Journal of Colloid and Interface Science 350 (2010) 90–98

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Journal of Colloid and Interface Science

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Synthesis of magnetic and fluorescent multifunctional hollow silicananocomposites for live cell imaging

Lei Sun a, Yang Zang b, Mingda Sun a, Hengguo Wang a, Xuanjing Zhu c, Shufei Xu a, Qingbiao Yang a,*,Yaoxian Li a, Yaming Shan b,**

a Department of Chemistry, Jilin University, Changchun 130021, People’s Republic of Chinab National Engineering Laboratory of AIDS Vaccine, College of Life Science, Jilin University, Changchun 130023, People’s Republic of Chinac School of Chemistry & Chemical Engineering, Shanghai University of Engineering Science, Pudong District, Shanghai, People’s Republic of China

a r t i c l e i n f o

Article history:Received 25 March 2010Accepted 16 June 2010Available online 20 June 2010

Keywords:MultifunctionalMagnetismFluorescenceHollow nanostructuresSilicaImaging

0021-9797/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jcis.2010.06.041

* Corresponding author. Fax: +86 431 88499845.** Corresponding author. Fax: +86 431 88499845.

E-mail address: yangqb@jlu.edu.cn (Q. Yang).

a b s t r a c t

In this paper, we report a synthesis of multifunctional core/shell silica nanocomposites in mixed water–eth-anol solvents at room temperature. Water-soluble CTAB-stabilized nanoparticles (Fe3O4 and quantum dots)are used as templates and tetraethoxysilane (TEOS) is used as a precursor to fabricate multifunctional hol-low silica nanocomposites. Owing to the high abundance of folate receptors in many cancer cells, folic acid isused as the targeting ligand. By coupling with folic acids, the multifunctional silica nanocomposites conju-gates are successfully used for tumor cell imaging. In vitro cellular uptakes of such SiO2 nanocomposites areinvestigated with fluorescence microscope, which demonstrate much higher internalization of the folate-decorated SiO2 nanocomposites by Hela cancer cells which are of over-expression of folate receptors thanthe cellular uptake by NIH 3T3 fibroblast cells which are of low expression of folate receptors. Magneticmanipulation, fluorescence imaging, hollow structure, and cell targeting are simultaneously possible usinga multifunctional silica nanocomposite. Our results demonstrate a robust hydrophobic nanoparticles-basedapproach for preparing multifunctional and biocompatible hollow silica composites, which could be alsosuitable for silica coating of other kinds of nanoparticles.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

In recent years, multifunctional nanoscaled particulates in a sin-gle entity have attracted a great deal of attention for their diagnosticand biomedical researches. Particularly, luminescent and magneticnanocomposites provide a new platform for both bioimaging andtreatment of disease due to their enhanced multifunctional proper-ties in contrast with their single use [1–5]. Among all of the lumines-cent particles and magnetic particles, iron oxide nanoparticles andquantum dots (QDs), interesting advanced nanomaterials, have beenextensively investigated. The unique optical properties of quantumdots make them apply in biolabeling and biomedical research. Com-pared with traditional organic fluorophores, quantum dots havesuperior optical properties, for example, a narrow, tunable, symmet-ric emission spectrum, broadband excitation, high photobleachingthreshold, and good chemical stability [6–8]. Meanwhile, magneticiron oxide nanoparticles have also been proved promising in a widerang of biomedical applications such as cell separation, magneticresonance imaging (MRI), magnetically assisted drug delivery, and

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tissue repair [9–11]. So far, various approaches have been developedto synthesize magnetic and fluorescent silica nanocomposites[12,13]. For example, Wang et al. reported a method to make varioustypes of nanoparticles, including CdTe quantum dots, Au nanoparti-cles and Fe3O4 nanoparticles, assemble on silica microspheres. Theproperties of the assembled nanoparticles were well retained inthe nanocomposite assemblies, and the controllable integration ofmagnetic and fluorescent properties was achieved [14]. Veronicaand his co-workers described a synthesis of composite silica sphereswith magnetic and luminescent functionalities by a modified Stö-ber’s method combined with layer-by-layer assembly technique[15]. Even now, progress in functionalization of silica composites ismade continuously. A lot of great efforts have been made to preparesilica spheres with more function.

Nowadays, the use of hollow structure silica nanocomposites as apromising nanostructured material is of intense interest owing totheir various applications, such as drug delivery and controlled stor-age and release [16,17]. The emulsion droplets are very effective tem-plates for obtaining hollow mesoporous silica spheres of differentstructures and sizes. The emulsion droplets systems include manymodes such as water-in-oil [18], oil-in-water [19–21], supercriticalCO2-in-water [22,23], and water–oil–water [24]. For example, Fowleret al. have reported a facile synthesis of hollow spherical shell with

L. Sun et al. / Journal of Colloid and Interface Science 350 (2010) 90–98 91

ordered mesoporous [25]. The structures were synthesized at roomtemperature by hydrolysis of TEOS in an aqueous solution of hexade-cyltrimethylammonium bromide (CTAB) as the template.

In this paper, we describe the synthesis of folate-conjugated mag-netic and luminescent hollow silica nanocomposites which are de-signed for cancer cells fluorescence imaging. CTAB-stabilized Fe3O4

nanoparticles and QDs are used as templates and tetraethoxysilane(TEOS) is used as a precursor to obtain hollow silica nanocomposites.Folic acids (FAs), high affinity ligand to folate receptors (FRs), are use-ful targeting agent for tumor specific delivery, because FRs are overex-pressed in many human cancers, including the malignancies of theovary, lung, brain, breast, nose, kidney, prostate, colon and throat[26]. Moreover, folate acids are highly stable, low-cost and nonimmu-nogenic. In vitro experiments on cellular uptake of the folate-conju-gated hollow silica nanocomposites are carried out by using Helacancer cells and NIH 3T3 fibroblast cells. Hela cells are used to testthe targeting effect due to the over-expression of folate receptors onthe cell surface. NIH 3T3 cells with low folate receptor are used as anegative control. Motivated by the previously described advantages,we combine several functions, magnetism, fluorescence, hollownanostructures and targeting agent, into one silica nanocomposites.The multifunctional nanomaterials cannot only show combinedproperties of the original components but also possess novel and col-lective performances not seen in the original components. The nano-composites have great potentials in bioimaging, drug delivery, andother biomedical areas, because it can be simultaneously manipu-lated by an applied external magnetic field and characterized by usingfluorescence microscopy. The hollow nanostructures also possessdrug loading and releasing capabilities. The use of multifunctionalmagnetic and luminescent hollow silica nanocomposites will im-prove further diagnostic effectiveness and reduce side effects in drugdelivery. In the following section, the synthesis, magnetic properties,optical characterization, morphology, and application in bioimagingof the silica nanocomposites are presented.

2. Materials and methods

2.1. Materials

FeCl3, FeCl2�4H2O, ammonium hydroxide (25 wt.%), tetraeth-oxysilane (TEOS), cetyltrimethylammonium bromide (CTAB), N,N’-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS),and folic acid were obtained from Shanghai Chemical ReagentCompany. Anhydrous ethanol, oleic acid, and toluene were pur-chased from Beijing Chemical Company. Three-aminopropyltrieth-oxysilane (APTS, 99%) was obtained from Acros Organics. Thehydrophobic CdSe/CdS quantum dots (CdSeS QDs) capped withoctadecylamine (ODA) ligands were provided by Dr. Y. AndrewWang, Principle Scientist of Ocean Nanotech, USA (quantum yield,QY: 42% in hexane versus Rhodamine 640). In all experiments, thechemicals were analytical grade and all reagents were used asreceived without any further purification.

2.2. Synthesis of Fe3O4 nanoparticles

Fe3O4 nanoparticles were prepared as reported previously byMassart [27], based on the coprecipitation of FeCl3 and FeCl2 (mo-lar ratio = 1: 2) by adding a concentrated ammonium hydroxidesolution. An aqueous solution (150 mL) containing FeCl2�4H2O(1.113 g) and FeCl3 (1.817 g) in a flask was heated to 50 �C withN2 bubbling for deaeration of O2, and then 12.5 mL of ammoniumhydroxide (25%) was added under vigorous stirring. After 30 min,precipitates were collected by a magnet and washed three timeswith deionized water. The Fe3O4 nanoparticles were redispersed

in water (170 mL) with the aid of ultrasound. After addition of oleicacid (2.0 mL), the dark suspension was stirred for 2 h at 75 �C un-der N2. The resulting black precipitates were washed with ethanolfive times to remove excess oleic acid and then dispersed in cyclo-hexane. After drying with N2, oleic acid modified Fe3O4 nanoparti-cles were obtained.

2.3. Synthesis of magnetic and fluorescent hollow silica nanocom-posites

Briefly, 4.0 mg of CdSeS QDs nanoparticles dispersed in 1 mL ofchloroform was added to a 5 mL of aqueous solution containing0.1 g of CTAB. After vigorous stirring of the resulting solution, ahomogeneous oil-in-water microemulsion was obtained. Heatingat 60 �C for 10 min induced evaporation of the chloroform, whichgenerated aqueous-phase dispersed nanoparticles. 1.4 mg of oleicacid stabilized magnetite nanoparticles in chloroform (2.5 mL)were transferred to 5 mL of aqueous solution using 0.1 g CTAB bythe same method. 0.3 mL of CTAB-stabilized magnetite nanoparti-cles aqueous solution and 0.3 mL of CTAB-stabilized CdSeS QDsaqueous solution were added simultaneously into mixed water(5 mL)-ethanol (5 mL) solvent. Then 0.3 mL of ammonium hydrox-ide was added to the solution and 50 lL of TEOS was slowly added.The resulting mixture was stirred for 1 min, and then aged for 4 h.The silica nanocomposites were collected by centrifugation andwashed with water and ethanol for three times.

2.4. Synthesis of the folate-conjugated SiO2 nanocomposites

The folate acid conjugated silica nanocomposites were preparedby reported method [28]. To attach folic acid to silica, 15 mg of sil-ica composites were washed with DMSO and resuspended inDMSO. In a flask, 0.1 mg of folic acid and 0.05 lL of APTS weremixed in 1 mL of DMSO. Next, NHS and DCC (molar ratio ofFAs/NHS/DCC = 1:1:2.5) were added into the mixture and stirredgently for 2 h at room temperature. In a separate flask containing4 mL of toluene and the silica nanocomposites-DMSO suspension,the folate-APTS solution was added, and the mixture was stirredfor 20 h at room temperature. The materials were obtained by cen-trifugation, and washed three times with ethanol.

2.5. Cytotoxicity of the SiO2 nanocomposites

The in vitro cytotoxicity was measured by using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)assay in Hela cells. Cells (1 � 105 well�1) were inoculated into a96-well cell-culture plate and then incubated at 37 �C in a 5%CO2-humidified incubator for 24 h. 10 lL of SiO2 nanocompositeswith different concentrations (10–100 lg mL�1, diluted in Dul-becco’s modified Eagle’s medium (DMEM)) were added to thewells, separately. After incubation for 24 h at 37 �C under 5% CO2,the supernatant was removed, and the cells were washed withPBS for three times. Subsequently, MTT (10 lL, 5 mg mL�1) solvedin DMEM (90 lL) were added and the plates were incubated at37 �C for another 4 h. Then supernatant was removed before DMSOwas added to each well to dissolve the formazan. The absorbanceat 570 nm was detected with spectrophotometric microplate read-er (BioRad Model 550). Each data point was collected by averagingthat of six wells, and the untreated cells were used as controls. Thefollowing equation was used to calculate the inhibition of cellviability.

Cell viability ð%Þ ¼ mean absorption of value treatment groupmean absorption value of control

� �

� 100

92 L. Sun et al. / Journal of Colloid and Interface Science 350 (2010) 90–98

2.6. Cell Labeling and fluorescent imaging

Hela cancer cells that overexpress folate receptor (positivecontrol) and NIH 3T3 fibroblast cells that have less folate receptor(negative control) were propagated in DMEM containing fetal bo-vine serum (FBS, 10%) and penicillin/streptomycin (1%). Then thecells were digested and resuspended in the DMEM medium (withoutFAs). 1 � 104 cells were transferred into a 24-well tissue cultureplates. After 24 h of incubation, the cells were carefully rinsed withPBS (0.01 mol L�1, pH 7.4). 200 lL of DMEM medium (without FBSand FAs) and the FAs–SiO2 conjugate (20 lL, 0.1 mg mL�1) wereadded to the Petri dishes and incubated for 4 h and no folate-modi-fied SiO2 nanocomposites were used as control. The labeled cellswere carefully rinsed with PBS to remove the unbound FAs–SiO2,and the fresh serum-free medium (200 lL) was added to the Petridishes. To preserve the cellular viability, the cells were further incu-bated for 10 min, then the cell nucleus were labeled by DAPI (20 lL,1 lg mL�1) for 10 min. The fluorescent imaging of Hela cells and NIH3T3 cells was performed on an IX71 inverted fluorescence micro-scope (Olympus).

2.7. Characterization

The structure and component of silica nanocomposites werecharacterized by field-emission scanning electron microscopy (FES-EM; FEI XL30), transmission electron microscopy (TEM; HitachiS-570), energy-dispersive X-ray analysis (EDAX, FEI XL30), andX-ray diffraction (XRD; DXP-18AHF diffractometer with Cu Ka radi-ation). The photoluminescent emission spectra were recorded atroom temperature with a Hitachi F-4500 spectrophotometerequipped with a continuous 150 W Xe-arc lamp. The measurementsof the X-ray photoelectron spectra (XPS) were performed by using aVG-Scientific ESCALAB 250 spectrometer with a monochromatic AlKa X-ray source at 1486.6 eV. The fluorescence images were per-formed on a fluorescence microscope (Olympus IX71) excited byan ultraviolet source.

3. Results and discussion

3.1. Synthesis and characterization of silica nanocomposites

The synthesis of monodisperse silica spheres was first describedby Stöber et al. employing a water–alcohol–ammonia–tetraalkox-ysilane system [29]. Stöber’s method was often further modifiedthrough introducing cationic surfactants such as CTAB [30]. Zhanget al. have reported a facile synthesis of mesoporous silica with avariety of morphologies through the ammonia-catalyzed hydroly-sis of TEOS in mixed water–ethanol solvent at room temperatureusing CTAB as the template [31]. Herein, we use CTAB-stabilizednanoparticles (Fe3O4 and CdSeS QDs) as the template to synthesizemultifunctional hollow silica. Scheme 1 shows the procedures forpreparation of nanoparticles embedded hollow silica spheres. Toconduct sol–gel reaction to form silica spheres, it is necessary totransfer these hydrophobic ligand-capped nanoparticles from or-ganic phase to aqueous phase. The hydrophobic nanoparticles dis-solved in organic solvent are transferred to water phase by mixingthem with an aqueous CTAB solution and evaporating the organicsolvent [32–34]. The water-soluble nanoparticles are obtained asshown in Fig. 1. Although the PL intensity of the CTAB-stabilizedCdSeS QDs in water has a slight decrease (Fig. S2), from theFig. 1e and f, we clearly observe the CdSeS QDs in water exhibitgood spectral quality. CTAB-stabilized nanoparticles acted as seedsfor the formation of spherical silica. The emulsion containing nano-particles (Fe3O4 and CdSeS QDs) is transferred into the mixed solu-tion of ethanol and water. The CTAB-stabilized oil (TEOS)-in-water

emulsion droplets are formed, which act as the template for theformation of the hollow silica shells. The surfactant CTAB playsthe role of interfacial stabilizer for stabilizing the emulsion dropletat the oil–water interface. CTAB also participates in the hydrolysisand condensation of TEOS, leading to formation of the hollow silicashells in the sol–gel reaction. Because of the catalysis of ammo-nium hydroxide, hydrolyzation and condensation of TEOS resultsin the formation of hollow nanostructures at oil/water interface.The synthesis procedures rely on two actions of CTAB: the stabi-lized surfactant for transfer of the nanoparticles to aqueous andthe organic template for the formation of the hollow silica sphere.Magnetite nanoparticles and quantum dots are simultaneouslyembedded in silica spheres.

The optical properties of the CdSe/CdS QDs and hollow silicananocomposites are investigated using the PL spectra. The PL spec-tra of the silica nanocomposites in water solution present a slightblue shift as compared with the CdSeS QDs in chloroform inFig. 2. The shift may be related to solvent effects (usually the sol-vent polarity), the presence of surface charges (e.g., the chargedCTAB), and the changing of QD environment (e.g., SiO2), whichhave been proven to cause the shift of QD spectra [35–37]. A slightblue shift is observed that could be attributed to a change in sur-face states of the QDs due to the immobilization in SiO2 nanocom-posite [38,39].

Diameters of the CTAB-stabilized Fe3O4 nanoparticles are in therange of 6–16 nm as shown by TEM observations in Fig. 3a. TheCTAB molecule can be confirmed by zeta potential measurements(Fig. S1). Because the Fe3O4 nanoparticles are stabilized with theCTAB, the zeta potential is obviously increased. Magnetic ironoxide nanoparticles have a large surface-tovolume ratio and there-fore possess high surface energies. Consequently, they tend toaggregate so as to minimize the surface energies. Moreover, thenaked iron oxide nanoparticles have high chemical activity, andare easily oxidized in air, generally resulting in loss of magnetismand dispersibility. Therefore, providing proper surface coating anddeveloping some effective protection strategies to keep the stabil-ity of magnetic iron oxide nanoparticles is very important. Oleicacid is often employed to passivate the surface of the iron oxidenanoparticles during or after the preparing procedure to avoidagglomeration. Oleic acid is also widely used in ferrite nanoparti-cles synthesis because it can form a dense protective monolayer,thereby producing highly uniform and monodisperse particles[40–44]. After Fe3O4 nanoparticles are surface modified with theuse of oleic acid and CTAB, the nanoparticles are dispersed wellin water. Moreover, CTAB also serves as organic template for theformation of the hollow silica spheres in the sol–gel reaction.

SEM imaging (Fig. 3b) shows that the silica nanocomposites arequite uniform in size with an average diameter of about 600 nm.The surface-functionalized 100 nm silica nanoparticles can be inter-nalized by Hela cells. The toxicity of amorphous silica is related toparticle size, and the amorphous silica has been found to inducecytotoxicity when their sizes are below 100 nm [45,46]. Smaller par-ticle size may lead to enhanced nonspecific cellular uptake accordingto above mentioned. We use folic acids as the targeting ligand. Helacells are used as model cancer cells, as Hela cells express the folatereceptor. To verify that the uptake is mediated via the folate receptor,nonspecific cellular uptake should be avoided. Thus, the size of silicananocomposites prepared by our method can be used in bioimages,whereas small particles shows enhanced unspecific cellular uptake.

In general, only a few deformed or cracked structures are ob-served but when imaged these, as well as samples that have beenmechanically fractured, indicates that the silica nanocompositespossess hollow structure, as shown for example in Fig. 3c. TEMimages (Fig. 3d and e) also indicate a core–shell structure and sug-gest that the hollow silica occurs at the interface of oil/water. TEMimages are consistent with the SEM observation and showed the

Scheme 1. The procedures for preparation of hollow silica spheres.

Fig. 1. CTAB-stabilized water-soluble Fe3O4 nanocrystals (a), oleate-capped Fe3O4

nanocrystals (b), ODA-capped CdSeS QDs in natural light (c), UV irradiation at365 nm (d), CTAB-stabilized CdSeS QDs in natural light (e), and UV irradiation at365 nm (f).

Fig. 2. PL spectra (kex = 232 nm) of SiO2 nanocomposites in water (red line,kem,max = 617 nm) and CdSeS QDs in chloroform (black line, kem,max = 620 nm).(For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

L. Sun et al. / Journal of Colloid and Interface Science 350 (2010) 90–98 93

hollow nanostructure spheres. Investigated from the images ofFig. 3d and e, the shell thickness is about 90 nm. The silica shellssuccessfully preserve the magnetic and luminescence propertiesand limit the toxicity and degradation in a biosystem.

The iron oxide nanoparticles and CdSeS QDs in the hollow silicananocomposites are investigated by energy-dispersive X-rayanalysis (EDAX) and the X-ray diffraction (XRD). As shown inFig. 4, the energy-dispersive X-ray (EDAX) microanalysis patternshows the presence of C, O, Si, S, Fe, Cd, and Se in the samples of hol-low silica nanocomposites, which effectively proves that the Fe3O4

nanoparticles and CdSeS are successfully coated by silica. XRD mea-surement confirms the presence of silica as the shell of the nanocom-posites. Fig. 5a and b exhibit the XRD spectra of Fe3O4 nanoparticlesand silica nanocomposites, respectively. As shown in Fig. 5, XRD pat-terns of the oleic modified iron oxide nanoparticles and the silicananocomposites show characteristic (2 2 0), (3 1 1), (4 0 0), (5 1 1),

Fig. 3. TEM (a) images of CTAB-stabilized Fe3O4 nanoparticles, and FESEM (b and c) and TEM (d and e) images of SiO2 nanocomposites.

94 L. Sun et al. / Journal of Colloid and Interface Science 350 (2010) 90–98

and (4 4 0) peaks. The XRD pattern is in good agreement with thedata for pure cubic Fe3O4, as reported in the JCPDS card (No.88–315, a = 8.375). This indicates that the Fe3O4 nanoparticles areobtained. Fe3O4 nanoparticles are successfully added to the hollowsilica microspheres. The CdSeS QDs of silica composite spheres arenot displayed clearly in the XRD pattern because the hollow silicamicrospheres only contain a few CdSeS QDs. The broad peak at22–28� in Fig. 5b corresponds to the silica, which indicates that thesilica is successfully coated on the surface of the nanoparticles.

The successful fabrication of the magnetic and fluorescent hol-low silica sphere is further verified by several photographs. Be-cause of the magnetic iron oxide, the composite silica can bedirected to specific locations when manipulated by an externalmagnetic field, which, in this case, can be easily monitored through

the intense luminescence of the composite particles. Fig. 6 shows aseries of photographs of an aqueous dispersion of silica compositesunder UV illumination (365 nm) and natural light and illustratesthe mentioned manipulation ability. While in the absence of amagnetic field the luminescence is observed from the whole dis-persion (Fig. 6a), when a handheld magnet is placed, the particlesaccumulate near it (Fig. 6b). Once the particles have been concen-trated close to the magnet, they can be easily dragged by movingthe magnet to a different position. Fig. 6a and b clearly suggest thatthe silica nanocomposites can be simultaneously manipulated byan applied external magnetic field and characterized by using fluo-rescence microscopy. The field-dependent magnetism of the silicananocomposites at 300 K shows no hysteresis (Fig. S3), demon-strating that they are superparamagnetic [47,48]. These materials

Fig. 4. EDAX results for Fe3O4/CdSeS/SiO2 nanocomposites.

Fig. 5. XRD patterns of (a) the oleate modified iron oxide nanoparticles and (b) thesilica nanocomposites.

Fig. 6. Photographs of magnetic and fluorescent silica spheres driven by external magnethandheld magnet placed (b).

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provide a robust framework and more components may be incor-porated to give multifunctional capabilities.

3.2. Cytotoxicity of the silica nanocomposites

By MTT assay, the cytotoxicity of the silica nanocomposites isdetermined on Hela cells. The results show that cell viability is great-er than 90% at concentration up to 70 lg mL�1 after 24 h of incuba-tion (Fig. 7). These datas show that the silica nanocomposites can beapplied in bioimaging and considered to possess low cytotoxicity.

3.3. Surface chemistry

Folic acid is used as the targeting component in this paper. Thecarboxyl group on the folic acid is conjugated with the amine groupon the aminopropyltriethoxysilane by dicyclohexylcarbodiimide(DCC) and N-hydroxysuccinimide (NHS) chemical strategy and thenthe folate-APTS is grafted onto the surface of the hollow silica nano-composites by the reported method [28,49]. The conjugation of folicacid on the SiO2 nanocomposites surface is proved via N1s signals(Fig. 8). Only one peak is detected from the no folate-modified SiO2

nanocomposites (Fig. 8a), which can be attributed to CTAB. TheN1s peak is now at 402.8 eV. This value is slightly higher that for pureCTAB (402 eV), indicative of the interaction between CTAB and thenanoparticles surface [50,51]. As shown in Fig. 8b and c, two peaks(left: the amide bond between APTS and folate, right: folic acid)are detected from the FAs–SiO2 nanocomposites [52–54]. Thereforeit can be used to confirm the successful conjugation of folic acid onthe SiO2 nanocomposites surface, which could be assumed that thetargeting ligands can be detected by corresponding receptors on cellmembranes. Because the folate-APTS are coated onto the surface ofthe silica nanocomposites, the N1s peaks of the CTAB (Fig. 8b) obvi-ously decrease in compare with the no folate-modified SiO2 nano-composites (Fig. 8a).

3.4. Application in fluorescent imaging

To prove the folate-receptor-mediated targeted delivery, weinvestigate FRs-positive Hela cells and FRs-negative NIH 3T3 cells.The effect of folic acid modification on the cellular uptake of silicananocomposites is studied with the cancer cells. As shown in Fig. 9,

field in natural light and UV irradiation at 365 nm. No magnet field applied (a) and a

Fig. 7. In vitro cell viability of Hela cells incubated with the silica nanocompositesat different concentrations for 24 h.

96 L. Sun et al. / Journal of Colloid and Interface Science 350 (2010) 90–98

Hela cells are labeled with FAs–SiO2 nanocomposites (Fig. 9a) andSiO2 nanocomposites without folic acid conjugated (Fig. 9b) after4 h of incubation at 37 �C. Confocal microscopy shows that theHela cells incubated with FAs–SiO2 nanocomposites are examinedto find a strong luminescence signal. In contrast, Fig. 9b shows thatsmall amount of SiO2 nanocomposites without folic acid conju-

Fig. 8. The XPS N1s spectra for the SiO2 nanocomposites

gated are internalized by Hela cells. Fig. 9d shows the Hela cellswithout any treatment. These results present strong evidencesabout target effects of FAs–SiO2 nanocomposites for Hela cells.

To further confirm the targeting effect of FAs–SiO2 nanocom-posites, the NIH 3T3 cells which lack folate receptors are treatedwith SiO2 nanocomposites and imaged under similar conditions.The reduced uptake of SiO2 nanocomposites by NIH 3T3 cells isclearly shown in Fig. 9c, when compare with Hela cells under sameconditions (Fig. 9b). The luminescence intensity obtained from theHela cells is higher than obtained from the NIH 3T3 cells. These re-sults implied that the Hela cells expressed more folate receptorsthan the NIH 3T3 cells did and that the FAs–SiO2 nanocompositesselectively accumulated on the surface of the FRs-positive Helacells. Hence, these results confirm that objective of increasingspecificity and sensitivity of FAs–SiO2 nanocomposites image bylabeling cancer cells with over-expression of folate receptors onthe surface was achieved.

4. Conclusions

In summary, we have synthesized multifunctional silica nano-composites that exhibited essential properties requisite for poten-tial biological applications, such as magnetism, fluorescence andhollow nanostructure. The silica nanocomposites are further con-jugated with folic acid for bioimaging in Hela cells. Fe3O4 magneticnanoparticles have great prospects in drug delivery, cell separationand magnetic resonance imaging and QDs have been attractedmore attention in biolabeling. The nanoparticles are protected by

(a), FAs–SiO2 nanocomposites (b), and folic acid (c).

Fig. 9. Hela cells treated with folic acid conjugated SiO2 (a), no folate-modified SiO2 (b), NIH 3T3 cells treated with folic acid conjugated SiO2 (c), and Hela cells without anytreatment (d).

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the hollow silica shell. Furthermore, the hollow nanostructures canbe applied in drug loading and controlling storage and releasing.Therefore, the nanocomposites are expected to find potential appli-cations in magnetically guiding and optically tracking the deliveryof drugs and genes.

Acknowledgments

The authors gratefully acknowledge the support of the NationalNatural Science Foundation of China (Nos. 20874033 & 30700998).We thank Mr. Y.A. Wang for providing the quantum dot sampleswith ODA ligands.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jcis.2010.06.041.

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