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Functionalized m

aCAS Key Laboratory of Separation Scien

Chromatographic R & A Center, Dalian

Academy of Sciences (CAS), Dalian 11602

Fax: +86-411-84379617; Tel: +86-411-84379bThe University of Chinese Academy of Scien

Cite this: RSC Adv., 2014, 4, 33986

Received 1st May 2014Accepted 22nd July 2014

DOI: 10.1039/c4ra03993a

www.rsc.org/advances

33986 | RSC Adv., 2014, 4, 33986–339

esoporous carbon nanoparticlesfor targeted chemo-photothermal therapy ofcancer cells under near-infrared irradiation

Guiju Xu,ab Shengju Liu,ab Huan Niu,ab Wenping Lva and Ren'an Wu*a

Chemo-photothermal therapy with the combination of chemotherapy and photothermal therapy has

emerged as a promising anticancer treatment for its synergistic effects. In this work, the functionalized

mesoporous carbon nanoparticles (FA/PEI/O-MCN) were constructed by modifying the mesoporous

carbon nanoparticles (MCN) with polyethylenimine (PEI) and folic acid (FA) for the targeted chemo-

photothermal therapy. The FA/PEI/O-MCN exhibited strong light absorption and high photothermal

conversion efficiency in the near-infrared (NIR) region due to the graphitic structure of MCN. Meanwhile,

FA/PEI/O-MCN displayed high drug loading capacity using doxorubicin hydrochloride (DOX) as a model

drug. Flow cytometry analysis and competitive binding experiments verified that the FA modification

could significantly enhance the uptake of FA/PEI/O-MCN by HeLa cells with folate receptors (FR) over-

expressing. Comparing with chemotherapy or photothermal therapy alone, the DOX-loaded FA/PEI/O-

MCN demonstrated the synergistic effects and resulted in the higher therapeutic efficacy. We believe

that the FA/PEI/O-MCN could be applied as an efficient chemo-photothermal platform to realize the

targeted synergistic therapy.

1. Introduction

Photothermal therapy is a physical treatment, in which light isconverted into cytotoxic heat to destroy tumor cells.1 In terms ofthe light source, the use of near-infrared (NIR) light is highlydesirable since NIR light (wavelength 700–1100 nm) is nonin-vasive for normal tissues and possesses a long penetrationdepth.2 As the efficacy of photothermal therapy could beenhanced by nanomaterials, a series of NIR-resonant nano-materials such as metal nanomaterials (e.g., gold nanorods,3

gold nanocages,4,5 gold nanoshells,6 gold nanostars7 and Pdnanosheets8) and carbon nanomaterials (e.g., carbon nano-tubes,9–11 carbon nanohorns,12,13 graphene oxide14 and grapheneshell15,16) have been developed for the photothermal treatmentof cancer cells. To improve the therapeutic efficacy, chemo-photothermal therapy with the combination of photothermaltherapy and chemotherapy has been developed, which caninduce the synergistic effects by delivering the cytotoxic heatand drugs to the tumor sites simultaneously and locally.17

Additionally, chemo-photothermal therapy can lower the drugdosage requirements and minimize systemic side-effects ofchemotherapeutic agents, since not only the cytotoxicity of

ce for Analytical Chemistry, National

Institute of Chemical Physics, Chinese

3, China. E-mail: wurenan@dicp.ac.cn;

828

ces, Beijing 100049, China

97

chemotherapeutic agents can be enhanced at elevated temper-atures18 but also photothermal therapy can sensitize the tumorto chemotherapeutic agents.10

To date, different types of NIR-resonant nanomaterials havebeen developed for chemo-photothermal therapy. The graphiticstructure (such as carbon nanotubes and graphene etc.) couldprovide the hydrophobic surface for drug-loading and alsoendow the optical absorption in near-infrared regions. Previ-ously, we have utilized carbon nanotubes to serve as the NIR-triggered drug-delivery nanosystem to overcome the drug-resistance of human leukemia cancer cells due to its efficientdrug loading capacity as well as NIR absorption.19 To combinedrug delivery and NIR photothermal therapy into one system,nanoscale graphene oxide with high optical absorbance in NIRregion has been used in chemo-photothermal therapy.20,21 Thesurface modication of graphene with stabilizing agents suchas PEG–lipid and PVP were applied, with the view to maintainthe stability of graphene in physiological solutions.22,23 On theother hand, metal-based NIR-resonant nanomaterials also havethe potential to combine drug delivery and NIR-resonant intoone system, such as gold nanostars.24 However, the improve-ment of drug-loading capacity usually required since thenonporous structure of most metal-based NIR-resonant nano-materials. Besides, the NIR absorption band will disappear forgold nanorods exposed to NIR laser because of the trans-formation tendency from gold nanorod to gold nanosphere.25

So, the hybrid nanocomposites with the integration of NIR-resonant nanomaterials (e.g., gold nanorods,26–29 gold

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nanocages,30 and Pd nanosheets31) and mesoporous silica weredeveloped. In these hybrid nanocomposites, the drugs arestored in nanopores of mesoporous materials, and the heat isgenerated by light on the NIR-resonant nanomaterials. Themesoporous structures not only improve the drug loadingperformance for the metal-based NIR-resonant nanomaterialsbut also provide the opportunity to achieve the controlledrelease of drugs by installing nanovalves on mesopores.32

Despite so much progress has been made for the NIR-resonantnanomaterials applied in chemo-photothermal therapy,seeking for a new platform possessing inherent high drug-loading capacity, good water-solubility and efficient NIRphoton-to-heat conversion is still meaningful for chemo-photothermal therapy.

Mesoporous carbon prepared by the hard or so templatingsynthetic methods have received signicant attention owing tolarge surface area, tunable pore size, good biocompatibility andwell-dened surface properties. Due to the strong hydropho-bicity of the internal surface of mesoporous carbon materials,we have demonstrated the highly efficient loading of endoge-nous peptides from human serum33 and N-linked glycans fromglycoprotein by ordered mesoporous carbon.34 Matching withthe targeted tumor therapy via the enhanced permeability andretention (EPR) effect, nanosized mesoporous carbon materialshave emerged potentials in drug delivery systems (DDS).35–37 Sofar, research attention on mesoporous carbon nanoparticles(MCN) has only been paid on drug-loading properties since thehydrophobicity and large surface area,38–40 the investigation of

Fig. 1 Schematic illustration of the preparation of FA/PEI/O-MCN and thePEI/O-MCN.

This journal is © The Royal Society of Chemistry 2014

using MCN as NIR-resonant nanomaterials combining with thedrug-loading for chemo-photothermal therapy has not beenreported, to the best of our knowledge. In this work, a MCN-based nanosystem has been developed to serve as an inte-grated system for combined drug delivery and NIR photo-thermal therapy. The functionalizedMCN (FA/PEI/O-MCN) wereconstructed via the modication of the pristine MCN withpolyethylenimine (PEI) and cancer-cell-specic ligand folic acid(FA). FA were incorporated for specic recognition of cancercells and enhance the cellular uptake of FA/PEI/O-MCN.41 Asexpected, the obtained FA/PEI/O-MCN exhibited strongabsorption of NIR light and efficient photothermal conversion,superior to that of reduced graphene oxide. Doxorubicinhydrochloride (DOX) was used as a model anticancer drug sinceDOX can emit uorescence, allowing for study of cellular uptakeusing ow cytometry. The inherent mesoporous structure of FA/PEI/O-MCN was desirable for efficient loading of DOX. Flowcytometry analysis and competitive binding experimentsdemonstrated that the FA modication could facilitate theinternalization of FA/PEI/O-MCN into HeLa cells with over-expressed folate receptor (FR). The combined NIR photo-thermal therapy and chemotherapy with the DOX-loaded FA/PEI/O-MCN complex showed excellent efficacy for the treat-ment of HeLa cells, superior to NIR photothermal therapy orchemotherapy alone. In summary, FA/PEI/O-MCN could effi-ciently combine NIR-induced hyperthermia, drug delivery andreceptor-specic targeting into one system for targeted chemo-photothermal therapy, as illustrated in Fig. 1.

chemo-photothermal targeted therapy based on the DOX-loaded FA/

RSC Adv., 2014, 4, 33986–33997 | 33987

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2. Experimental section2.1 Materials and apparatus

Triblock copolymer Pluronic F127 and folic acid werepurchased from Sigma-Aldrich (St. Louis, MO). Formaldehyde,phenol, sodium hydroxide and sodium borohydride werepurchased from Sinopharm Chemical Reagent Co. Ltd.(Shanghai, China). N-Hydroxysuccinimide (NHS), 1-[3-(dime-thylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC)and branched polyethylenimine (PEI, MW ¼ 600) werepurchased from Alfa Aesar (Ward Hill, MA). 2-(4-Morpholino)ethanesulfonic acid was purchased from Aladdin (Shanghai,China). Polyvinylpyrrolidone (PVP, MW ¼ 58 000) werepurchased from Bailingwei Chemical Regant Co. Ltd.(Shanghai, China). Doxorubicin hydrochloride (DOX) waspurchased from Meilun Biology Technology Co. Ltd. (Dalian,China). RPMI-1640 cell culturing medium and penicillin/streptomycin solution (100�) were purchased from GibcoInvitrogen Corporation (Carlsbad, CA). Cell Counting Kit-8 waspurchased from Dojindo laboratory (Kumamoto, Japan). TheLDH Assay Kit was acquired from the Beyotime Institute ofBiotechnology (Haimen, China). Sulfuric acid (H2SO4), nitricacid (HNO3), concentrated ammonia and ethanol were ofanalytical grade. Deionized water was puried with a Milli-Qwater system (Millipore, USA).

Transmission electron microscopy (TEM) measurementswere carried out on a JEM-2000 EX (JEOL) microscope operatedat 120 kV. UV-Vis-NIR spectra were measured on a Double BeamUV-Vis spectrophotometer (UV-8000S) (Metash) at a wavelengthof 190–1100 nm. Dynamic light scattering (DLS) and zetapotential measurements were made on a Zetasizer nano ZS(ZEN3600) instrument (Malvern). Fourier transform infrared(FTIR) spectra were taken in KBr disks on a Tensor 27 spec-trometer (Bruker). Nitrogen sorption isotherms were measuredat 77 K with BK122W (JWGB). Raman spectra were taken atroom temperature on a Renishaw invia spectrometer with anargon-ion laser at an excitation wavelength of 514 nm. Flowcytometry analysis were performed with a FACS Vantage SE owcytometer (BD).

2.2 Experimental details

2.2.1 Synthesis of mesoporous carbon nanoparticles(named as MCN). The MCN were synthesized according to thelow-concentration hydrothermal route.37 Briey, phenol (0.6 g),formalin aqueous solution (2.1 mL, 37 wt%) and NaOH aqueoussolution (15 mL, 0.1 M) were mixed and stirred at 70 �C for 0.5 hto obtain the phenolic resols. Aer the addition of triblockcopolymer Pluronic F127 (0.96 g) dissolved in H2O (15 mL), themixture was stirred at 340 rpm at 66 �C for 2 h. Then, water(50 mL) was added, and the solution was further reacted for16–18 h. Aer that, the obtained solution was diluted by waterin a volume ratio of one to three, transferred into an autoclaveand heated at 130 �C for 24 h. The products were collected bycentrifugation and washed with water for three times and driedunder vacuum. The resulting powders were then heated at700 �C for 3 h in nitrogen ow to remove the template.

33988 | RSC Adv., 2014, 4, 33986–33997

2.2.2 Oxidization of MCN (named as O-MCN). Theobtained MCN were added to a mixed solution of the concen-trated sulfuric acid (98%) and concentrated nitric acid (70%)with the ratio of 3 : 1 (v/v), and sonicated for 4 h at 35–40 �C. Theoxidized MCN (O-MCN) were collected by centrifugation andwashed with water till the pH neutral of the washed water. Theobtained O-MCN were nally dried under vacuum overnight.

2.2.3 Procedures for PEI functionalization of O-MCN(named as PEI/O-MCN). The graing of PEI onto O-MCN wascarried out by covalently bonding polyethylenimine (PEI, MW¼600) onto O-MCN via the diimide-activated amidation. Briey,O-MCN (80 mg) were rst dissolved in a 40 mL aqueous buffersolution of 2-(4-morpholino) ethanesulfonic acid (MES) (50mM, pH ¼ 6.0), and then activated with stirring gently at 25 �Cfor 0.5 h aer the addition of EDC (192 mg, 1 mmol) and NHS(287 mg, 2.5 mmol). Aer that, the PEI (600 mg, 1 mmol)dispersed in the MES solution (5 mL) was added to the activatedO-MCN solution and stirred for another 24 h at 25 �C. Finally,the excess EDC, NHS and PEI were removed by washing thematerials repeatedly with water for several times. At last, the PEIgraed O-MCN (PEI/O-MCN) were dried under vacuum for 12 hat 60 �C.

2.2.4 Synthesis of folic-acid-conjugated PEI/O-MCN(named as FA/PEI/O-MCN). Firstly, folic acid (220 mg, 0.5mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-chloride (EDC, 96 mg, 0.5 mmol) and N-hydroxysuccinimide(NHS, 145 mg, 1.25 mmol) were mixed in DMSO (25 mL) andstirred gently at 25 �C for 0.5 h. Then, the PEI/O-MCN (50 mg)pre-dispersed in DMSO was added and stirred at roomtemperature for 24 h. The resulting solids were centrifuged andwashed with DMSO, water, and ethanol, successively. Theobtained products (FA/PEI/O-MCN) were then dried undervacuum at 60 �C for 12 h.

2.2.5 Synthesis of PVP-modied reduced graphene oxide(named as rGOpvp). Graphene oxide (GO) was purchased fromXFnano (Nanjing, China). The reduced graphene oxide (rGO)was reduced from GO with sodium borohydride using a methodreported elsewhere.42 Briey, 30 mg GO was rst immersed in adiluted ammonia solution to form a solution of GO with theconcentration of 1 mg mL�1 (pH 11.8–12.8) under a 30 minsonication. Aer the following addition of 60 mg sodiumborohydride into this suspension, the reduction process of theGO was performed by stirring and reuxing for 12 h. Theresulting reduced graphene oxide (rGO) was further modied bypolyvinylpyrrolidone (PVP) to prepare a stable water suspensionfollowing a literature protocol.43

2.2.6 DOX loading and loading yield measurement. 1mg ofFA/PEI/O-MCN nanoparticles were suspended in 5 mL of DOXaqueous solution (180 mg mL�1) with pH values of 9, 7.4 and 5.5,respectively, in Tris–HCl, phosphate and acetate buffers. Aer24 h stirring under dark, the FA/PEI/O-MCN nanoparticles werecollected by centrifugation, and carefully washed with the cor-responding buffer till the supernatant turned colourless. Theamount of DOX loaded on FA/PEI/O-MCN was estimated bymonitoring the concentrations of DOX in the initial solutionand the supernatant by UV-Vis spectrometry at 480 nm.

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2.2.7 DOX release. To examine the release of DOX from FA/PEI/O-MCN, the DOX-loaded FA/PEI/O-MCN nanoparticles wererst dispersed in 5 mL buffer at various pH (5.5 and 7.4) in a20 mL transparent glass bottle. Then, the bottle was placed intoa shaker and shook for a certain time under dark with 150 rpmat 37 �C. At predetermined time intervals, the nanomaterialssolution was centrifuged (11 000 rpm, 10 min), and the super-natant was withdrawn. Aer the samples were redispersed in5 mL fresh buffer and irradiated with NIR laser centered at 808nm at an output power of 15 W cm�2 for 5 min under magneticstirring, the nanomaterials solution was centrifuged (11 000rpm, 10 min) and supernatant was withdrawn. The concentra-tions of DOX in the supernatant before and aer NIR laserirradiation were analyzed by UV-Vis spectrometry. The releasebehavior was also performed without NIR laser irradiation atdifferent pH values.

2.2.8 CCK8 and the LDH activity assay for measuring cellviability. The impact of FA/PEI/O-MCN on cell proliferation wasdetermined by CCK8 and LDH activity assays. Unless otherwisestated, HeLa cells (a human cervical carcinoma cell line) werecultured in complete culture media (RPMI 1640 supplementedwith 10% bovine serum and 0.1% penicillin/streptomycin) in5% CO2 atmosphere at 37 �C in a humidied incubator. For cellviability measurements, HeLa cells were plated into 96-wellplates and cultured until a conuency of 80% was reached.HeLa cells were treated with FA/PEI/O-MCN at differentconcentrations in culture media. Cells cultured in blankcomposites medium were taken as the control. Aer 12 h, 24 hand 48 h, the viability of HeLa cells were determined by theCCK8 assay and LDH activity assay, according to the manufac-turer suggested procedures.

2.2.9 Assessment of targeting ability of FA/PEI/O-MCN. Foroptical microscope images observation, HeLa cells were pre-grown in 6-well culture plates using folate-decient RPMI1640 medium (named as folate-free medium) and cultured untila conuency of 80% was reached. The cell medium wasremoved, and then cells were incubated with fresh cell mediumcontaining 25 mg mL�1 of FA/PEI/O-MCN for 12 h. For folic acidcompetition experiments, HeLa cells were cultured in themedium containing 3 mM free folic acid (named as folatemedium). Aer removal the cells medium, the cells were rinsedand viewed live with the Olympus CKX 41 microscope.

For ow cytometry analysis, HeLa cells were pre-grown in6-well culture plates using folate-decient RPMI 1640 medium(named as folate-free medium) and cultured until a conuencyof 80% was reached. Next, the DOX-loaded PEI/O-MCN or FA/PEI/O-MCN was added at a concentration of 25 mg mL�1 inthe same medium and incubated for 2 h. Then the cells werewashed with PBS buffer for 3 times and collected. Themeasurement of intracellular DOX levels was fullled by a FACSVantage SE ow cytometer from BD (Franklin Lakes, NJ).

The inuence of target unit on the efficiency of cell killing byFA/PEI/O-MCN combined with NIR laser irradiation was inves-tigated. The cells cultured in different medium (folate mediumand folate-free medium) were treated with the same concen-tration of FA/PEI/O-MCN for 8 h. Then, the cell culture was

This journal is © The Royal Society of Chemistry 2014

washed three times with PBS and replaced by 100 mL freshculture media. Aer that, the cells on plate were exposed to 808nm laser irradiation (15 W cm�2 for 5 min per treatment, threetreatments), and incubated for another 12 h at 37 �C. Cellviability was measured by the CCK8 assay.

2.2.10 Chemo-photothermal therapy of HeLa cells. HeLacells seeded on 96-well plates with a conuency of 80% weretreated FA/PEI/O-MCN and DOX-loaded FA/PEI/O-MCN atvarious concentrations for 8 h. Cellular unbound nanoparticleswere removed by rinsing with PBS. Aer the addition of freshculturemedia intowells, the cellswere irradiatedby 808nm laser(15 W cm�2 for 5 min per treatment, three treatments) for pho-tothermal and chemo-photothermal treatments, respectively.For chemotherapy alone, the cells were not exposed to NIR irra-diation. Aerwards, the cellswere incubated at 37 �C for a further12 h. Cell viability was measured by the CCK8 assay. The datareported represented the means of triplicate measurements.

To monitor the changes of temperature in the culturechamber arising from NIR laser irradiation, HeLa cells seededon 96-well plates with a conuency of 80% were rst incubatedwith FA/PEI/O-MCN at various concentrations for 8 h. Then,cellular unbound nanoparticles were removed by rinsing withPBS. Aer the addition of fresh culture media into wells, thecells were irradiated by 808 nm laser (15 W cm�2 for 5 min). Thetemperature changes were measured by a Fluke thermometerwith a thermocouple suspended in the growth medium.

3. Results and discussion3.1 Preparation and characterization of FA/PEI/O-MCN

The synthesis of FA/PEI/O-MCN is shown in Fig. 1. Firstly, themesoporous carbon nanoparticles (MCN) were synthesizedaccording to the low-concentration hydrothermal route.37 Tofurther improve the water-solubility of the as-synthesized MCN,MCN were oxidized by a mixture of the concentrated HNO3 andthe concentrated H2SO4 (v/v, 1/3) with bath sonication (denotedas O-MCN). For the sake of subsequent conjugation of folic acid,the low-molecular-weight and hyper branched poly-ethylenimine (PEI) with high surface concentration of amino-groups was covalently linked on the surface of O-MCNthrough carbodiimide coupling (denoted as PEI/O-MCN). Folicacid (FA) was conjugated through a covalent amide linkagebetween the carboxyl group in FA and the amino group in thePEI chain (denoted as FA/PEI/O-MCN) to endow the MCN withthe targeting ability.

The shape and porous structure were characterized byTransmission electron microscopy (TEM). As shown in Fig. 2A,the pristine MCN are roughly spherical in shape with a diameterof ca. 100 nm. TEM images showed that there were no obviouschanges in themesoporous structure of O-MCN before and aerthe conjugation with PEI (Fig. 2A-2 and A-3). The hydrodynamicdiameter of the functionalized MCN (FA/PEI/O-MCN) wasmeasured by the dynamic light scattering (DLS) analysis withnanoparticles dispersed in phosphate buffered saline (PBS, pH¼ 7.4) by sonication. The average hydrodynamic diameter of theFA/PEI/O-MCN was about 120 nm (Fig. 2B), close to the particlesize observed by TEM. The polydispersity index (PDI), reecting

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Fig. 2 (A) TEM images of O-MCN (1), PEI/O-MCN (2) and FA/PEI/O-MCN (3), the scale bar is 100 nm; (B) particle diameter distribution of FA/PEI/O-MCN; (C) Raman spectra (excitation at 514 nm) with the G and D bands of graphitic carbon. (D) N2 adsorption–desorption isotherm and poresize distribution (inset) curves of O-MCN and FA/PEI/O-MCN.

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the dispersity of nanoparticles, was 0.172 which indicated themonodisperse distribution of the FA/PEI/O-MCN. The surfacearea and pore size distribution were 517 m2 g�1 and 3.2 nm forthe O-MCN, and 312 m2 g�1 and 2.6 nm for the FA/PEI/O-MCN,as characterized by N2 adsorption–desorption at 77 K (Fig. 2D).The structural information of the pristine and functionalizedMCN was investigated by Raman spectroscopy. As shown inFig. 2C, a graphite-like band (G-band) at �1600 cm�1 and adisorder-induced band (D-band) at �1380 cm�1 were observed.The D-band was used to characterize the amorphous or disor-dered carbon. The G-band was related to the vibration of sp2-hybridized carbon atoms, which veried the presence ofgraphitic domains.44 The existence of the G-band in all samplessuggests that the well dened graphitic domains are indeeddeveloped. It has been reported that the G/D-band ratio is nearlyproportional to graphitization degree.45 As observed, the ratiosof G/D-band are 1.41, 1.38, and 1.33 for samples MCN, O-MCNand FA/PEI/O-MCN, respectively. The almost unchanged G/D-band ratio for the pristine and functionalized MCN suggestedthat the graphitic structure is well preserved.

To evaluate the functionalization of the MCN-based vectorsby branched PEI and folic acid, the resulting products werecharacterized by FTIR spectroscopy, with the data presented inFig. 3A. A band of O–H stretching vibrations due to the existenceof surface hydroxyl groups or chemisorbed water was observedin all the recorded spectra in the range of 3600–3200 cm�1.46

The bands at 3448 and 1723 cm�1, representing the typicalstretching vibrations of O–H and C]O attributed to theformation of carboxylic structures were observed in the IRspectrum of O-MCN. The band at 1587 cm�1 was correspondingto the aromatic ring stretching coupled to highly conjugated

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keto groups. The band at 1250 cm�1 might be attributed toC–O–C vibrations.47 Two additional bands were observed at1400 and 750 cm�1 in the IR spectra of PEI/O-MCN and FA/PEI/O-MCN. The bands at 1400 cm�1 could be assigned to thestretching vibrations of C–N. The new intense band at 750 cm�1

was attributed to the –NH2 vibrations.48 The FTIR technique wasinsufficient to distinguish FA signals in FA/PEI/O-MCN fromthose in PEI/O-MCN. The UV-Vis spectrum was recorded tofurther conrm the successful conjugation of FA on MCN.49 Wedetected the UV-Vis-NIR spectra of PEI/O-MCN@PEI and FA/PEI/O-MCN with the same concentration in PBS. Then spectrasubtraction was applied by subtracting the spectrum of PEI/O-MCN from the spectrum of FA/PEI/O-MCN, and the obtainedspectrum was named as residual spectrum. As shown in Fig. 3B,the conjugation of FA on the nanospheres was demonstratedfrom the spectrum of the FA/PEI/O-MCN and residual spectrum,which showed the characteristic absorption peaks (280 nm) ofFA.50 Meanwhile, the UV-Vis-NIR spectrum indicated that theFA/PEI/O-MCN exhibited broad absorption from the UV to theNIR region, which was similar to carbon nanotubes and gra-phene reported in previous studies.11,14

Moreover, the surface modications on MCN could bereected by the change of the zeta potential. Fig. 4 showed thezeta potential of functionalized MCN at phosphate bufferedsaline (PBS, pH ¼ 7.4). As the existence of hydroxyl and carboxylgroups on O-MCN, the zeta potential of O-MCN was �39.7 mV.Aer graing with PEI, the zeta potential of PEI/O-MCN wasincreased to +2.5 mV, which indicated the existence of a greatamount of amino groups. Due to the successful functionaliza-tion with FA, the zeta potential of FA/PEI/O-MCN was decreasedto �16.1 mV.

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Fig. 3 (A) The FT-IR spectra of MCN (1), O-MCN (2), PEI/O-MCN (3)and FA/PEI/O-MCN (4); (B) UV-Vis-NIR spectra of PEI/O-MCN, FA/PEI/O-MCN and FA.

Fig. 4 Zeta potentials of O-MCN, PEI/O-MCN and FA/PEI/O-MCN inPBS. Error bars were based on triplet samples.

Fig. 5 (A) Photothermal heating curves of FA/PEI/O-MCN at variousconcentrations with NIR laser irradiation; (B) temperature changeswith FA/PEI/O-MCN and rGOpvp at various concentrations under NIRlaser irradiation (t ¼ 2.5 min) and UV-Vis-NIR spectra (inset) of FA/PEI/O-MCN and rGOpvp at the same concentration of 50 mg mL�1. Errorbars were based on triplet samples.

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3.2 Photothermal effect of FA/PEI/O-MCN

To test the feasibility of FA/PEI/O-MCN as photothermal agents,we chose an 808 nm laser to evaluate the photothermal

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conversion capability of FA/PEI/O-MCN. The FA/PEI/O-MCNwere dispersed in phosphate buffered saline (PBS, pH ¼ 7.4)at concentrations ranging from 6.25 to 75 mg mL�1, and irra-diated with an 808 nm laser at a power density of 15 W cm�2 for5 min. PBS was used as a negative control. As illustrated inFig. 5A, no obvious temperature increase was observed for PBSalone aer 5 min NIR laser irradiation. In contrast, the tempera-ture was increased with irradiation time for all FA/PEI/O-MCNsolutions. Furthermore, temperature evolution of FA/PEI/O-MCNat increasing concentrations from 6.25 to 75 mg mL�1 revealedan obvious concentration-dependent temperature increase underNIR laser irradiation. It was vital that no sedimentation of theFA/PEI/O-MCN suspension was observed even for temperaturehigher than 37 �C, and the heating rate was not affected by theNIR irradiation times. The ratios of G/D-band for the FA/PEI/O-MCN were 1.30 and 1.33 aer and before the NIR laser irra-diation, which conrmed that the FA/PEI/O-MCN not only couldconvert NIR photon energy into thermal energy but alsowere thermostable. The existence of graphitic structure on

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FA/PEI/O-MCN may be related to explain the infrared-absorption mechanism in functionalized MCN.15,51

As the reduced graphene oxide exhibited excellent NIRabsorbance and photothermal heating effect,52,53 the compar-ison of the photothermal efficiency between the FA/PEI/O-MCNand the reduced graphene oxide was carried out. To preventreduced graphene oxide aggregation in aqueous dispersions,polyvinylpyrrolidone (PVP) has to be introduced as stabilizingagents. Comparing the UV-Vis-NIR spectra of FA/PEI/O-MCNand rGOpvp with the concentration of 50 mg mL�1, we foundthat FA/PEI/O-MCN exhibited stronger absorbance than rGOpvp

at 808 nm (Fig. 5B, inset). A series of PVP-modied reduced gra-phene oxide (rGOpvp) solutions with different concentrations wereirradiated with an 808 nm laser at a power density of 15 W cm�2

for 2.5 min. The rGOpvp showed a concentration-dependenttemperature increase in response to the NIR laser irradiation(Fig. 5B). It was observed that heat could be generated moreefficiently by FA/PEI/O-MCN than rGOpvp with the same concen-tration. These data indicated that the photothermal sensitivityof FA/PEI/O-MCN was superior to that of rGOpvp. The excellentNIR absorption and photothermal conversion efficiency ofFA/PEI/O-MCN prompted us to evaluate their feasibility asNIR-resonant materials for cancer therapy.

Fig. 6 (A) The loading capacity of DOX on O-MCN and FA/PEI/O-MCN at different pH values; (B) NIR-triggered release of DOX atdifferent pH values. The inset shows the release profile of DOX atacidic condition (pH 5.5) in the absence and presence of NIR laser.Error bars were based on triplet samples.

3.3 Doxorubicin loading and release properties of FA/PEI/O-MCN

The structural features of FA/PEI/O-MCN are highly desirablefor drug delivery because of the large specic surface area andmesopores. To evaluate the loading performance of FA/PEI/O-MCN for drugs, doxorubicin hydrochloride (DOX), anaromatic anticancer agent, was used as the model drug, and theFA/PEI/O-MCN were mixed with DOX at varied pH for drugloading. Fig. 6A showed that the loading efficiency of DOX onFA/PEI/O-MCN increased with an increase in the pH value.Speaking concretely, the loading amount of DOX on FA/PEI/O-MCN was 100 mg mg�1 at pH 5.5, 520 mg mg�1 at pH 7.4, and750 mg mg�1 at pH 9.0. The pH-dependent DOX loading on FA/PEI/O-MCN was similar to that with carbon nanotubes andgraphene oxide.54,55 The existence of hydrophobic interiorsurface and graphite structure of the FA/PEI/O-MCN and thepH-dependant solubility of DOX made this phenomenonreasonable.54 That is the decreased hydrophilicity of DOX at ahigher pH and the resultant enhanced hydrophobic interactionbetween DOX and FA/PEI/O-MCN. To be convenient, the DOX-loaded FA/PEI/O-MCN refers to the products of loading at pHvalues of 7.4 in the following descriptions unless speciedotherwise. Comparing the loading capacity of FA/PEI/O-MCNand O-MCN (Fig. 6A), the conjugation of PEI and FA on theFA/PEI/O-MCN exhibited negligible inuence on the loadedamount of DOX, suggesting that the subsequent modicationdid not compromise the loading efficiency of DOX.

In order to mimic the approximate neutral environment ofblood circulation system and the acidic condition in cellularendosome, the release prole of DOX from FA/PEI/O-MCN wasexamined at pH 7.4 and 5.5, respectively. As shown in Fig. 6B,the cumulative release of DOX from FA/PEI/O-MCN

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demonstrated a much rapid release of DOX at acidic condition(6.8% at pH 5.5) than the neutral condition (1.2% at pH 7.4).The observed higher release rate of DOX from FA/PEI/O-MCN atacidic condition than basic condition could be attributed to theincreased hydrophilicity of DOX at acidic condition, whichweakened the p–p stacking and hydrophobic interactionsbetween DOX and FA/PEI/O-MCN and made the dissociation ofDOX from FA/PEI/O-MCN easier.

To examine whether the NIR laser irradiation would affectthe release behavior of DOX from FA/PEI/O-MCN, the releasekinetics of DOX was also investigated with the assistance of NIRlaser irradiation. As shown in Fig. 6B inset, the release prole ofDOX at acidic conditions (pH 5.5) indicated that no burstrelease of drugs occurred in the absence of NIR laser irradia-tion. In contrast, a sudden release of DOX from FA/PEI/O-MCNcould be observed, once the NIR light switched on. As revealedby the bar chart in Fig. 6B, the NIR laser irradiation couldincrease the release of DOX from FA/PEI/O-MCN regardless of

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the acidic or basic conditions. In detail, the release rate of DOXreached 3.8% at pH 7.4 and 15.7% at pH 5.5 within 9 h. Theaccelerated release of DOX from FA/PEI/O-MCN with NIR laserirradiation could be ascribed to the laser-converted heat whichweakened the interactions between DOX and FA/PEI/O-MCN.56

3.5 In vitro cytotoxicity of FA/PEI/O-MCN

The cytotoxicity of FA/PEI/O-MCN to HeLa cells was investigatedby Cell Counting Kit-8 (CCK8) assay and lactate dehydrogenase(LDH) assay. It could be seen from Fig. 7 that the FA/PEI/O-MCNshowed no obvious cytotoxicity to the HeLa cells at concentra-tions of 10–75 mg mL�1 with incubation time of 12 h, 24 h and48 h. Both in vitro CCK8 and LDH assays clearly indicated theFA/PEI/O-MCN showed low cytotoxicity and goodbiocompatibility.

3.6 Targeted ability of FA/PEI/O-MCN

The ow cytometry analysis was used to study the cellular uptakeefficiency of FA/PEI/O-MCN in FR-positive HeLa cells. The HeLacells were incubated with DOX-loaded FA/PEI/O-MCN and DOX-loaded PEI/O-MCN for 2 h at 37 �C at a dose of 25 mg mL�1,

Fig. 7 Cytotoxicity detection with CCK8 assay (A) and LDH activityassay (B) for HeLa cells treated with different concentrations of FA/PEI/O-MCN for the indicated times.

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respectively. As shown in Fig. 8A, much greater uorescenceintensity of DOX was observed in HeLa cells treated with DOX-loaded FA/PEI/O-MCN than that treated with DOX-loaded PEI/O-MCN. Because the only difference between these two sets ofnanocarriers was the FA functionalization, this proved that theincreased internalization of FA/PEI/O-MCN into HeLa cells is dueto FA functionalization.

The FR blocking experiment further evidenced the highlyspecic FR targeting by FA/PEI/O-MCN.57 As discussed previ-ously, the existence of free FA had negative impacts on theexpression of folate receptors on the surface of HeLa cells.58 FA/PEI/O-MCN was placed in two different media: (1) folate-freemedium (the cells in this medium are considered as highfolate expressing HeLa cells); (2) folate medium (the cells in thismedium are considered as folate-receptor blocking HeLa cells,because it contains 3 mM FA). The semi-qualitative indicationof the interactions between FA/PEI/O-MCN and cells via anoptical microscopy could be observed based on the intensity ofdark signal (from FA/PEI/O-MCN) and its association withcells.55 Fig. 8B showed that the FA/PEI/O-MCN dispersed infolate-free medium were remarkably internalized and existed asblack granules in the cells. While FA/PEI/O-MCN with the sameconcentration (25 mg mL�1) dispersed in folate medium wereinternalized with a lower efficiency by HeLa cells. This resultdemonstrated that with free FA serving as a competitive inhib-itor, the uptake amount of FA/PEI/O-MCN was reduced due toloss of availability of the folate receptors on the cancer cellsurface. This in turn veried that the FA functionalized FA/PEI/O-MCN could target HeLa cells via the folate receptors.

In addition to the optical microscope images described above,we tested the viability of HeLa cells incubated with FA/PEI/O-MCNdispersed in different medium under NIR laser irradiation. HeLacells cultured in two different media (folate medium and folate-free medium) were incubated with FA/PEI/O-MCN (25 mg mL�1)for 8 h, washed to remove nanoparticles, and then exposed to an808 nm laser at a power density of 15 W cm�2 for 5 min. 50% ofHeLa cells treated by FA/PEI/O-MCN dispersed in folate-freemedium were killed aer NIR laser irradiation (Fig. 8C), whileFA/PEI/O-MCN dispersed in folate medium treated cells showedmuch less cell death aer exposure to the NIR laser. As theaforementioned, the photothermal effect was concentrationdependent. For free folate in the culture media competitivelybound to the folate receptors on the cell surface, the uptakeamount of FA/PEI/O-MCN was negligible, and thereby insuffi-cient heat was transformed into cells, which accounted for theirhigh survival of HeLa cells under NIR laser irradiation. Theselective thermal ablation of HeLa cells using FA/PEI/O-MCNwith the assistance of NIR laser irradiation further conrmedthe targeted uptake of FA/PEI/O-MCN.

3.7 Chemo-photothermal therapy based on FA/PEI/O-MCN

To investigate the efficiency of NIR-photothermal therapy basedon FA/PEI/O-MCN, HeLa cells were incubated with FA/PEI/O-MCN at concentrations of 6.25, 12.5 and 25 mg mL�1 for 8 h.The cell viabilities were measured by cell-counting kit-8 (CCK8)assay with or without NIR laser irradiation. As shown in Fig. 9A,

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Fig. 8 (A) Analysis of cellular uptake of DOX-loaded nanocompositesby flowcytometry (from left to right: control, DOX-loaded PEI/O-MCN,DOX-loaded FA/PEI/O-MCN); (B) cellular uptake of FA/PEI/O-MCN byopticalmicroscope. HeLa cells were incubatedwith 25 mgmL�1 FA/PEI/O-MCN for 12 h in folate medium (1 and 2) and folate-free medium (3and 4); Scale bars represent 50 mm; (C) cytotoxicity of FA/PEI/O-MCNonHeLa cells incubated at different culturemediawith andwithoutNIRlaser irradiation (15 W cm�2 for 5 min per treatment, three treatments).

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FA/PEI/O-MCN produced negligible toxicity to HeLa cells in theabsence of NIR laser irradiation. In contrast, the viabilityshowed a dramatic dose-dependent decrease for cells incubatedwith FA/PEI/O-MCN and exposed to NIR laser. It was reportedthat the temperature higher than 42 or 43 �C would begin toinduce cellular death.56 To determine the cancer cells deathinduced by the temperature increase of FA/PEI/O-MCN underNIR laser irradiation, a thermocouple was suspended in thegrowth medium of the culture chamber to monitor the changeof temperature during the process of NIR laser irradiation. Forthe control experiment, the cells were cultured in the mediumwith the absence of FA/PEI/O-MCN. As shown in Fig. 9B, theincrease of temperature depended upon the concentration ofFA/PEI/O-MCN. The DT for control cells, which did not contactany FA/PEI/O-MCN, was merely 3.8 �C for 5 min irradiation. Incontrast, for cells cultured with 50 mg mL�1 FA/PEI/O-MCN, theDT was elevated to 39.9 �C under 5 min exposure to NIR laser.The signicant increase of temperature induced by NIR laserirradiation demonstrated that the FA/PEI/O-MCN would be ahighly efficacious platform to perform the photothermaltreatment.

Fig. 9 (A) The cell viability of HeLa cells treated with FA/PEI/O-MCN,NIR + FA/PEI/O-MCN, DOX-loaded FA/PEI/O-MCN and NIR + DOX-loaded FA/PEI/O-MCN, NIR represents irradiated by 808 nm laser withpower of 15 W cm�2 for 5 min, three treatments; (B) temperaturechanges of HeLa cells incubated with various concentrations of FA/PEI/O-MCN after exposed to 808 nm laser for 5 min.

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To evaluate the efficiency of FA/PEI/O-MCN for targetedchemo-photothermal therapy, HeLa cells were incubated withdifferent concentrations of DOX-loaded FA/PEI/O-MCN and FA/PEI/O-MCN (6.25, 12.5 and 25 mg mL�1) for 8 h and exposed toNIR light. Fig. 9A showed the cytotoxicity of these treatmentsincreased with the increase of their concentrations. When thecells were treated with FA/PEI/O-MCN (25 mgmL�1) and exposedto NIR laser irradiation, 50% of HeLa cells were killed. Theinhibition rate of DOX-loaded FA/PEI/O-MCN (25 mg mL�1, withan equivalent of 13 mg mL�1 DOX) in the absence of NIR laserirradiation was 64%. Upon NIR laser irradiation, the inhibitionrate of DOX-loaded FA/PEI/O-MCN was increased to 74%.Comparing the cell killing efficiency, it was obvious that thecombination of chemotherapy and NIR-photothermal therapybased on DOX-loaded FA/PEI/O-MCN was superior to thechemotherapy or photothermal therapy alone. It demonstratedthat DOX-loaded FA/PEI/O-MCN under NIR laser irradiationcould selectively carry heat and drug to cancer cells andsignicantly enhance the therapeutic efficacy of chemo-photothermal.

4. Conclusion

In summary, the efficient nanocarriers based on the function-alized mesoporous carbon nanoparticles (FA/PEI/O-MCN) weredesigned to perform the drug delivery and NIR photon-to-heatconversion for the chemo-photothermal synergistic therapy ofHeLa cells. The FA/PEI/O-MCN showed promising features ofthe ease of synthesis and functionalization, good water-solubility and stability in physiological solutions, as well astheir biocompatibility. The inherent mesoporous structuremade FA/PEI/O-MCN a favorable drug delivery nanocarriers forchemotherapy. The efficient NIR photon-to-heat conversion andgood thermal stability made FA/PEI/O-MCN an ideal platformfor NIR photothermal therapy. The conjugation of FA providedFA/PEI/O-MCN with the targeting ability to cancer cells withover-expressed folate receptor. Moreover, DOX-loaded FA/PEI/O-MCN under NIR laser irradiation exhibited the highest cyto-toxicity to HeLa cells, comparing with chemotherapy or photo-thermal treatment alone. Taken together, the mesoporouscarbon nanocarrier has demonstrated the promising feasibilityof the targeted chemo-photothermal therapy for cancer cells bythe combination of the receptor-specic targeting, the NIR-induced hyperthermia and the drug delivery.

Acknowledgements

The nancial supports from the National Natural ScienceFoundation of China (nos 21175134, 21375125) and the CreativeResearch Group Project of National Natural Science Foundationof China (21321064) are greatly acknowledged.

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