and then by surface modication withPEGylation. Because the Cu2�xSe nanoparticles core can effec-tively convert NIR light into fatal heat due to the strong surfaceplasmon resonance (SPR) absorption, and the biocompatiblemesoporous silica shell can provide the carrier for loading theanticancer drug, as-prepared Cu2�xSe@mSiO2–PEG core–shellnanoparticles can be used as a low-toxic difunctional nanoplat-form integrating both photothermal therapy and chemotherapyfor killing cancer cells under NIR radiation. In particular, the lowpH in cellular endosomes and the heat generated by the NIRirradiation can lead to rapid release of the DOX moleculesloaded inside the mesoporous SiO2 shells and enhance thechemotherapeutic effects of DOX. Cu2�xSe@mSiO2–PEG core–shell nanoparticles as a difunctional nanoplatform for chemo-photothermal therapy have at least three important features: (1)unlike chemo-photothermal therapy nanoplatforms based onnoble-metal nanostructures that are limited by the high cost ofthe noble-metal, the Cu2�xSe@mSiO2–PEG nanoplatform isbeneted from its low cost and simple synthesis route. (2) TheCu2�xSe@mSiO2–PEG nanoplatform has an absorbance peakclose to 980 nm, and guarantees high photothermal conversionability when driven by the 980 nm laser. Also, the 980 nm lighthas deeper penetration depth in biological tissues than 808 nmlight, and the Cu2�xSe@mSiO2–PEG nanoplatform shows a goodPTT effect when is driven at a safe power density of 0.72 W cm�2

(higher than the 808 nm laser limit intensity set).23,24,37,38 (3) TheCu2�xSe@mSiO2 composites are modied by PEG, improvingtheir colloidal stability and biocompatibility and decreasingimmunogenicity in in vivo therapy.28,29 In in vitro and in vivoexperiments, the combination of photothermal therapy andchemotherapy signicantly improves the therapeutic efficacy,and displays better therapeutic effects for cancer treatment thanindividual therapy.

2 Experimental section2.1 Chemicals and reagents

All reagents were used without further purication. Copper (I)chloride (CuCl), cetyltrimethylammonium bromide (CTAB),

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sodium hydroxide (NaOH) and anhydrous ethanol are analyti-cally pure and were purchased from Sinopharm ChemicalReagent Co. (Shanghai, China), and tetraethylorthosilicate(TEOS, GR), selenium powders, oleic acid, and oleylamine(approximate C18 from 80–90%) were obtained from Aladdin,and 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (PEG–silane, MW ¼ 596–725 g mol�1, 9–12 EO) was obtainedfrom Gelest (Morrisville, PA) and doxorubicin hydrochloride(DOX) was obtained from Huafeng United Technology CO., Ltd.(Beijing, China).

2.2 Characterization

Sizes, morphologies, and microstructures of the nanoparticleswere determined by using a transmission electron microscope(TEM; JEM-2100F). Powder X-ray diffraction (XRD) was con-ducted by using a D/max-2550 PCX-ray diffractometer (Rigaku,Japan). The surface area, pore size, and pore-size distribution ofthe products were determined by Brunauer–Emmett–Teller(BET), nitrogen adsorption–desorption and Barrett–Joyner–Halenda (BJH) methods (Micromeritics, ASAP2020). Fouriertransform infrared (FTIR) spectra were measured using anIRPRESTIGE-21 spectrometer (Shimadzu) using the KBr pressedpellets. UV-visible absorption spectra were measured on aUV-vis 1901 spectrophotometer (Phoenix) using quartz cuvetteswith an optical path of 1 cm. The content of copper ions and Auions in the solution was determined by using a Leeman Labo-ratories Prodigy high-dispersion inductively coupled plasmaatomic emission spectrometer.

2.3 Synthesis of Cu2�xSe nanocrystals

Cu2�xSe nanocrystals were prepared by a modied thermalinjection method described previously.35 In a typical procedure,39.5 mg of selenium powders and 5 mL of oleic acid (OA) in aask were heated at 120 �C with owing nitrogen for 30 min inorder to remove any moisture and oxygen and subsequentlywere heated to 280 �C under magnetic stirring for 30 min. ThenSe–OA precursor was synthesized for the following use. Inanother ask, mixtures of 5 mL of OA, 5 mL of oleylamine(OAM) and 49.5 mg of CuCl were heated at 120 �C with owingnitrogen for 30 min and subsequently were heated to 220 �Cunder magnetic stirring. Then 5 mL of the Se–OA precursor wasinjected into the above copper precursors, forming a darksolution immediately, and then was maintained at 220 �C for 5min. Subsequently the reaction solution rapidly cooled to 60 �C.Then ethanol was added to the reaction solution followed bycentrifuging at 12 000 rpm for 10 min. This procedure wasrepeated twice to remove residual surfactants. Finally, theCu2�xSe nanocrystals were dispersed in 10 mL of chloroform forlater use.

2.4 Synthesis of CTAB-stabilized Cu2�xSe nanocrystals(Cu2�xSe–CTAB)

A phase transfer of as-prepared Cu2�xSe nanocrystals fromchloroform to water proceeded by a modied method describedpreviously.29,39 First, 10 mL of the above chloroform solutioncontaining the as-prepared Cu2�xSe nanocrystals was mixed

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with CTAB solution (1.5 g CTAB in 75 mL of water). By vigorousstirring at 40 �C for 36 h, a brown oil-in-water microemulsionformed, and then the chloroform was completely boiled off byrotary evaporation. The above solution was ltered through a0.22 mm of millipore lter to remove impurities and thendiluted to 75 mL with deionized water, forming a transparentdark green solution of Cu2�xSe–CTAB. In order to preventprecipitation of CTAB, the ltration and storage of Cu2�xSe–CTAB solution should be done above 25 �C.

2.5 Synthesis of Cu2�xSe@mSiO2–PEG core–shellnanoparticles

The above 50 mL of the Cu2�xSe–CTAB solution was treated byultrasonication and further continuously stirred for 1 h at 40 �C.Then, 3 mL of ethanol and 100 mL of NaOH solution (30 mgmL�1) were added to the above solution. Aer stirring forseveral minutes, 100 mL of TEOS was dropped into this mixture.The mixture was allowed to react for 1.5 h at 40 �C undercontinuous stirring. Subsequently, 100 mL of PEG–silane wasadded under stirring for another 6.5 h and a Cu2�xSe@mSiO2–

PEG core–shell nanoparticle mixture was obtained. Thesynthesized product was centrifuged (12 000 rpm, 10 min) andwashed with ethanol three times. Then the products weretransferred to the ethanol solution of NH4NO3 (50 mL, 10 mgmL�1) and stirred at 50 �C for 2 h, the template of CTAB wasremoved through ion exchange. Aer that, as-prepared Cu2�x-Se@mSiO2–PEG nanoparticles were washed with ethanol threetimes. The nal products were dispersed into deionized waterand stored at 4 �C for further use.

2.6 Measurement of photothermal performance

Measurement of photothermal performance was referenced toour previous method.29 The Cu2�xSe@mSiO2–PEG nano-particles and Au nanorod aqueous dispersions (0.3 mL) ofdifferent concentrations (10, 25, 50, 100, 200, and 400 mgmL�1)were irradiated by laser (980 nm) for 8 min. The light source wasan external adjustable power (0–0.3 W) 980 nm semiconductorlaser device with a 5 mm diameter laser module (Xi'an ToursRadium Hirsh Laser Technology Co., Ltd. China). The outputpower density was independently calibrated using a handyoptical power meter (Newport model 1918-C, CA, USA) and wasfound to be �0.72 W cm�2. The temperature of the solutionswas measured by using a digital thermometer (with an accuracyof 0.1 �C) with a thermocouple probe every 5 s.

2.7 DOX loading and in vitro release

5 mg of Cu2�xSe@mSiO2–PEG nanoparticles were dispersed in10 mL of PBS solution (pH 7.4) containing DOX (0.15 mg mL�1).The mixture was stirred at room temperature for 48 h underdark conditions. The DOX-loaded Cu2�xSe@mSiO2–PEG(Cu2�xSe@mSiO2–PEG/DOX) nanocomposites were collected bycentrifugation at 12 000 rpm for 12 min and were washedseveral times with water until the supernatant became colorless.All supernatants were collected together. The amount of loadedDOX for Cu2�xSe@mSiO2–PEG was analyzed by using a UV-visspectrophotometer at 482 nm and calculated as below.

This journal is © The Royal Society of Chemistry 2014

Encapsulation efficiency ¼ (weight of DOX loaded into theCu2�xSe@mSiO2–PEG)/(initial weight of DOX), loading content¼ (weight of DOX loaded into the Cu2�xSe@mSiO2–PEG)/(weight of the Cu2�xSe@mSiO2–PEG + DOX loaded into theCu2�xSe@mSiO2–PEG). The release behaviors were monitoredat room temperature. Above-prepared Cu2�xSe@mSiO2–PEG/DOX complexes were dispersed in 5 mL of buffer solution withultrasonication at pH values of 7.4 and 4.8 respectively, andstirred at room temperature. At selected time intervals, thenanomaterial solutions were centrifuged (12 000 rpm, 10 min)and supernatants were withdrawn for analysis of DOX and thesame volume of fresh buffers was added back to the residualnanomaterials. The amounts of the released DOX in thesupernatant solutions were analyzed by using a UV-vis spec-trometer at 482 nm. To study if the DOX release from thenanoparticles could increase under laser irradiation, releasemedia solutions were irradiated with the NIR laser (980 nm,0.72 W cm�2) for 5 min at predetermined time intervals, andthen centrifuged and supernatants were withdrawn for analysisof DOX.

2.8 Cell culture

Cell culture was a referenced method reported in the litera-ture.22,23,40 Human cervical carcinoma cell lines (HeLa) werecontinuously grown in 50 mL culture asks, incubated byDMEM medium supplemented with 10% heat-inactivated fetalbovine serum, 100 mg mL�1 streptomycin, and 100 U mL�1

penicillin in a humidied incubator under 5% CO2 at 37 �C.

2.9 In vitro cytotoxicity of the Cu2�xSe@mSiO2–PEGnanoparticles

The in vitro cytotoxicity was measured using the MTT assay inthe human cervical carcinoma cell line.22,28 HeLa cells wereplated into a 96-well plate (1 � 104 cells per well) in a completemedium at 37 �C and 5% CO2 for 24 h before the experiments.The culture mediumwas replaced and cells were incubated withthe complete medium containing the Cu2�xSe@mSiO2–PEGcore–shell nanoparticles at a series of concentrations at 37 �Cwith 5% CO2 for further 24 h. Then the culture medium wasreplaced with fresh medium and MTT solution (10 mL, 5 mgmL�1) was added to each well of the culture plate, and the cellswere incubated in the CO2 incubator for another 4 h. The cellsthen were lysed by the addition of 100 mL of DMSO. The spec-trophotometric absorbance of formazan was measured using aplate reader at 570 nm. Four replicates were done for eachtreatment group.

2.10 In vitro chemo-photothermal therapy

HeLa cells were plated into a 96-well plate (1 � 104 cells perwell) in a complete medium at 37 �C and 5% CO2 for 24 h beforethe experiments. The culture medium was replaced withcomplete medium containing DOX, Cu2�xSe@mSiO2–PEG/DOXand Cu2�xSe@mSiO2–PEG at an equivalent DOX concentration.Cu2�xSe@mSiO2–PEG and Cu2�xSe@mSiO2–PEG/DOX haveequivalent Cu+ concentration. Aer 4 h of incubation, the cellswere washed with PBS to remove the unbound material and

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Fig. 1 Schematic illustration of Cu2�xSe@mSiO2–PEG core–shellnanoparticle preparation and application as a difunctional treatmentplatform for cancer treatment.

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replaced with fresh medium. The cells were treated with orwithout 980 nm laser (0.72 W cm�2) irradiation for 5 min,respectively, and then incubated at 37 �C with 5% CO2 forfurther 20 h. MTT assay was also carried out to quantify the cellviabilities. Four replicates were done for each treatment group.

2.11 In vivo chemo-photothermal therapy

The chemo-photothermal therapy test of cancer cells in vivo wasrespectively carried out by a modied method similar to ref. 20and 29. The osteosarcoma bearing mice were from ShanghaiTenth People's Hospital. When the tumors inside the mice hadgrown to 5–10 mm in diameter, the mice were randomly allo-cated into three groups. The mice were rst anaesthetized bytrichloroacetaldehyde hydrate (10%) at a dosage of 40 mg kg�1

body weight. Then the mouse in group A was intratumorallyinjected with 0.10 mL of phosphate-buffered saline (PBS) solu-tion containing Cu2�xSe@mSiO2–PEG nanoparticles (400 mgmL�1), the mouse in group B was intratumorally injected with0.10 mL PBS solution containing Cu2�xSe@mSiO2–PEG/DOXnanocomposites (400 mg mL�1), while group C was intra-tumorally injected with 0.1 mL saline solution. Aer 1 h, theinjected areas of mice from three groups were perpendicularlyirradiated by the 980 nm wavelength laser devices at 0.72 Wcm�2 for 5 min. The temperature of the tumor surface wasmeasured by a photothermal therapy-monitoring system GX-A300 (Shanghai Guixin Corporation). The mice were killed 8 haer photothermal therapy, and tumors were removed,embedded in paraffin, and cryo-sectioned into 4 mm slices. Theslides were stained with hematoxylin–eosin (H&E). The sliceswere examined under a Zeiss Axiovert 40 CFL inverted uores-cence microscope, and images were captured with a Zeiss Axi-oCam MRc5 digital camera.

2.12 In vivo antitumor effect

The osteosarcoma bearing mice were from Shanghai TenthPeople's Hospital. When the tumor volume inside the micereached �85 mm3, the mice were randomly allocated into fourgroups and intratumorally injected with 150 mL of PBS, DOX,Cu2�xSe@mSiO2–PEG, and Cu2�xSe@mSiO2–PEG/DOX solu-tions (DOX 5 0 mg mL�1, Cu2�xSe@mSiO2–PEG 320 mg mL�1).Aer injection, tumors were irradiated with NIR light (980 nm,0.72 W cm�2) for 10 min. Tumor sizes were monitored every twodays for 10 days. The length and width of the tumors weremeasured by using a digital vernier caliper. The tumor volumewas calculated according to the following formula: width2 �length/2. All animal experiments were carried out according toprotocols approved by the institutional committee for animalcare and also in accordance with the policy of the NationalMinistry of Health.

3 Results and discussion

Fig. 1 illustrates the synthesis process of the Cu2�xSe@mSiO2–

PEG core–shell nanoparticles and the theranostic principlebased on the unique properties of Cu2�xSe@mSiO2–PEG forcancer treatment. First, Cu2�xSe nanocrystals as photothermal

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agents were synthesized, and coated by mesoporous silicaforming Cu2�xSe@mSiO2 core–shell nanoparticles. In order toenhance the biocompatibility and stability of Cu2�xSe@mSiO2 inaqueous solution, Cu2�xSe@mSiO2 nanoparticles were furthermodied by PEG–silane on their outmost surfaces, formingCu2�xSe@mSiO2–PEG core–shell nanoparticles. The anticancerdrug DOX was loaded on the mesoporous silica shell within theCu2�xSe@mSiO2–PEG core–shell nanoparticles, forming Cu2�x-Se@mSiO2–PEG/DOX nanocomposites. The Cu2�xSe@mSiO2–

PEG/DOX nanocomposites were internalized by cancer cells dueto their good features. The Cu2�xSe cores inside nanocompositesoperate as NIR light absorbing agents, which can effectivelyconvert NIR light into heat resulting in rapid release of DOX, thusdual therapeutic modes (photothermal therapy and chemo-therapy) can be achieved by NIR radiation and result in a syner-gistic effect for killing cancer cells.

The hydrophobic Cu2�xSe nanoparticles were rst synthe-sized according to a reported protocol35 with some modica-tions. The transmission electron microscopy (TEM) images ofthe obtained Cu2�xSe nanocrystals showed an average diameterof �12 nm and high crystallinity (Fig. 2a and S1a†). Then,hydrophobic Cu2�xSe nanoparticles were transferred into anaqueous phase by utilizing cetyltrimethylammonium bromide(CTAB) to endow the hydrophilic property and facilitate SiO2

coating. In the silica-coating process, CTAB formed a bilayeraround the Cu2�xSe nanocrystals and acted as an organictemplate for preparing Cu2�xSe@mSiO2 core–shell nano-particles. The Cu2�xSe@mSiO2–PEG core–shell nanoparticles(x is 0.42 measured by ICP) were obtained by modifyingCu2�xSe@mSiO2 nanoparticles with PEG–silane. Based on TEMimages of the fabricated Cu2�xSe@mSiO2–PEG core–shellnanoparticles (Fig. 2b and c and S1b and c†), the Cu2�xSenanocrystals are completely encapsulated into a mesoporousSiO2 shell with an estimated thickness of �10 nm, and Cu2�x-Se@mSiO2-PEG nanoparticles have a diameter of �32 nm.

The phase structures of the as-obtained Cu2�xSe nano-crystals and Cu2�xSe@mSiO2–PEG nanoparticles were

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Fig. 2 TEM images of (a) Cu2�xSe and (b and c) Cu2�xSe@mSiO2–PEG.(d) XRD patterns of the standard Cu2�xSe (lower), as-synthesizedCu2�xSe (middle) and Cu2�xSe@mSiO2-PEG (upper). (e) Absorptionspectra of Cu2�xSe-CTAB and Cu2�xSe@mSiO2–PEG, the inset showsa photograph of Cu2�xSe@mSiO2–PEG dispersion solution. (f) N2

adsorption–desorption isotherms (inset: the pore diameter distribu-tion) of Cu2�xSe@mSiO2–PEG core–shell nanoparticles.

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examined by the XRD pattern, as shown in Fig. 2d. Several well-dened characteristic peaks such as (1, 1, 1), (2, 2, 0) and (3, 1, 1)exhibit the cubic phase, referenced by the standard Cu2�xSephase (JCPDS card no: 06-0680), while Cu2�xSe@mSiO2–PEGnanoparticles have a wide and weak peak at about 23�which isclearly due to amorphous silica coating. The Cu2�xSe andCu2�xSe@mSiO2–PEG nanoparticles were further investigatedby high-resolution TEM (HRTEM). As shown in Fig. S1a and d,†the interplane-distance of (111) crystal planes is 0.330 nm,corresponding to the standard cubic Cu2�xSe phase and otherresearch studies.41 It was proved that the Cu2�xSe had a cubicphase both before and aer the silica coating. Since the cubicphase is more stable for Cu2�xSe,42 it is advantageous to useCu2�xSe to acquire a stable photothermal effect in the photo-thermal therapy process. Absorption spectra of as-obtainedCu2�xSe–CTAB and Cu2�xSe@mSiO2–PEG are shown in Fig. 2e.Both Cu2�xSe–CTAB and Cu2�xSe@mSiO2–PEG nanoparticlesshow strong NIR region absorption. The as-prepared Cu2�xSe–CTAB nanoparticles have an absorbance peak at 1012 nm. Aersilica-shell coating, they exhibit a small red-shi (�24 nm). Thisphenomenon could be explained by the fact that the refractiveindex of the silica shell is larger than water.28 The absorbancepeak of Cu2�xSe@mSiO2–PEG is close to the wavelength (980nm) of excitation laser, which is advantageous for full usage ofthe SPR effect of materials, and enhancing photothermal effi-ciency.43 Fig. 2f shows the N2 adsorption–desorption isothermand pore size distribution (inset) of Cu2�xSe@mSiO2–PEG. The

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isotherm of Cu2�xSe@mSiO2–PEG aer CTAB extractiondisplays a typical type IV feature. The BET (Brunauer–Emmett–Teller) surface area and total pore volume of the core–shellnanoparticles were measured to be 579 m2 g�1 and 1.15 m3 g�1

respectively. The pore size distribution (Fig. 2f, inset) exhibits asharp peak centered at a mean value of 2.8 nm. Thus, Cu2�x-Se@mSiO2–PEG core–shell nanoparticles have large a surfacearea and appropriate pore size, which are favorable for drugloading.

PEG modication of the silica shells can improve colloidalstability, decrease immunogenicity and enhance the tumortargeting efficiency for in vivo applications,28,44 thus the Cu2�x-Se@mSiO2 core–shell nanoparticles were further graed withPEG–silane on their outmost surfaces via covalent bonding,forming the Cu2�xSe@mSiO2–PEG core–shell nanoparticles.The Fourier transform infrared (FTIR) spectra of the Cu2�x-Se@mSiO2–PEG core–shell nanoparticles and the PEG–silanereactant are shown in Fig. S2.† The adsorption bands at 2980–2845 cm�1 and adsorption peaks at 1390 cm�1 are assigned tothe stretching vibrations and deformation vibration of methy-lene (CH2) respectively, in the long alkyl chain of the backboneof PEG.45 Compared to the PEG–silane reactant, the Cu2�x-Se@mSiO2–PEG nanoparticles have similar adsorption bands atthe same area, suggesting that the PEG is successfully graedon the mesoporous silica surface. Besides, the Cu2�xSe@mSiO2–PEG core–shell nanoparticles show excellent colloidalstability. Aer a period of 7 days, there is no aggregation andnegligible absorption reduction in the NIR region during thestorage of Cu2�xSe@mSiO2–PEG core–shell nanoparticles inwater (Fig. S3†).

As-prepared Cu2�xSe@mSiO2–PEG nanoparticles show astrong NIR absorption feature, making them a potential pho-tothermal therapy agent. To investigate the photothermal effectgenerated by NIR laser irradiation, the temperatures of thesolutions containing various concentrations of Cu2�xSe@mSiO2–PEG were measured under irradiation of 980 nm laserwith a safe power density (0.72W cm�2). As shown in Fig. 3a, thetemperature of Cu2�xSe@mSiO2–PEG aqueous solution at aconcentration of 200 mg mL�1 (48.9 ppm Cu+) was raised from25.0 �C to 45.3 �C aer 480 s NIR irradiation. In comparison, thetemperature of the control experiment of pure water (0 mgmL�1)and Au nanorods (50 ppm Au) was only increased respectively by4.3 �C and 14.2 �C aer irradiation by the NIR laser under thesame conditions (Fig. S4†), which indicates that Cu2�xSe@mSiO2–PEG nanoparticles exhibit a better phototherml effectthan the widely used Au nanorods when they are driven by980 nm laser at the same metal ion concentration. Moreover,the higher the concentration of Cu2�xSe@mSiO2–PEG nano-particles, the higher the temperature elevated (Fig. 3b). Thesedata conrm that the Cu2�xSe@mSiO2–PEG can effectivelyconvert NIR light into fatal heat due to the strong SPR absorp-tion of the Cu2�xSe core.25

Based on the mesoporous shell structures, the Cu2�x-Se@mSiO2–PEG particles are expected to be suitable for anti-cancer drug delivery. DOX was loaded into theCu2�xSe@mSiO2–PEG nanoparticles by simply mixing DOXaqueous solutions for 48 h, and then repeated washing twice to

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Fig. 3 (a) Temperature change of the aqueous solution containingCu2�xSe@mSiO2–PEG nanoparticles with different concentrationsunder irradiation of the 980 nm laser at a safe power density (0.72 Wcm�2). (b) The plot of temperature elevation over a period of 480 sversus the aqueous dispersion of the Cu2�xSe@mSiO2–PEGnanoparticles.

Fig. 4 (a) Absorption spectra of the DOX, Cu2�xSe@mSiO2–PEG andCu2�xSe@mSiO2–PEG/DOX solutions. (b) The cumulative releasekinetics from the Cu2�xSe@mSiO2–PEG/DOX nanocomposites inphosphate-buffer saline (pH 7.4), phosphate-buffer saline (pH 6.5) andacetate buffer (pH 4.8) at room temperature with or without NIR laserirradiation by a safe power density (0.72 W cm�2).

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remove unbound DOX, forming Cu2�xSe@mSiO2–PEG/DOXnanocomposites, and aqueous solution with a bronze color(Fig. 4a, inset). Aer loading with DOX, the Cu2�xSe@mSiO2–

PEG/DOX complex still exhibits strong NIR absorbance ofCu2�xSe@mSiO2–PEG, and shows a typical absorption peak ofDOX near the 480 nm region, which indicates that DOX hasbeen successfully incorporated into the Cu2�xSe@mSiO2–PEGcore–shell nanoparticles (Fig. 4a). The encapsulation efficiencyand loading capacity of DOX on the Cu2�xSe@mSiO2–PEGparticles reached up to 61.27% and 15.53% (by weight) asdetermined by UV-vis, respectively. The loading content ishigher than the present core–shell nanocomposites (Au@mSiO2

core–shell nanostructures 14.5%,27 Cu9S5@mSiO2–PEG core–shell nanocomposites 13.76%29). The high encapsulation effi-ciency and loading capacity of DOX can be assigned to the largesurface area and appropriate pore size of Cu2�xSe@mSiO2–PEG.

The DOX release from the Cu2�xSe@mSiO2–PEG nano-composites against buffer solution at pH 7.4, 6.5 and 4.8 wasresearched to simulate the normal physiological environment,tumor cell environment and acidic cellular endosomes,respectively. As shown in Fig. 4b, the release of DOX from the

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Cu2�xSe@mSiO2–PEG nanoparticles strongly depended on thepH of the medium and the releasing time. Because DOX becamemore water soluble at a low pH value due to the protonateddaunosamine group,46 DOX released more rapidly from Cu2�x-Se@mSiO2–PEG nanoparticles at a low pH value. At pH 7.4, DOXmolecules are barely released, indicating that the Cu2�x-Se@mSiO2–PEG nanoplatform releases very little DOX mole-cules in a normal physiological environment and normal cellscan be effectively avoided from damage by DOX, but in thetumor cell environment, DOX releases more rapidly and reaches11.33% in 10 h. At pH 4.8, DOX releases as high as 31.67%,which always occurs when the nanocomposites are endocytosedby tumor cells. Thus, such a pH-sensitive release of DOX fromCu2�xSe@mSiO2–PEG is benecial for cancer therapy.

To investigate whether the NIR photothermal effect offeredby Cu2�xSe nanoparticles could trigger the DOX release, therelease kinetics of DOX from the Cu2�xSe@mSiO2–PEG/DOXunder NIR laser irradiation was studied. During the DOX releaseprocess from the Cu2�xSe@mSiO2–PEG nanoparticles in thebuffer solution at pH ¼ 4.8, 6.5 and 7.4, 5 min NIR laser irra-diations (980 nm, 0.72 W cm�2) were given at 1.0, 2.0, 3.0, 4.0,

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Fig. 5 (a) Viabilities of HeLa cells incubated for 24 h with differentconcentrations of the Cu2�xSe@mSiO2–PEG nanoparticles. (b)Viabilities of HeLa cells after incubation with different concentrationsof free DOX, Cu2�xSe@mSiO2–PEG and Cu2�xSe@mSiO2–PEG/DOXwith or without 5 min of NIR irradiation (0.72 W cm�2, 980 nm).Cu2�xSe@mSiO2–PEG and Cu2�xSe@mSiO2–PEG/DOX have equiva-lent Cu+ concentration at corresponding equal DOX points.

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6.0, 8.0 and 10.0 h. As shown in Fig. 4b, the rst 5 min of NIRirradiation at 1.0 h increased the cumulative release of DOXfrom 11.6% to 19.5% at pH 4.8 and from 2.1% to 3.9% at pH 6.5.In other cycles, the same phenomenon with a rapid release rateupon NIR irradiation and a slow release rate without NIR irra-diation was observed. At pH 4.8, the DOX cumulative releasereached to 56.5% within 10 h aer seven cycles of 5 min irra-diation at given time intervals, while only 31.7% DOX wasreleased without irradiation. At pH 6.5 and aer 10 h, anincrease of DOX release from 10.3 to 20.1% was also achieved byNIR irradiation. The enhanced drug release under NIR irradia-tion can be attributed to heat generated by the photothermaleffect of Cu2�xSe@mSiO2–PEG nanoparticles. The heat disso-ciates the strong interactions between DOX and SiO2,28 thusmore DOX molecules are released. These results demonstratethat the Cu2�xSe@mSiO2–PEG have successfully achieved thedrug controllable release by pH and NIR light.

As discussed above, Cu2�xSe@mSiO2–PEG nanoparticlesdisplay excellent photothermal and drug delivery effects. Butthe ideal photothermal agent also should be nontoxic or havelow toxicity for biological applications. So we rst incubatedHeLa cells with the Cu2�xSe@mSiO2–PEG particles at differentconcentrations for 24 h and then tested the cell viabilities byusing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetr azoliumbromide (MTT) assay. No signicant differences in cell prolif-eration were observed in the absence or presence of Cu2�x-Se@mSiO2–PEG nanoparticles (Fig. 5a). Even at the highesttested dose of Cu2�xSe@mSiO2–PEG nanoparticles (500 mgmL�1), cell viability still remained approximately 96.3%. Thesedata show that the aqueous dispersion of Cu2�xSe@mSiO2–PEGnanoparticles (<500 mg mL�1) can be considered to have lowcytotoxicity.

To investigate the effect of photothermal therapy, chemo-therapy and their combination in vitro, HeLa cells were incu-bated with different concentrations of free DOX,Cu2�xSe@mSiO2–PEG/DOX, Cu2�xSe@mSiO2–PEG and Cu2�x-Se@mSiO2–PEG/DOX treated with or without NIR irradiation atsafe power density (0.72 W cm�2), and then the MTT assay wascarried out to test the cell viability. The Cu2�xSe@mSiO2–PEG/DOX nanoparticles demonstrated higher cytotoxicity than freeDOX at the same concentrations (Fig. 5b). The Cu2�xSe@m-SiO2–PEG/DOX nanocomposites at an equivalent DOX concen-tration of 40 mg mL�1 killed about 46% of cells, but free DOXkilled only 37.3% cells under the same conditions. This resultexplains why DOX-loaded nanoparticles can enter cancer cellsmore easily than free DOX.30,31 Aer uptake by cancer cells, theCu2�xSe@mSiO2–PEG/DOX nanocomposites experience a low-pH that induces rapid release of the loaded DOX inside thecells. Aer NIR laser irradiation (0.72 W cm�2, 980 nm), theCu2�xSe@mSiO2–PEG/DOX particles exhibited a higher cell-killing effect in HeLa cells at all tested concentrations than thechemotherapy (without NIR irradiation) or the photothermaltherapy (without DOX) alone. For example, 63.5% of the cellswere killed by the Cu2�xSe@mSiO2–PEG/DOX nanoparticleswith NIR irradiation at an equivalent DOX concentration of20 mg mL�1. However, 43.9% of the cells were killed by theCu2�xSe@mSiO2–PEG/DOX nanoparticles without NIR

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irradiation. In the absence of DOX, the Cu2�xSe@mSiO2–PEG(Cu+ concentration is identical as Cu2�xSe@mSiO2–PEG/DOX)under NIR irradiation killed only 34.4% of the cells. Therefore,these results suggest that as-prepared Cu2�xSe@mSiO2–PEGnanoparticles can act as a difunctional nanoplatform forchemo-photothermal therapy to effectively kill cancer driven byNIR irradiation at safe power density, and more importantly,the combination of photothermal therapy and chemotherapydemonstrates better therapeutic effects for cancer treatmentthan individual therapy.

The Cu2�xSe@mSiO2–PEG core–shell nanoparticles display agood photothermal conversion effect, high drug loadingcapacity, pH-dependency and NIR light-triggered drug releaseabilities, dual therapeutic modes (DOX release for chemo-therapy and hyperthermia), and in vitro characteristics. Becauseof the complexity of the in vivo environment, the efficacy shouldbe examined in vivo before clinical trials in humans. The oste-osarcoma bearing mice were from Shanghai Tenth People'sHospital. When the tumors inside the mice had grown to 5–10mm in diameter, the mice were randomly allocated into three

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groups. The mouse in group A was intratumorally injected with0.10 mL of the Cu2�xSe@mSiO2–PEG nanoparticles' dispersionsolution (400 mg mL�1), the mouse in group B was intra-tumorally injected with 0.10 mL of the Cu2�xSe@mSiO2–PEG/DOX (400 mg mL�1) nanocomposites' dispersion solution, whilegroup C was intratumorally injected with 0.1 mL saline solu-tion. Aer 1 h, the injected areas of the mice from three groupswere perpendicularly irradiated by the 980 nm wavelength laserdevices (0.72 W cm�2) for 5 min. For the mouse treated withsaline solution, the surface temperature of the tumor increasedonly 3.5 �C during the entire irradiation process (Fig. 6a). Incontrast, as for the Cu2�xSe@mSiO2–PEG injected mouse, thetumor surface temperature increased rapidly from 31.1 �C to48.1 �C, and reached 51.8 �C aer 300 s. The tumor surfacetemperature of Cu2�xSe@mSiO2–PEG/DOX injected mouseincreased to 47.1 �C, a little lower than that of the Cu2�x-Se@mSiO2–PEG injected mouse, but it was still high enough tokill the cancer cells in vivo. More importantly, the anticancerdrug DOX could rapidly release from nanoparticles for chemo-therapy because of the thermal effect. These results reveal thatboth Cu2�xSe@mSiO2–PEG and Cu2�xSe@mSiO2–PEG/DOXwithin the tumor can effectively adsorb and convert the NIRlight to heat due to the deep penetration depth of 980 nm-wavelength laser light in biological tissues.20,37,38

The mice were killed 8 h aer photothermal therapy, andtumors were removed, embedded in paraffin, and cryo-sectioned into 4 mm slices. The slides were stained withhematoxylin–eosin (H&E). The histopathological images of themouse treated by saline (Fig. 6b, S5a†) show almost no cellnecrosis aer irradiation. Because saline solution merely con-verted very little light to heat and temperature of the tumorsurface only increased 3.5 �C, which is not high enough to killthe cancer cells. The histopathological images of the Cu2�x-Se@mSiO2–PEG treatment are shown in Fig. 6c and S5b.†

Fig. 6 (a) Plots of the temperature within the irradiated tumor area inthree mice injected respectively with saline solution, Cu2�xSe@mSiO2–PEG and Cu2�xSe@mSiO2–PEG/DOX solution as a function ofirradiation time, respectively, H&E-stained histological images of (b)injected saline solution, (c) injected Cu2�xSe@mSiO2–PEG solution,and (d) injected Cu2�xSe@mSiO2–PEG/DOX solution groups of miceafter 5 minute laser irradiation, respectively.

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Clearly, the common features of thermonecrosis are presentedon most areas of the examined tumor slide, such as cellshrinkage, loss of contact, coagulation, nuclear damage.Further, the histopathological images of the Cu2�xSe@mSiO2–

PEG/DOX treatment showed that almost the entire tumor tissuewas necrotized, exhibiting degradation and corruption of theextracellular matrix of the tumor in the examined tumor slide(Fig. 6d, and S5c†). The enhanced cell necrosis proportion of theCu2�xSe@mSiO2–PEG/DOX treatment could probably beattributed to synergistic interaction between chemotherapy andphotothermal therapy due to enhanced toxicity of DOX andhyperthermia,47,48 both of which were activated simultaneouslyby the 980 nm laser.

In order to further verify the enhanced anticancer effect ofchemo-photothermal therapy by Cu2�xSe@mSiO2–PEG/DOXnanocomposites in vivo, we then compared the in vivo thera-peutic efficiency of saline, DOX, Cu2�xSe@mSiO2–PEG andCu2�xSe@mSiO2–PEG/DOX by measuring tumor growth rates.The mice of the osteosarcoma tumor model were randomlydivided into four groups. The four groups were intratumorallyinjected into the tumor site, and then subjected to laser illu-mination (980 nm, 0.72 W cm�2) for 10 min. The tumor sizes

Fig. 7 (a) The osteosarcoma tumor growth curves of different groupsafter treatments. (b) Body weights of different groups of mice aftervarious treatments.

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and body weights of each group aer treatments were thenmeasured. The results demonstrated that the tumors treatedwith PBS grew obviously over time, suggesting that the osteo-sarcoma tumor growth was not affected by laser irradiation(Fig. 7a and S6†). The growth of tumors was inhibited to acertain degree by free DOX as compared with PBS. The meantumor size in the Cu2�xSe@mSiO2–PEG group was muchsmaller than that of the PBS group, which demonstrates that thetumor growth could be obviously inhibited by just the photo-thermal effect of the Cu2�xSe@mSiO2–PEG composites. Thecombination of photothermal therapy and chemotherapyoffered by Cu2�xSe@mSiO2–PEG/DOX nanocomposites underlaser irradiation showed signicantly an enhanced antitumoractivity and even resulted in complete eradication of tumor(Fig. S6†). These results could probably be attributed to asynergistic effect between the chemotherapy and photothermaltherapy due to enhanced cytotoxicity of DOX at elevatedtemperatures and higher heat sensitivity for the cells exposed tothe DOX,47,48 and continuous release of the DOX from theCu2�xSe@mSiO2–PEG/DOX nanocomposites, inhibiting tumorgrowth for a long time. During the treatments, we alsomeasured the body weight of the mice for all groups, because ahigh toxicity usually leads to weight loss. For all groups, noobvious weight loss was found (Fig. 7b), implying that thetoxicity of treatments was low. Therefore, the as-preparedCu2�xSe@mSiO2–PEG nanoparticles driven by NIR irradiationat the safe power density not only have good photothermaltreatment ability, but also act as drug delivery carriers for cancerchemotherapy. Importantly, the combination of photothermaltherapy and chemotherapy signicantly improves the thera-peutic efficacy, and demonstrates better therapeutic effects forcancer treatment than individual therapy in vivo. To the best ofour knowledge, this is the rst time that a combination ofphotothermal therapy and chemotherapy based on Cu2�xSenanomaterials has been reported. These results revealed thatthe DOX loaded Cu2�xSe@mSiO2–PEG core–shell nanoparticlesdriven by NIR irradiation at the safe power density are apowerful agent for combining chemotherapy and photothermaltherapy of cancer in vivo.

4 Conclusions

In summary, the hydrophilic Cu2�xSe@mSiO2–PEG core–shellnanoparticles were successfully prepared, which show a highphotothermal conversion effect and excellent biocompatibilityin vitro. Moreover, the outer biocompatible mesoporous SiO2

shell can protect the photothermal utilization of the Cu2�xSecore in complex biological environments and serve as a drugcarrier for loading DOX for chemotherapy of cancer.

The release of DOX from the Cu2�xSe@mSiO2–PEG core–shell nanoparticles can be triggered by pH and NIR light,resulting in a synergistic effect for killing cancer cells. Impor-tantly, we have demonstrated that Cu2�xSe@mSiO2–PEG/DOXnanocomposites driven by NIR radiation with a safe powerdensity showed signicant enhancement of therapy effects oncancer cells than individual therapy approaches in vitro and invivo. Therefore, Cu2�xSe@mSiO2–PEG/DOX core–shell

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nanocomposites integrate photothermal therapy and chemo-therapy and have great potential for use in cancer therapy.

Acknowledgements

This work was nancially supported by the National NaturalScience Foundation of China (grant no. 21171035 and51302035), the Key Grant Project of Chinese Ministry ofEducation (grant no. 313015), the PhD Programs Foundation ofthe Ministry of Education of China (grant no. 20110075110008and 20130075120001), the National 863 Program of China(grant no. 2013AA031903), the Science and TechnologyCommission of Shanghai Municipality (grant no.13ZR1451200), the Fundamental Research Funds for theCentral Universities, Program for Changjiang Scholars andInnovative Research Team in University (grant no. IRT1221), theShanghai Leading Academic Discipline Project (grant no. B603),and the Program of Introducing Talents of Discipline toUniversities (no. 111-2-04).

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