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Colloids and Surfaces B: Biointerfaces 143 (2016) 224–232

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fb

heranostic MUC-1 aptamer targeted gold coated superparamagneticron oxide nanoparticles for magnetic resonance imaging andhotothermal therapy of colon cancer

orteza Azhdarzadeha,b, Fatemeh Atyabia,b, Amir Ata Saei c,ehrang Shiri Varnamkhasti a,b, Yadollah Omidid, Mohsen Fatehe, Mahdi Ghavamif,aeed Shanehsazzadehg, Rassoul Dinarvanda,b,∗

Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417614411, IranDepartment of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417614411, IranDepartment of Medical Biochemistry & Biophysics, Karolinska Institutet, Stockholm, SwedenResearch Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, IranMedical Laser Research Center, Academic Center for Education, Culture, and Research (ACECR), Tehran, IranDepartment of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Health Science Faculty, Blegdamsvej 3c, 2200openhagen N, DenmarkNuclear Science and Technology Research Center (NSTRI), Tehran, Iran

r t i c l e i n f o

rticle history:eceived 4 November 2015eceived in revised form 6 February 2016ccepted 25 February 2016vailable online 27 February 2016

eywords:UC1 aptamerRI

rotein coronaPIONheranostics

a b s t r a c t

Favorable physiochemical properties and the capability to accommodate targeting moieties make super-paramegnetic iron oxide nanoparticles (SPIONs) popular theranostic agents. In this study, we engineeredSPIONs for magnetic resonance imaging (MRI) and photothermal therapy of colon cancer cells. SPIONswere synthesized by microemulsion method and were then coated with gold to reduce their cytotoxic-ity and to confer photothermal capabilities. Subsequently, the NPs were conjugated with thiol modifiedMUC-1 aptamers. The resulting NPs were spherical, monodisperse and about 19 nm in size, as shown bydifferential light scattering (DLS) and transmission electron microscopy (TEM). UV and X-ray photoelec-tron spectroscopy (XPS) confirmed the successful gold coating. MTT results showed that Au@SPIONs haveinsignificant cytotoxicity at the concentration range of 10–100 �g/ml (P > 0.05) and that NPs covered withprotein corona exerted lower cytotoxicity than bare NPs. Furthermore, confocal microscopy confirmedthe higher uptake of aptamer-Au@SPIONs in comparison with non-targeted SPIONs. MR imaging revealed

anoparticles that SPIONs produced significant contrast enhancement in vitro and they could be exploited as contrastagents. Finally, cells treated with aptamer-Au@SPIONs exhibited a higher death rate compared to controlcells upon exposure to near infrared light (NIR). In conclusion, MUC1-aptamer targeted Au@SPIONs couldserve as promising theranostic agents for simultaneous MR imaging and photothermal therapy of cancercells.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

Colon cancer is the third most common type of cancer and is

ssociated with a high mortality rate [1]. Similar to other types ofancer, effective treatment of colon cancer depends on early detec-ion and treatment. Among different nanoscale contrast agents,

∗ Corresponding author at: Faculty of Pharmacy, Tehran University of Medicalciences, Tehran 1417614411, Iran.

E-mail address: dinarvand@tums.ac.ir (R. Dinarvand).

ttp://dx.doi.org/10.1016/j.colsurfb.2016.02.058927-7765/© 2016 Elsevier B.V. All rights reserved.

SPIONs have attracted a great deal of attention because of theirunique superparamagnetism, desirable physiochemical properties,biocompatibility and biodegradability [2]. SPIONs are the mostattractive MRI contrast agents owing to their superparamegneticbehavior [3].

However, non-targeted SPIONs are nonspecific and cannot accu-mulate inside the tumor and thus, they are only suitable for general

imaging applications. To improve the localization of SPIONs in thevicinity of cancer cells, they are usually conjugated with tumortargeting moieties such as monoclonal antibodies, antibody frag-ments, aptamers, cell penetrating peptides and small molecules

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M. Azhdarzadeh et al. / Colloids and Su

uch as folic acid. Not only targeting increases the tumor accu-ulation of NPs, but it also decreases the off target side effects.hile antibodies are the most specific targeting moieties, they

ntail a complicated manufacturing process and can be potentiallymmunogenic [4]. These issues can make their regulatory approvalrocess a real challenge for the engineered imaging contrast agentsunctionalized with monoclonal antibodies. Furthermore, antibod-es increase the hydrodynamic size of the NPs, reducing the chancesf cellular internalization [5,6]. The increase in size is also knowno offset the NPs’ “stealth” characteristics, leading to higher uptakeith cells of the reticuloendothelial system [7]. Antibody fragments

lso suffer from molecule and manufacturing complexities. Thisight be the reason why antibody fragments have been rarely used

or functionalization of SPIONs for imaging applications [8]. On thether hand, cell penetrating peptides and small molecules are read-ly available, but they are usually not very specific for a particularumor. For example, folate receptor is generally overexpressed inancer cells and is not specific to a certain type of cancer [9].

In this context, aptamers are arguably the most attractive tar-eting agents. Aptamers are relatively small oligonucleotides thatecognize specific cellular targets. For example, highly specificptamers have been shown to differentiate between subtypes ofon-small cell lung cancer [10]. Another advantage of aptamersver larger targeting moieties is the fact that a higher number ofptamers can be conjugated onto the surface of a given SPION,hich also allows for multiple binding and synergistic affinity [11].

To increase the biocompatibility of SPIONs, the NPs cores aresually coated. Inorganic coatings such as gold and silica can sta-ilize the SPION core and reduce the effect of environment on NPsegradation and corrosion [12]. The added benefits of a gold coatingre that the engineered NPs can be used for photothermal therapyf tumor [13] and the gold coated NPs can be tracked using Fourierransform infrared spectroscopy (FTIR) [14], making the whole NPystem a multimodal imaging probe.

In the current study, SPIONs were synthesized using aicroemulsion method and were then modified by a gold coat-

ng. The thiol modified oligonucleotide MUC-1 aptamer was furtheronjugated onto Au@SPIONs as a targeting agent. The aberrant gly-osylated form of mucin serves as a good epithelial tumor cellurface marker. The targeting efficiency of NPs was investigatedn vitro by confocal microscopy and flow cytometry. The appli-ability of photothermal therapy was assessed by comparing theiability of aptamer-Au@SPIONs-treated cells with untreated cells.

. Materials and methods

.1. Materials

FeCl2·4H2O, FeCl3·6H2O, cethyltrimethylammonium bro-ide (CTAB), 1-butanol, gold chloride solution (HAuCl4),H2OH·HCl, dithiotreitol (DTT), sodium citrate, DAPI dye, Trisuffer acetate-EDTA (TAE) and MTT (3-(4,5-dimethylthiazol-2-yl)-,5-diphenyltetrazolium bromide) were purchased from Sigma,SA. HT-29 human colon cancer cell line, L929 mouse fibroblastell line and CHO (Chinese hamster ovarian) cell line were obtainedrom Pasteur Institute, Iran. DMEM medium and fetal bovine serumFBS) were provided by Gibco (Grand Island, USA). Penicillin andtreptomycin were purchased from Sigma, USA. MUC-1 Aptameras obtained from TAG Copenhagen A/S, Denmark. LysoTrackered DND-99 and ethidium bromide were supplied by Life Tech-ologies, USA. Amicon ultracentrifugal tube-Millipore (10 KDa),

garose, gelatin, toluene, sodium dodecyl sulphate (SDS), dimethylulfoxide (DMSO), 2-mercaptoethanol (2-ME) and bromophenollue were purchased from Merck, Germany. All other chemicalsere of analytical grade.

B: Biointerfaces 143 (2016) 224–232 225

2.2. SPION synthesis

SPIONs were synthesized by a microemulsion method accord-ing to Panahifar et al. [15] with minor modifications. Briefly, twomicroemulsions (A and B) were prepared. Toluene (29 ml) 1.8 g andCTAB were added to beaker A as oil phase and as surfactant, respec-tively. FeCl3 (202 mg) and FeCl2 (75 mg) were added at a molarratio of 2:1 to 2 ml of deionized (DI) water and poured as aqueousphase into beaker A. Beaker B was composed of toluene in the sameamount as oil phase plus 1.8 g CTAB and 25% ammonium hydroxidesolution (2.65 ml). Subsequently, each beaker was separately mixedat 7000 rpm using a homogenizer and titrated by approximately1.5 ml 1-butanol (as co-surfactant) until the color became trans-parent. Afterwards, microemulsion A was added to a three-neckedflask while it was homogenized at 7000 rpm under constant flowof N2 gas at 50 ◦C. Then, microemulsion B was added to the set andhomogenized for 60 min. The reaction was stopped and the blackproduct was cooled down to room temperature and 25 ml ethanolwas added to the flask. After collection of the prepared SPIONs usinga magnet, they were washed with boiling ethanol (4X), acetone (2X)and DI water (2X) to remove non-reacted agents. Finally, the SPIONswere precipitated by centrifugation at 5000 rpm and redispersed inDI water.

2.3. Gold coating of SPIONs

Prepared SPIONs were coated by gold according to a methodby Lyon et al. [16] with little modifications. By considering thatfreshly prepared Fe3O4 NPs have very little tendency for gold coat-ing, Fe3O4 NPs were oxidized to �-Fe2O3 by heating and exposingthem to air under stirring for 30 min. Then, the �-Fe2O3 NP solu-tion was diluted by water to the concentration of 1.1 mM and afteraddition of an equal volume of 0.1 M sodium citrate, the mixturewas stirred for 10 min. Afterwards, the �-Fe2O3 NPs solution wasdiluted five times with water and an aliquot of 0.2% HAuCl4 and0.2 M NH2OH·HCl were incrementally added with 10 min inter-vals according to Table S1. After addition of HAuCl4 solution andNH2OH·HCl, the solution color turned into purple.

2.4. Conjugation of MUC-1 aptamer

Aptamer-NPs were prepared by adding thiol modified MUC-1aptamer (5′-HS-C6-GAG/ACA/AGA/ATA/AAC/GCT/CAA/GAA/GTG/AAA/ATG/ACA/GAA/CAC/AAC/ATT/CGA/CAG/GAG/GCT/CAC/AAC/AGGC-3′) to the NP solution. For this purpose, thiol modifiedaptamers were first activated by adding DTT and activatedaptamers were separated using a 10 KDa Amicon tube and resus-pended in Tris Buffer (50 mM Tris, 100 mM NaCl). Subsequently,25 �M activated aptamer solution was added to 0.5 ml of NPs(50 �g/ml) and incubated under shaking for 16 h. The solutionwas centrifuged at 16,000g for 25 min to separate aptamer-NPconjugates from unreacted aptamer. The precipitated aptamer-NPswere resuspended in Tris buffer and aptamer attachment wasevaluated by agarose gel electrophoresis.

2.5. Cell culture

Cells were grown in DMEM supplemented with 10% FBS plus100 units/mL penicillin/streptomycin and incubated at 37 ◦C in 5%CO2.

2.6. MTT assay

Upon entrance to a biological fluid such as plasma, NPs arerapidly covered with existing proteins, giving rise to the formationof a so called “protein corona” around NPs [17]. Therefore, in this

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26 M. Azhdarzadeh et al. / Colloids and Su

tudy, we evaluated the cellular viability upon exposure to bothare and protein corona coated SPIONs. Two type of protein coronaoated SPIONs were prepared by incubating Au@SPIONs with 10%nd 100% FBS at 37 ◦C for 1 h. Then, the dispersions were centrifugedt 12000 rpm at 15 ◦C for 30 min [18]. The supernatants were sep-rated and the formed NP-protein complexes were resuspendedn PBS and centrifuged twice at 12000 rpm at 15 ◦C for 30 min toemove loosely bound proteins (soft corona). Finally, hard coronaoated NPs were resuspended in PBS to reach the desired concen-ration. HT-29, CHO and L929 cells were seeded in 96 well plates at

seeding density of 104 per well. After 24 h, media were removednd cells were exposed to SPIONs in concentrations of 10, 100 and00 �g/ml for 24 and 48 h, in line with the normally used concentra-ions for SPIONs in biomedical applications. Media were removednd wells were washed with PBS. 100 �l of a 1 mg/ml MTT solutionas added to each well and the plate was incubated for 2 h. Then,MSO solution was added to each well to dissolve the formed for-azan crystals, and absorbance was read at 540 and 630 nm using

n ELISA reader (ELx800, BioTek instruments, USA). Four replicatesere used for each SPION and data were shown as mean ± SD.ell viability was calculated as mean absorbance in treated cellsivided by the mean absorbance of untreated controls (backgroundbsorbance is subtracted).

.7. NP uptake evaluation by confocal microscopy and flowytometry

For confocal imaging, cover glasses were placed in 6 well platesnd a 0.1% gelatin solution was poured into it and after 1 h, theemaining gelatin was discarded. Then, CHO and HT-29 cells wereeeded in wells and after 48 h, the media were removed and NPs50 �g/ml) (non-targeted Au@SPIONs and aptamer-Au@SPIONs)ere added to each well. After 6 h, media were discarded and

he cover glass was washed with phosphate buffered saline (PBS).or evaluation of NP uptake, the acidic organelles were stainedsing LysoTracker Red. The higher uptake of NPs will lead toigher staining of endosomes and lysosomes [19]. For staining step,00 nM LysTtracker Red dye was added to each well for 1 h andhen the solution was discarded and the cover glass was washedhree times with PBS, fixed by adding 0.4% formaldehyde solutionSigma) for 20 min and stained by DAPI solution for 5 min to stainhe nucleus. Finally, cover glasses were transferred onto the glasslides and visualized by Nikon confocal microscope A1 (Nikon Inc.,witzerland) armed with an A1 scan head and a standard detectorsing a 405 nm diode laser with DAPI filter and 543 nm diode laserMelles Griot, USA) with TRITC filter.

Flow cytometry was also used for quantitative measurement ofP uptake. Briefly, cells were seeded in 6 well plates and treatedith non-targeted Au@SPIONs and aptamer-Au@SPIONs for 6 h.

ubsequently, the NP suspension was discarded and the cells weretained by LysoTracker Red for 1 h. The cells were then trypsinized,entrifuged and resuspended in PBS and analyzed by flow cytome-ry (Partec PASII, Germany).

.8. Photothermal therapy with Au@SPIONs

HT-29 cells were seeded in a 6 well plate in a density of 4 × 105ell per each well and incubated in 1 ml of the medium with 100,00 and 500 �g/ml SPIONs at 37 ◦C with 5% CO2 for 12 h. Then theedium was removed and cells were washed with water and fresh

PMI medium was added. Subsequently, cells were irradiated with.7 W/cm2 NIR light (820 nm) via LED for 2, 4 and 8 min for pho-othermal treatments. After treatment, cell viability was assessedy MTT assay.

B: Biointerfaces 143 (2016) 224–232

3. Results

3.1. Size, morphology and zeta potential of SPIONs andAu@SPIONs

The average size of the bare SPIONs was around 19 nm, whichincreased to approximately 24 nm upon coating with gold, asassessed by TEM (Fig. 1(A)–(B)). Particle size distribution was givenby DLS measurements (Fig. 1(C)–(D)). The NPs possessed a ratherspherical morphology and after gold coating a narrow layer of goldcoating about 5 nm was deposited on NPs and the morphology stillremained spherical. Zeta potential of bare SPIONs was −13 mV andafter coating by gold dropped to −22 mV because of sodium citrateconsumption at this method.

3.2. Confirmation of gold coating via UV–vis spectroscopy andXPS

To evaluate gold coating, in the first step, a magnet was used toseparate Au@SPIONs from possibly formed AuNPs. The separatedAu@SPIONs were then resuspended in DI water and analyzed byUV–vis spectroscopy. While no peak was detected between 520and 590 nm for bare SPIONs in the acquired spectra (Fig. S1(A)),there was a peak at 580 nm (Fig. S2(B)) in Au@SPION spectra whichbelongs to the gold coating. Moreover, after each Au iteration, theintensity of the peak increased, which confirms an increase in Auto SPION ratio.

XPS is another technique which detects the surface elements ofparticles up to a 9 nm thickness. Fig. 2(A) presents the XPS surveyscan of SPIONs and Au@SPION. The Fe2p3 peak (715 eV) and Fe2p1(724 eV) peak (Fig. 2(B)) which belong to Fe3O4 are clearly observedfor both NPs. The Au binding energy for Au@SPION (Fig. 2(C)) at83 eV (Au 4f7/2) and 87 eV (Au 4f5/2) confirmed the formation ofthe gold coating around SPIONs, while no peak could be detectedat this points for bare SPIONs. These results confirm the successfuldeposition of gold onto SPIONs.

3.3. Evaluation of aptamer attachment by gel electrophoresis

Free aptamers, Au@SPIONs and aptamer-Au@SPIONs were runthrough agarose gel electrophoresis to confirm aptamer attach-ment onto the NPs. While no band was observed for Au@SPIONs,a wide band was noted for aptamer-Au@SPIONs at the agarosewell. The less movement of aptamer-Au@SPIONs compared to freeaptamers is expected, since NPs cannot freely move through the gelas free aptamer molecules do. Therefore, this experiment confirmsaptamer conjugation onto the NPs (Fig. S2).

3.4. SPION cytotoxicity

The cytotoxicity of SPIONs and SPIONs with hard corona atdifferent concentrations was assessed by MTT assay (Fig. 3). Expect-edly, all treated cell lines were more viable at low concentrations ofNPs and cytotoxicity increased by increasing NP concentration. NPscoated with protein corona exerted less cytotoxicity in comparisonwith bare NPs. (Fig. 3(A)–(F)).

3.5. Confirmation of SPION uptake using confocal microscopy

The cellular uptake of Au@SPIONs and aptamer-Au@SPIONs was

assessed in the MUC-1-positive HT-29 and MUC-1-negative CHOcell lines. After incubation of cells with NPs, their uptake wastracked by staining with LysoTracker Red. As shown in Fig. 4, HT-29 cells treated with aptamer-Au@SPIONs have higher fluorescent

M. Azhdarzadeh et al. / Colloids and Surfaces B: Biointerfaces 143 (2016) 224–232 227

Fig 1. TEM images of (A) bare and (B) Au@SPIONs. Histograms corresponding to NP size distribution have been shown in (C) for bare and in (D) for Au@SPIONs.

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Fig. 2. XPS spectra of bare SPIONs and Au@SPION. (A) Full-scan spectra of bare SPIONs and Au@SPION; (B) High resolution spectrum of Fe at Au@SPION and (C) High resolutionspectrum of Au 4f at Au@SPION which confirm the formation of gold coating.

228 M. Azhdarzadeh et al. / Colloids and Surfaces B: Biointerfaces 143 (2016) 224–232

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ntensity compared to those treated with Au@SPIONs, which con-rms the higher uptake of targeted SPIONs in these cells. This ishile there is no difference between the uptake of Au@SPIONs and

ptamer-Au@SPIONs in CHO cells (Fig. 4).

.6. Flow cytometry measurements

Additionally, we investigated the cellular uptake of aptamer-u@SPIONs in comparison with non-targeted Au@SPIONs in vitrosing flow cytometry. After treatment of HT-29 and CHO cells withPIONs, fluorescence intensity of cells was analyzed (Fig. 5) as com-ared to their non-targeted counterparts. In the HT-29 cell lines MUC-1 positive cell line, the mean fluorescence intensity for

ptamer-Au@SPIONs was 11.4 compared to 7.5 for non-targetedu@SPIONs. While in the MUC-1 negative CHO cells, the meanuorescence intensity for aptamer-Au@SPIONs and non-targetedu@SPIONs was comparable (8.88 versus 7.50, respectively).

over (A) 24 and (B) 48 h, CHO cell line over (C) 24 and (D) 48 h, and HT-29 cell line

3.7. MRI measurements

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed to determine iron concentration after NPsuptake by digesting samples with boiling HNO3. Iron concentra-tion results have been shown in Table 1. The aptamer-Au@SPIONswere internalized to a higher extent than non-targeted SPIONs.

To investigate the MRI contrast enhancement effect of NPs, thesignal intensity of HT-29 cell line was measured after treatmentwith NPs. As shown in Fig. 6(A) and (B), NPs decrease the signalintensity compared to the control untreated sample. In Tables 1and S2, the signal intensity of samples at different TR and TE hasbeen shown (P < 0.05).

3.8. Photothermal therapy application

Finally, the therapeutic effect of NPs for photothermal therapywas evaluated by applying LED. HT-29 cells were incubated with

M. Azhdarzadeh et al. / Colloids and Surfaces B: Biointerfaces 143 (2016) 224–232 229

Fig. 4. Confocal images showing lysosomes stained by LysoTracker Red as marker of NP uptake (nuclei stained blue by DAPI): HT-29 and CHO cell line treated by Au@SPIONsor aptamer-Au@SPIONs (all scale bars are 50 �m). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Table 1ICP results after treatment of cells with NPs and signal intensities (MRI) after treatment with NPs in various echo times (TR = 3000 ms, TE = 102 ms) (Mean ± SD).

Sample Iron content (mg/L)(Mean ± SD, n = 3)

Uptake ratio (sampleuptake/control uptake)

Signal intensity(Mean ± SD, n = 3)

Percentage of signaldecrements comparedto control group

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Control 0.790 ± 0.054 1

Au@SPIONs 1.875 ± 0.154 2.37

Aptamer-Au@SPIONs 2.925 ± 0.164 3.70

hree different concentrations of aptamer-Au@SPIONs and subse-uently irradiated by NIR light via a light emitting diode (LED)or 2, 4 and 8 min. Cells not exposed to irradiation were con-idered as controls. Results indicated that at high concentrations200–500 �g/ml) of SPIONs, cancerous cell were eradicated at 2, 4nd 8 min of irradiation, while at low concentrations (100 �g/ml),ytotoxicity was 94%, 90% and 79% after 2, 4 and 8 min of irradia-ion, respectively (Fig. 7). Furthermore, irradiating NP-free cells haslmost no effect on their viability. The most effective concentrationf aptamer-Au@SPIONs was 500 �g/ml, which killed about 80% ofancerous cells (Fig. 7).

. Discussion

In the recent decades, versatile SPION based formulations haveeen developed for numerous biomedical applications such as MRIontrast enhancement [20], stem cell tracking [21], hyperthermiaherapy of cancer [22], drug delivery [23], gene transfection [24]nd cell separation [25]. All these delicate applications requireppropriately sized and monodisperse SPIONs. The superparam-gnetism of SPIONs is largely dependent on size properties, asmall SPIONs act as mono-domain magnets and this characteris-ic enables them to lose their magnetic properties after removal

f the external magnetic field [26]. With these considerations,icroemulsion method was employed to prepare monodisperse

PIONs. In this method, the nanodroplets of microemulsion func-ion as nano-reactors, within which molecules interact and SPIONs

1326.49 ± 89.2 –972.88 ± 78.6 26.66778.30 ± 65.3 41.33

develop [15]. Since the prepared NPs have a size of 19 nm, theymust exhibit superparamagnetic properties.

The large surface to volume ratio as well as the magnetic proper-ties of SPIONs make them susceptible to aggregation. To avoid suchphenomena, SPIONs are usually stabilized with coatings such asgold [12], silica [27], dextran [28], chitosan [29], polyethylene gly-col (PEG) [30] and other polymers [31]. The coating also providesanchorage points for potential targeting moieties to be attached.Gold coating has several specific advantages. Not only it has verylow chemical reactivity, it also facilitates the attachment of thiol-modified targeting agents or drugs and protects the iron oxide coreagainst oxidation events [32]. Furthermore, gold coating can reduceSPION cytotoxicity and can be exploited as a photothermal agent toproduce heat upon exposure to a LED source NIR light and eradicatenearby cancerous cells [13]. Since the ions released from SPIONsare incorporated in the natural iron metabolism in human body,SPIONs are generally regarded as safe [33]. A 5 nm increase in sizewas noted upon gold coating of SPIONs, as evaluated by TEM. Fur-thermore, UV–vis spectroscopy and XPS were used to confirm thesuccessful coating of SPIONs with gold.

Upon entrance of NPs into the blood, proteins rapidly surroundits surface, giving rise to a protein corona [34]. The composi-tion of protein corona is largely dictated by the size and surfacecharge of NPs [35,36]. In some cases, protein corona has been

shown to mitigate the toxicity of nanomaterials [37]. Therefore,in the current study, we compared the cytotoxicity of bare SPIONsand those covered with protein corona on HT-29, CHO and L929cell lines. The protein corona coated SPIONs demonstrated lower

230 M. Azhdarzadeh et al. / Colloids and Surfaces B: Biointerfaces 143 (2016) 224–232

Fig. 5. Flow cytometry results of cells treated with NPs after staining by LysoTrackerRat

ctgcNatcpt

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Fig. 6. The T2* weighted image of the samples: (A) axial view and (B) lateral view.(sample 1 = aptamer-Au@SPIONs, sample 2 = Au@SPIONs and sample 3 = control).

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ed as a marker of NP uptake in (A) MUC-1 positive HT-29 cells and (B) MUC-1 neg-tive CHO cells. (For interpretation of the references to colour in this figure legend,he reader is referred to the web version of this article).

ytotoxicity compared to bare SPIONs, especially at high concentra-ions. Presumably, bare NPs have high surface energy and a muchreater affinity for the cell surface compared to protein coronaoated NP [35]. This results in more energy transfer when barePs interact with cells and this phenomenon can be more criticalt higher concentrations of NPs. Furthermore, research has shownhat the uptake of bare NPs is much higher than protein coronaoated NPs, which leads to more cytotoxicity [38]. Adsorption ofrotein corona decreases the non-specific interaction of NPs withhe cell surface and this can reduce NP uptake and cytotoxicity [39].

ICP-AES measurements confirmed that cells treated withptamer-Au@SPIONs had a higher iron content, indicating theigher uptake of targeted SPIONs vs. identical non-targeted ones40]. The in vitro cellular internalization of targeted aptamer-u@SPIONs and non-targeted Au@SPIONs was studied in HT-29nd CHO cell lines by confocal microscopy. LysoTracker Red, aarker for acidic medium (lysosome and endosome), was chosen

o track NPs. When SPIONs are internalized by cells, endosome andysosome staining increases and helps to estimate SPION uptake19]. The difference in the ability of MUC-1 positive and negative

ell lines to internalize aptamer-Au@SPIONs was obvious in con-ocal images as shown in Fig. 4. Due to the expression of MUC-1lycoprotein on its surface, HT-29 cell line is highly capable of inter-alizing aptamer-Au@SPIONs. In order to quantitatively confirm

Fig. 7. Viability of cells treated with different aptamer-Au@SPION concentrationsin different durations of laser irradiation (Mean ± SD,n = 3).

the confocal microscopy data, flow cytometry was used. As shownin Fig. 5(A), the mean fluorescence intensity of cells treated withaptamer-Au@SPIONs was much higher than those treated withAu@SPIONs. This is while the mean fluorescence intensity of tar-geted and non-targeted SPIONs is comparable in MUC-1 negativeCHO cells, as shown in Fig. 5(B), confirming that the internalizationof NPs is in part mediated by the aptamer functionality.

The high T2 relaxivity enables SPIONs to generate strong neg-ative T2 contrast in MR imaging [3]. The prepared SPIONs wereincubated with HT-29 cells in vitro and imaged using a 3T MRscanner. Results (Fig. 6) indicated that tubes containing SPIONsappeared dark (negative enhancement) in the T2* weighted imageand this confirms the efficiency of prepared NPs as potential con-trast agents. Expectedly, aptamer-Au@SPIONs generated a highernegative enhancement on T2* weighted image. This result also indi-

rectly confirms the targeting efficiency of aptamer-Au@SPIONs.

Finally the therapeutic effect of photothermal drug-free SPIONswas evaluated. The NIR irradiation of cancer cells treated with

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M. Azhdarzadeh et al. / Colloids and Su

ifferent concentrations of NPs was cytotoxic, while untreated cellsetained their viability. These observations confirm the photother-al effect of Au@SPIONs and the low toxicity of laser by itself.

urthermore, it has been demonstrated that normal cells are lessusceptible to heat compared to cancerous cells [41,42] and thisharacteristic makes photothermal therapy a safe treatment strat-gy for cancer. However, the exact mechanism through which celleath occurs after photothermal therapy has not been elucidated

n detail and different mechanisms such as disruption of plasmaembrane [43], ROS mediated apoptosis [44], depolarization ofitochondrial membrane [45] and DNA damage [46] have been

roposed.

. Conclusion

In this study, we fabricated MUC-1 aptamer targetedu@SPIONs for MR imaging and photothermal therapy of colonancer. A relatively simple and cost-effective methodology wasmployed for coating SPIONs with gold for photothermal therapypplications. MR imaging confirmed that the engineered NPsould potentially serve as efficient contrast agents. Moreover,e demonstrated that protein corona significantly offsets the

ytotoxicity of SPIONs and that this effect is more significant atigher SPION concentrations. Aptamer-Au@SPIONs were showno have higher uptake in MUC-1 positive cells compared to MUC-1egative cells. However, the effect of protein corona on targetingfficiency should be evaluated. Finally, we have shown that theultifunctional gold coating can be used for photothermal therapy

f cancer. The engineered aptamer-Au@SPIONs have high poten-ial to be used as actively-targeted dual-purpose agents for MRmaging and photothermal therapy of colon cancer in a drug-freepproach.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.02.58.

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