Theranostic MUC-1 aptamer targeted gold coated superparamagnetic iron oxide nanoparticles for magnetic resonance imaging and photothermal therapy of colon cancer

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

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Theranostic MUC-1 aptamer targeted gold coated superparamagnetic iron oxide nanoparticles for magnetic resonance imaging and photothermal therapy of colon cancer Morteza Azhdarzadeh a,b , Fatemeh Atyabi a,b , Amir Ata Saei c , Behrang Shiri Varnamkhasti a,b , Yadollah Omidi d , Mohsen Fateh e , Mahdi Ghavami f , Saeed Shanehsazzadeh g , Rassoul Dinarvand a,b,∗ a

Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417614411, Iran Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417614411, Iran c Department of Medical Biochemistry & Biophysics, Karolinska Institutet, Stockholm, Sweden d Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran e Medical Laser Research Center, Academic Center for Education, Culture, and Research (ACECR), Tehran, Iran f Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Health Science Faculty, Blegdamsvej 3c, 2200 Copenhagen N, Denmark g Nuclear Science and Technology Research Center (NSTRI), Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 4 November 2015 Received in revised form 6 February 2016 Accepted 25 February 2016 Available online 27 February 2016 Keywords: MUC1 aptamer MRI Protein corona SPION Theranostics Nanoparticles

a b s t r a c t Favorable physiochemical properties and the capability to accommodate targeting moieties make superparamegnetic iron oxide nanoparticles (SPIONs) popular theranostic agents. In this study, we engineered SPIONs for magnetic resonance imaging (MRI) and photothermal therapy of colon cancer cells. SPIONs were synthesized by microemulsion method and were then coated with gold to reduce their cytotoxicity and to confer photothermal capabilities. Subsequently, the NPs were conjugated with thiol modified MUC-1 aptamers. The resulting NPs were spherical, monodisperse and about 19 nm in size, as shown by differential light scattering (DLS) and transmission electron microscopy (TEM). UV and X-ray photoelectron spectroscopy (XPS) confirmed the successful gold coating. MTT results showed that Au@SPIONs have insignificant cytotoxicity at the concentration range of 10–100 ␮g/ml (P > 0.05) and that NPs covered with protein corona exerted lower cytotoxicity than bare NPs. Furthermore, confocal microscopy confirmed the higher uptake of aptamer-Au@SPIONs in comparison with non-targeted SPIONs. MR imaging revealed that SPIONs produced significant contrast enhancement in vitro and they could be exploited as contrast agents. Finally, cells treated with aptamer-Au@SPIONs exhibited a higher death rate compared to control cells upon exposure to near infrared light (NIR). In conclusion, MUC1-aptamer targeted Au@SPIONs could serve as promising theranostic agents for simultaneous MR imaging and photothermal therapy of cancer cells. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Colon cancer is the third most common type of cancer and is associated with a high mortality rate [1]. Similar to other types of cancer, effective treatment of colon cancer depends on early detection and treatment. Among different nanoscale contrast agents,

∗ Corresponding author at: Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417614411, Iran. E-mail address: [email protected] (R. Dinarvand). http://dx.doi.org/10.1016/j.colsurfb.2016.02.058 0927-7765/© 2016 Elsevier B.V. All rights reserved.

SPIONs have attracted a great deal of attention because of their unique superparamagnetism, desirable physiochemical properties, biocompatibility and biodegradability [2]. SPIONs are the most attractive MRI contrast agents owing to their superparamegnetic behavior [3]. However, non-targeted SPIONs are nonspecific and cannot accumulate inside the tumor and thus, they are only suitable for general imaging applications. To improve the localization of SPIONs in the vicinity of cancer cells, they are usually conjugated with tumor targeting moieties such as monoclonal antibodies, antibody fragments, aptamers, cell penetrating peptides and small molecules

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such as folic acid. Not only targeting increases the tumor accumulation of NPs, but it also decreases the off target side effects. While antibodies are the most specific targeting moieties, they entail a complicated manufacturing process and can be potentially immunogenic [4]. These issues can make their regulatory approval process a real challenge for the engineered imaging contrast agents functionalized with monoclonal antibodies. Furthermore, antibodies increase the hydrodynamic size of the NPs, reducing the chances of cellular internalization [5,6]. The increase in size is also known to offset the NPs’ “stealth” characteristics, leading to higher uptake with cells of the reticuloendothelial system [7]. Antibody fragments also suffer from molecule and manufacturing complexities. This might be the reason why antibody fragments have been rarely used for functionalization of SPIONs for imaging applications [8]. On the other hand, cell penetrating peptides and small molecules are readily available, but they are usually not very specific for a particular tumor. For example, folate receptor is generally overexpressed in cancer cells and is not specific to a certain type of cancer [9]. In this context, aptamers are arguably the most attractive targeting agents. Aptamers are relatively small oligonucleotides that recognize specific cellular targets. For example, highly specific aptamers have been shown to differentiate between subtypes of non-small cell lung cancer [10]. Another advantage of aptamers over larger targeting moieties is the fact that a higher number of aptamers can be conjugated onto the surface of a given SPION, which also allows for multiple binding and synergistic affinity [11]. To increase the biocompatibility of SPIONs, the NPs cores are usually coated. Inorganic coatings such as gold and silica can stabilize the SPION core and reduce the effect of environment on NPs degradation and corrosion [12]. The added benefits of a gold coating are that the engineered NPs can be used for photothermal therapy of tumor [13] and the gold coated NPs can be tracked using Fourier transform infrared spectroscopy (FTIR) [14], making the whole NP system a multimodal imaging probe. In the current study, SPIONs were synthesized using a microemulsion method and were then modified by a gold coating. The thiol modified oligonucleotide MUC-1 aptamer was further conjugated onto Au@SPIONs as a targeting agent. The aberrant glycosylated form of mucin serves as a good epithelial tumor cell surface marker. The targeting efficiency of NPs was investigated in vitro by confocal microscopy and flow cytometry. The applicability of photothermal therapy was assessed by comparing the viability of aptamer-Au@SPIONs-treated cells with untreated cells.

2. Materials and methods 2.1. Materials FeCl2 ·4H2 O, FeCl3 ·6H2 O, cethyltrimethylammonium bromide (CTAB), 1-butanol, gold chloride solution (HAuCl4 ), NH2 OH·HCl, dithiotreitol (DTT), sodium citrate, DAPI dye, Tris buffer acetate-EDTA (TAE) and MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) were purchased from Sigma, USA. HT-29 human colon cancer cell line, L929 mouse fibroblast cell line and CHO (Chinese hamster ovarian) cell line were obtained from Pasteur Institute, Iran. DMEM medium and fetal bovine serum (FBS) were provided by Gibco (Grand Island, USA). Penicillin and streptomycin were purchased from Sigma, USA. MUC-1 Aptamer was obtained from TAG Copenhagen A/S, Denmark. LysoTracker Red DND-99 and ethidium bromide were supplied by Life Technologies, USA. Amicon ultracentrifugal tube-Millipore (10 KDa), agarose, gelatin, toluene, sodium dodecyl sulphate (SDS), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME) and bromophenol blue were purchased from Merck, Germany. All other chemicals were of analytical grade.

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2.2. SPION synthesis SPIONs were synthesized by a microemulsion method according to Panahifar et al. [15] with minor modifications. Briefly, two microemulsions (A and B) were prepared. Toluene (29 ml) 1.8 g and CTAB were added to beaker A as oil phase and as surfactant, respectively. FeCl3 (202 mg) and FeCl2 (75 mg) were added at a molar ratio of 2:1 to 2 ml of deionized (DI) water and poured as aqueous phase into beaker A. Beaker B was composed of toluene in the same amount as oil phase plus 1.8 g CTAB and 25% ammonium hydroxide solution (2.65 ml). Subsequently, each beaker was separately mixed at 7000 rpm using a homogenizer and titrated by approximately 1.5 ml 1-butanol (as co-surfactant) until the color became transparent. Afterwards, microemulsion A was added to a three-necked flask while it was homogenized at 7000 rpm under constant flow of N2 gas at 50 ◦ C. Then, microemulsion B was added to the set and homogenized for 60 min. The reaction was stopped and the black product was cooled down to room temperature and 25 ml ethanol was added to the flask. After collection of the prepared SPIONs using a magnet, they were washed with boiling ethanol (4X), acetone (2X) and DI water (2X) to remove non-reacted agents. Finally, the SPIONs were precipitated by centrifugation at 5000 rpm and redispersed in DI water. 2.3. Gold coating of SPIONs Prepared SPIONs were coated by gold according to a method by Lyon et al. [16] with little modifications. By considering that freshly prepared Fe3 O4 NPs have very little tendency for gold coating, Fe3 O4 NPs were oxidized to ␥-Fe2 O3 by heating and exposing them to air under stirring for 30 min. Then, the ␥-Fe2 O3 NP solution was diluted by water to the concentration of 1.1 mM and after addition of an equal volume of 0.1 M sodium citrate, the mixture was stirred for 10 min. Afterwards, the ␥-Fe2 O3 NPs solution was diluted five times with water and an aliquot of 0.2% HAuCl4 and 0.2 M NH2 OH·HCl were incrementally added with 10 min intervals according to Table S1. After addition of HAuCl4 solution and NH2 OH·HCl, the solution color turned into purple. 2.4. Conjugation of MUC-1 aptamer Aptamer-NPs were prepared by adding thiol modified MUC-1 aptamer (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 modified aptamers were first activated by adding DTT and activated aptamers were separated using a 10 KDa Amicon tube and resuspended 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 solution was centrifuged at 16,000g for 25 min to separate aptamer-NP conjugates from unreacted aptamer. The precipitated aptamer-NPs were resuspended in Tris buffer and aptamer attachment was evaluated by agarose gel electrophoresis. 2.5. Cell culture Cells were grown in DMEM supplemented with 10% FBS plus 100 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 are rapidly covered with existing proteins, giving rise to the formation of a so called “protein corona” around NPs [17]. Therefore, in this

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study, we evaluated the cellular viability upon exposure to both bare and protein corona coated SPIONs. Two type of protein corona coated SPIONs were prepared by incubating Au@SPIONs with 10% and 100% FBS at 37 ◦ C for 1 h. Then, the dispersions were centrifuged at 12000 rpm at 15 ◦ C for 30 min [18]. The supernatants were separated and the formed NP-protein complexes were resuspended in PBS and centrifuged twice at 12000 rpm at 15 ◦ C for 30 min to remove loosely bound proteins (soft corona). Finally, hard corona coated NPs were resuspended in PBS to reach the desired concentration. HT-29, CHO and L929 cells were seeded in 96 well plates at a seeding density of 104 per well. After 24 h, media were removed and cells were exposed to SPIONs in concentrations of 10, 100 and 500 ␮g/ml for 24 and 48 h, in line with the normally used concentrations for SPIONs in biomedical applications. Media were removed and wells were washed with PBS. 100 ␮l of a 1 mg/ml MTT solution was added to each well and the plate was incubated for 2 h. Then, DMSO solution was added to each well to dissolve the formed formazan crystals, and absorbance was read at 540 and 630 nm using an ELISA reader (ELx800, BioTek instruments, USA). Four replicates were used for each SPION and data were shown as mean ± SD. Cell viability was calculated as mean absorbance in treated cells divided by the mean absorbance of untreated controls (background absorbance is subtracted).

2.7. NP uptake evaluation by confocal microscopy and flow cytometry For confocal imaging, cover glasses were placed in 6 well plates and a 0.1% gelatin solution was poured into it and after 1 h, the remaining gelatin was discarded. Then, CHO and HT-29 cells were seeded in wells and after 48 h, the media were removed and NPs (50 ␮g/ml) (non-targeted Au@SPIONs and aptamer-Au@SPIONs) were added to each well. After 6 h, media were discarded and the cover glass was washed with phosphate buffered saline (PBS). For evaluation of NP uptake, the acidic organelles were stained using LysoTracker Red. The higher uptake of NPs will lead to higher staining of endosomes and lysosomes [19]. For staining step, 300 nM LysTtracker Red dye was added to each well for 1 h and then the solution was discarded and the cover glass was washed three times with PBS, fixed by adding 0.4% formaldehyde solution (Sigma) for 20 min and stained by DAPI solution for 5 min to stain the nucleus. Finally, cover glasses were transferred onto the glass slides and visualized by Nikon confocal microscope A1 (Nikon Inc., Switzerland) armed with an A1 scan head and a standard detector using a 405 nm diode laser with DAPI filter and 543 nm diode laser (Melles Griot, USA) with TRITC filter. Flow cytometry was also used for quantitative measurement of NP uptake. Briefly, cells were seeded in 6 well plates and treated with non-targeted Au@SPIONs and aptamer-Au@SPIONs for 6 h. Subsequently, the NP suspension was discarded and the cells were stained by LysoTracker Red for 1 h. The cells were then trypsinized, centrifuged and resuspended in PBS and analyzed by flow cytometry (Partec PASII, Germany).

2.8. Photothermal therapy with Au@SPIONs HT-29 cells were seeded in a 6 well plate in a density of 4 × 105 cell per each well and incubated in 1 ml of the medium with 100, 200 and 500 ␮g/ml SPIONs at 37 ◦ C with 5% CO2 for 12 h. Then the medium was removed and cells were washed with water and fresh RPMI medium was added. Subsequently, cells were irradiated with 0.7 W/cm2 NIR light (820 nm) via LED for 2, 4 and 8 min for photothermal treatments. After treatment, cell viability was assessed by MTT assay.

3. Results 3.1. Size, morphology and zeta potential of SPIONs and Au@SPIONs The average size of the bare SPIONs was around 19 nm, which increased to approximately 24 nm upon coating with gold, as assessed by TEM (Fig. 1(A)–(B)). Particle size distribution was given by DLS measurements (Fig. 1(C)–(D)). The NPs possessed a rather spherical morphology and after gold coating a narrow layer of gold coating about 5 nm was deposited on NPs and the morphology still remained spherical. Zeta potential of bare SPIONs was −13 mV and after coating by gold dropped to −22 mV because of sodium citrate consumption at this method. 3.2. Confirmation of gold coating via UV–vis spectroscopy and XPS To evaluate gold coating, in the first step, a magnet was used to separate Au@SPIONs from possibly formed AuNPs. The separated Au@SPIONs were then resuspended in DI water and analyzed by UV–vis spectroscopy. While no peak was detected between 520 and 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 which belongs to the gold coating. Moreover, after each Au iteration, the intensity of the peak increased, which confirms an increase in Au to SPION ratio. XPS is another technique which detects the surface elements of particles up to a 9 nm thickness. Fig. 2(A) presents the XPS survey scan of SPIONs and Au@SPION. The Fe2p3 peak (715 eV) and Fe2p1 (724 eV) peak (Fig. 2(B)) which belong to Fe3 O4 are clearly observed for both NPs. The Au binding energy for Au@SPION (Fig. 2(C)) at 83 eV (Au 4f7/2) and 87 eV (Au 4f5/2) confirmed the formation of the gold coating around SPIONs, while no peak could be detected at this points for bare SPIONs. These results confirm the successful deposition of gold onto SPIONs. 3.3. Evaluation of aptamer attachment by gel electrophoresis Free aptamers, Au@SPIONs and aptamer-Au@SPIONs were run through agarose gel electrophoresis to confirm aptamer attachment onto the NPs. While no band was observed for Au@SPIONs, a wide band was noted for aptamer-Au@SPIONs at the agarose well. The less movement of aptamer-Au@SPIONs compared to free aptamers is expected, since NPs cannot freely move through the gel as free aptamer molecules do. Therefore, this experiment confirms aptamer conjugation onto the NPs (Fig. S2). 3.4. SPION cytotoxicity The cytotoxicity of SPIONs and SPIONs with hard corona at different concentrations was assessed by MTT assay (Fig. 3). Expectedly, all treated cell lines were more viable at low concentrations of NPs and cytotoxicity increased by increasing NP concentration. NPs coated with protein corona exerted less cytotoxicity in comparison with 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 CHO cell lines. After incubation of cells with NPs, their uptake was tracked by staining with LysoTracker Red. As shown in Fig. 4, HT29 cells treated with aptamer-Au@SPIONs have higher fluorescent

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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 resolution spectrum of Au 4f at Au@SPION which confirm the formation of gold coating.

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Fig. 3. The results of MTT assay for bare and protein corona coated NPs in L929 cell line over (A) 24 and (B) 48 h, CHO cell line over (C) 24 and (D) 48 h, and HT-29 cell line over (E) 24 and (F) 48 h (n = 3, data is shown as mean ± SD).

intensity compared to those treated with Au@SPIONs, which confirms the higher uptake of targeted SPIONs in these cells. This is while there is no difference between the uptake of Au@SPIONs and aptamer-Au@SPIONs in CHO cells (Fig. 4).

3.6. Flow cytometry measurements Additionally, we investigated the cellular uptake of aptamerAu@SPIONs in comparison with non-targeted Au@SPIONs in vitro using flow cytometry. After treatment of HT-29 and CHO cells with SPIONs, fluorescence intensity of cells was analyzed (Fig. 5) as compared to their non-targeted counterparts. In the HT-29 cell line as MUC-1 positive cell line, the mean fluorescence intensity for aptamer-Au@SPIONs was 11.4 compared to 7.5 for non-targeted Au@SPIONs. While in the MUC-1 negative CHO cells, the mean fluorescence intensity for aptamer-Au@SPIONs and non-targeted Au@SPIONs was comparable (8.88 versus 7.50, respectively).

3.7. MRI measurements Inductively coupled plasma atomic emission spectroscopy (ICPAES) was employed to determine iron concentration after NPs uptake by digesting samples with boiling HNO3. Iron concentration results have been shown in Table 1. The aptamer-Au@SPIONs were internalized to a higher extent than non-targeted SPIONs. To investigate the MRI contrast enhancement effect of NPs, the signal intensity of HT-29 cell line was measured after treatment with NPs. As shown in Fig. 6(A) and (B), NPs decrease the signal intensity compared to the control untreated sample. In Tables 1 and S2, the signal intensity of samples at different TR and TE has been shown (P < 0.05).

3.8. Photothermal therapy application Finally, the therapeutic effect of NPs for photothermal therapy was evaluated by applying LED. HT-29 cells were incubated with

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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@SPIONs or 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 1 ICP 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)

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– 26.66 41.33

three different concentrations of aptamer-Au@SPIONs and subsequently irradiated by NIR light via a light emitting diode (LED) for 2, 4 and 8 min. Cells not exposed to irradiation were considered as controls. Results indicated that at high concentrations (200–500 ␮g/ml) of SPIONs, cancerous cell were eradicated at 2, 4 and 8 min of irradiation, while at low concentrations (100 ␮g/ml), cytotoxicity was 94%, 90% and 79% after 2, 4 and 8 min of irradiation, respectively (Fig. 7). Furthermore, irradiating NP-free cells has almost no effect on their viability. The most effective concentration of aptamer-Au@SPIONs was 500 ␮g/ml, which killed about 80% of cancerous cells (Fig. 7).

4. Discussion In the recent decades, versatile SPION based formulations have been developed for numerous biomedical applications such as MRI contrast enhancement [20], stem cell tracking [21], hyperthermia therapy of cancer [22], drug delivery [23], gene transfection [24] and cell separation [25]. All these delicate applications require appropriately sized and monodisperse SPIONs. The superparamagnetism of SPIONs is largely dependent on size properties, as small SPIONs act as mono-domain magnets and this characteristic enables them to lose their magnetic properties after removal of the external magnetic field [26]. With these considerations, microemulsion method was employed to prepare monodisperse SPIONs. In this method, the nanodroplets of microemulsion function as nano-reactors, within which molecules interact and SPIONs

develop [15]. Since the prepared NPs have a size of 19 nm, they must exhibit superparamagnetic properties. The large surface to volume ratio as well as the magnetic properties of SPIONs make them susceptible to aggregation. To avoid such phenomena, SPIONs are usually stabilized with coatings such as gold [12], silica [27], dextran [28], chitosan [29], polyethylene glycol (PEG) [30] and other polymers [31]. The coating also provides anchorage points for potential targeting moieties to be attached. Gold coating has several specific advantages. Not only it has very low chemical reactivity, it also facilitates the attachment of thiolmodified targeting agents or drugs and protects the iron oxide core against oxidation events [32]. Furthermore, gold coating can reduce SPION cytotoxicity and can be exploited as a photothermal agent to produce heat upon exposure to a LED source NIR light and eradicate nearby cancerous cells [13]. Since the ions released from SPIONs are incorporated in the natural iron metabolism in human body, SPIONs are generally regarded as safe [33]. A 5 nm increase in size was noted upon gold coating of SPIONs, as evaluated by TEM. Furthermore, UV–vis spectroscopy and XPS were used to confirm the successful coating of SPIONs with gold. Upon entrance of NPs into the blood, proteins rapidly surround its surface, giving rise to a protein corona [34]. The composition of protein corona is largely dictated by the size and surface charge 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 SPIONs and those covered with protein corona on HT-29, CHO and L929 cell lines. The protein corona coated SPIONs demonstrated lower

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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).

120 No parcle +laser

Fig. 5. Flow cytometry results of cells treated with NPs after staining by LysoTracker Red as a marker of NP uptake in (A) MUC-1 positive HT-29 cells and (B) MUC-1 negative CHO cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

cytotoxicity compared to bare SPIONs, especially at high concentrations. Presumably, bare NPs have high surface energy and a much greater affinity for the cell surface compared to protein corona coated NP [35]. This results in more energy transfer when bare NPs interact with cells and this phenomenon can be more critical at higher concentrations of NPs. Furthermore, research has shown that the uptake of bare NPs is much higher than protein corona coated NPs, which leads to more cytotoxicity [38]. Adsorption of protein corona decreases the non-specific interaction of NPs with the cell surface and this can reduce NP uptake and cytotoxicity [39]. ICP-AES measurements confirmed that cells treated with aptamer-Au@SPIONs had a higher iron content, indicating the higher uptake of targeted SPIONs vs. identical non-targeted ones [40]. The in vitro cellular internalization of targeted aptamerAu@SPIONs and non-targeted Au@SPIONs was studied in HT-29 and CHO cell lines by confocal microscopy. LysoTracker Red, a marker for acidic medium (lysosome and endosome), was chosen to track NPs. When SPIONs are internalized by cells, endosome and lysosome staining increases and helps to estimate SPION uptake [19]. The difference in the ability of MUC-1 positive and negative cell lines to internalize aptamer-Au@SPIONs was obvious in confocal images as shown in Fig. 4. Due to the expression of MUC-1 glycoprotein on its surface, HT-29 cell line is highly capable of internalizing aptamer-Au@SPIONs. In order to quantitatively confirm

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the confocal microscopy data, flow cytometry was used. As shown in Fig. 5(A), the mean fluorescence intensity of cells treated with aptamer-Au@SPIONs was much higher than those treated with Au@SPIONs. This is while the mean fluorescence intensity of targeted and non-targeted SPIONs is comparable in MUC-1 negative CHO cells, as shown in Fig. 5(B), confirming that the internalization of NPs is in part mediated by the aptamer functionality. The high T2 relaxivity enables SPIONs to generate strong negative T2 contrast in MR imaging [3]. The prepared SPIONs were incubated with HT-29 cells in vitro and imaged using a 3T MR scanner. Results (Fig. 6) indicated that tubes containing SPIONs appeared dark (negative enhancement) in the T2* weighted image and this confirms the efficiency of prepared NPs as potential contrast agents. Expectedly, aptamer-Au@SPIONs generated a higher negative enhancement on T2* weighted image. This result also indirectly confirms the targeting efficiency of aptamer-Au@SPIONs. Finally the therapeutic effect of photothermal drug-free SPIONs was evaluated. The NIR irradiation of cancer cells treated with

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different concentrations of NPs was cytotoxic, while untreated cells retained their viability. These observations confirm the photothermal effect of Au@SPIONs and the low toxicity of laser by itself. Furthermore, it has been demonstrated that normal cells are less susceptible to heat compared to cancerous cells [41,42] and this characteristic makes photothermal therapy a safe treatment strategy for cancer. However, the exact mechanism through which cell death occurs after photothermal therapy has not been elucidated in detail and different mechanisms such as disruption of plasma membrane [43], ROS mediated apoptosis [44], depolarization of mitochondrial membrane [45] and DNA damage [46] have been proposed. 5. Conclusion In this study, we fabricated MUC-1 aptamer targeted Au@SPIONs for MR imaging and photothermal therapy of colon cancer. A relatively simple and cost-effective methodology was employed for coating SPIONs with gold for photothermal therapy applications. MR imaging confirmed that the engineered NPs could potentially serve as efficient contrast agents. Moreover, we demonstrated that protein corona significantly offsets the cytotoxicity of SPIONs and that this effect is more significant at higher SPION concentrations. Aptamer-Au@SPIONs were shown to have higher uptake in MUC-1 positive cells compared to MUC-1 negative cells. However, the effect of protein corona on targeting efficiency should be evaluated. Finally, we have shown that the multifunctional gold coating can be used for photothermal therapy of cancer. The engineered aptamer-Au@SPIONs have high potential to be used as actively-targeted dual-purpose agents for MR imaging and photothermal therapy of colon cancer in a drug-free approach. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.02. 058. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics 2015, CA, Cancer J. Clin. 65 (2015) 5–29. [2] S. Laurent, A.A. Saei, S. Behzadi, A. Panahifar, M. Mahmoudi, Superparamagnetic iron oxide nanoparticles for delivery of therapeutic agents: opportunities and challenges, Expert Opin. Drug Deliv. 11 (2014) 1449–1470. [3] S. Sharifi, H. Seyednejad, S. Laurent, F. Atyabi, A.A. Saei, M. Mahmoudi, Superparamagnetic iron oxide nanoparticles for in vivo molecular and cellular imaging, Contrast Media Mol. Imaging 10 (2015) 329–355. [4] K. Kuus-Reichel, L. Grauer, L. Karavodin, C. Knott, M. Krusemeier, N. Kay, Will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies? Clin. Diagn. Lab. Immunol. 1 (1994) 365–372. [5] K.A. Chester, R.E. Hawkins, Clinical issues in antibody design, Trends Biotechnol. 13 (1995) 294–300. [6] L.X. Tiefenauer, G. Kuehne, R.Y. Andres, Antibody-magnetite nanoparticles: in vitro characterization of a potential tumor-specific contrast agent for magnetic resonance imaging, Bioconjugate Chem. 4 (1993) 347–352. [7] L. Brannon-Peppas, J.O. Blanchette, Nanoparticle and targeted systems for cancer therapy, Adv. Drug Deliv. Rev. 64 (2012) 206–212. [8] K.L. Vigor, P.G. Kyrtatos, S. Minogue, K.T. Al-Jamal, H. Kogelberg, B. Tolner, K. Kostarelos, R.H. Begent, Q.A. Pankhurst, M.F. Lythgoe, Nanoparticles functionalised with recombinant single chain Fv antibody fragments (scFv) for the magnetic resonance imaging of cancer cells, Biomaterials 31 (2010) 1307–1315. [9] J. Sudimack, R.J. Lee, Targeted drug delivery via the folate receptor, Adv. Drug Deliv. Rev. 41 (2000) 147–162. [10] Z. Zhao, L. Xu, X. Shi, W. Tan, X. Fang, D. Shangguan, Recognition of subtype non-small cell lung cancer by DNA aptamers selected from living cells, Analyst 134 (2009) 1808–1814. [11] Y.-F. Huang, H.-T. Chang, W. Tan, Cancer cell targeting using multiple aptamers conjugated on nanorods, Anal. Chem. 80 (2008) 567–572.

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