Journal of Colloid and Interface Science 434 (2014) 89–97

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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

Doxorubicin-loaded magnetic gold nanoshells for a combination therapy of hyperthermia and drug delivery Faruq Mohammad a,b,⇑, Nor Azah Yusof a a b

Institute of Advanced Technology, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia Environmental Toxicology, Southern University and A&M College, Baton Rouge, LA 70813, USA

a r t i c l e

i n f o

Article history: Received 27 May 2014 Accepted 18 July 2014 Available online 2 August 2014 Keywords: Core-shell type Superparamagnetism SPIONs@Au Hyperthermia Drug delivery Doxorubicin

a b s t r a c t In the present work, nanohybrid of an anticancer drug, doxorubicin (Dox) loaded gold-coated superparamagnetic iron oxide nanoparticles (SPIONs@Au) were prepared for a combination therapy of cancer by means of both hyperthermia and drug delivery. The Dox molecules were conjugated to SPIONs@Au nanoparticles with the help of cysteamine (Cyst) as a non-covalent space linker and the Dox loading efficiency was investigated to be as high as 0.32 mg/mg. Thus synthesized particles were characterized by HRTEM, UV–Vis, FT-IR, SQUID magnetic studies and further tested for heat and drug release at low frequency oscillatory magnetic fields. The hyperthermia studies investigated to be strongly influenced by the applied frequency and the solvents used. The Dox delivery studies indicated that the drug release efficacy is strongly improved by maintaining the acidic pH conditions and the oscillatory magnetic fields, i.e. an enhancement in the Dox release was observed from the oscillation of particles due to the applied frequency, and is not effected by heating of the solution. Finally, the in vitro cell viability and proliferation studies were conducted using two different immortalized cell lines containing a cancerous (MCF-7 breast cancer) and non-cancerous H9c2 cardiac cell type. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction In recent years, the involvement of nanotechnology-based appliances have significantly advanced the traditional medical sector due to the possibility of treating the degenerative diseases such as cancer, diabetes, cardiovascular, and arthritis by means of applying the ‘‘combination therapy or polytherapy’’. The emerging applications of nanotechnology with advanced drug delivery for example, offer the possibilities for an early detection of disease, its diagnosis and treatment in addition to disease prevention by making use of the multifunctional nanomaterials [1]. The various kinds of nanomaterials involved in the disease diagnosis and treatment purposes include the polymeric (liposomes, microspheres, micelles, etc), metallic particles (core–shell, onion type, composite, alloy, magnetic), quantum dots, carbon nanotubes, etc [2]. The nanostructured magnetic materials in combination with thermally-sensitive polymers form magnetothermally-responsive systems and the use of these systems for drug delivery applications allows for an outer controlled triggered release of encapsulated or conjugated drug molecules to the site specific region of interest. ⇑ Corresponding author at: Institute of Advanced Technology, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia. E-mail address: [email protected] (F. Mohammad). http://dx.doi.org/10.1016/j.jcis.2014.07.025 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.

The use of magnetothermally-responsive systems (or magnetically-triggered, thermally-sensitive) significantly improves the treatment efficiency especially for a disease such as cancer [3,4]. With this approach, the diffusion and activated release of drug mechanism achieved through the incidence of a phase change in the host material by means of alternating magnetic field triggers the drug externally to the body, in addition to localized heating in vivo. Since, most of the cancer therapeutic drugs are associated with several side effects and thus the benefits of applying the magnetically-triggered formulations for treating cancerous diseases are a reduction in the total amount of drug required to reach an effective dose, less frequency of drug administration, targeted and outer controlled delivery, possibility for an inclusion of other imaging agents and multiple modes of therapy (combination therapy or polytherapy) [3,5]. The therapeutic heating can be easily achieved by the use of superparamagnetic particles (of typically less than 20 nm size) and the applied field causes the magnetic particles to release heat by means of two different mechanisms; Néel relaxation (rotation of magnetizations within the particle) and Brownian relaxation (rotation of particles against the dispersed medium). In contrast to many other magnetic materials (Mn, Co, Cu, Gd, etc), the superparamagnetic iron oxide nanoparticles (SPIONs) are of highest priority due to their biocompatibility, biodegradability, magnetic behavior, and image enhancing (MRI) capabilities [3,6,7].

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One of the main advantages of employing magnetic fluid hyperthermia technique to cancerous diseases is that the possibility of destroying only the cancer diseased cells through localized heating of tumor while simultaneous protection of healthy normal cells. The recent developments such as the availability of various targeting strategies (antibody, protein, etc), particle formation into nanosize (allowing the easy passage across biological membranes), easy administration, effective and outer control of heat, more efficient and homogeneous heat release compared to macrosized particles, made the magnetic hyperthermia as one of the preferred ways of treating the tumors which are located far inside the body (liver, brain, pancreas) [6,8]. Also, in our recent study, we observed that various cell types, i.e. cancer and non-cancer cells respond differently to the SPIONs-induced heat stress when applied using the oscillatory magnetic fields operating at low frequencies. The study indicated that the normal GT1-7 neurons are more resistant to the heat induced stress than the LNCap prostate cancer cells by means of expressing molecular chaperones in the form of heat shock protein-70 only in GT1-7 neurons [9]. Similarly, some of the anticancer drugs which have been tested for external magnetic field-induced targeted drug release studies includes paclitaxel [10,11], doxorubicin (Dox) [10], hydroxycamptothecin [12], 5-fluorouracil [13] and docetaxel [14]. It was observed from these studies that the presence of external magnetic field significantly influenced the drug release kinetics, targeting ability, magnetic hyperthermia and all these contributes finally to the efficiency of combination therapy. In the majority of studies related to hyperthermia-based therapy and hyperthermia-based drug delivery, the main challenge in the design of magnetic particles is that the high frequency of oscillatory magnetic fields i.e. lies in the range of several kHz and MHz [15,16]. Further, to influence the specific power loss (SPL) which is directly correlated with the hyperthermia effect, the changes made in the composition of magnetic particles are negatively affecting their use for most of the biomedical applications. For example, even though the presence of Mn, Co, Pt, Ni in SPIONs composition significantly enhancing the SPL, the oxidative instability and free radical induced toxic mechanisms (apoptosis mostly) with these elements are still a concern [6,17–19]. Similarly, during the development of magnetically influenced drug delivery systems, several attempts have been made for the use of SPIONs while retaining their inherent magnetic and image enhancing properties. In that view, the commonly used approach involves the conjugation or encapsulation of a drug of interest to the polymers such as chitosan, dextran, starch, poly(ethylene glycol), polylactide, and poly(DL-lactide-co-glycolide). The presence of thermally responsive polymers in this approach requires the development of complex conjugation chemistry for drug loading (often results in limited drug association with SPIONs) in addition to the formation of large-sized microparticles with limited drug encapsulation [10,20]. Finally, all these factors adversely influencing the SPIONs biodistribution, imaging characteristics and drug targeting/releasing efficiency in vivo due to the occurrences of the changes in the physical and/or surface characteristics of original SPIONs (zeta potential, hydrodynamic size, charge, stability and magnetization) [10]. In a similar approach, with the use of liposomal or emulsion formulations for the magnetic drug delivery and tumor imaging applications, the observed drug loading efficiency is as low as only 2–3% [21]. Therefore, construction of an ideal drug delivery system fulfilling all the specifications such as superior magnetic behavior, no toxicity, and efficient drug loading capability is still remaining as a challenge. Inspired by our recent review article based on the applications of magnetic nanomaterials for dual application sites of diagnosis as well as therapy [22], the present work is aimed to develop a multiple application probe with improved drug loading efficiency and

possesses an externally controlled drug releasing system for cancer. For that, we constructed a SPIONs probe with an ability to function as MRI contrast enhancing agent for the diagnosis and therapy by two different means. In one way, the probe generates local hyperthermia in accordance with the external magnetic field while in the second way, and the outer controlled delivery of conjugated drug can be achieved from the oscillation of particles with the help of same field. In the study, we selected the SPIONs (Fe3O4) coated with gold (Au) i.e. SPIONs@Au, due to the non-toxic, enhanced stability, and superior magnetic behavior compared to the naked SPIONs. Also, the anticancer drug, Doxorubicin hydrochloride (DoxHCl) was selected for the study, as it already proved to be cancer therapeutic and its mechanism of action is via intercalation with cell’s DNA and inhibition of macromolecular biosynthesis [10]. For the conjugation of Dox drug to the SPIONs@Au particles, cysteamine (Cyst) biomolecule was used as a space linker and the synthetic methodology is shown in the Fig. 1. The particles were characterized at various stages using HRTEM studies, UV–Vis, FT-IR, magnetization and further used for testing the heat and drug release studies. In addition, we also performed the in vitro toxicity test by using two different cell types which includes a cancerous (MCF-7 breast carcinoma) and the non-cancerous cell type (H9c2 cardiomyoblast). 2. Materials and methods 2.1. Chemicals Iron(III) acetylacetonate (Fe(C5H7O2)3, 99.9%), 1,2 hexadecanediol (C16H34O2), 90%), oleylamine (C18H37N, 70%), oleic acid (C18H34O2, 99%), phenyl ether (C12H10O, 99%), Cysteamine hydrochloride (C2H7NSHCl, 98%), and Doxorubicin hydrochloride (C27H29NO11HCl, 98%) were purchased from Sigma Aldrich (St. Louis, MO). Gold acetate (Au(C2H3O2)3) was purchased from Alfa Aesar (Ward Hill, MA). All chemicals were used as received. 2.2. Synthesis of Dox-loaded SPIONs@Au nanoparticles (NPs) To prepare Dox-conjugated magnetic SPIONs formulation, we first synthesized SPIONs@Au (gold coated iron oxide NPs) as described by Mohammad et al., i.e. the reduction of organometallics in the presence of oleic acid and oleylamine surfactants. The biocompatible Au coating on SPIONs expected to provide the oxidative stability for the iron oxide core and protects from Fe-induced free radical generation, in addition to allowing the easy conjugation with biomolecules [6]. The following step involves the stabilization of hydrophobic oleic acid/oleylamine coated SPIONs@Au NPs with Cyst molecules to form a water-dispersible system [23]. Thus obtained SPIONs@Au–Cyst NPs are stable, water dispersible and further used for the loading of Dox molecules (of hydrophilic behavior) by using the method described by Jain et al. [10]. Briefly, 0.7 g of iron acetylacetonate was mixed with 20 mL of phenyl ether, 2 mL of oleic acid, and 2 mL of oleylamine under inert atmosphere with vigorous stirring in a three necked round bottomed flask. To this, 2.6 g of 1,2-hexadecanediol was added and heated to 210 °C with reflux for 2 h in a nitrogen atmosphere. Following the period, the reaction mixture was cooled, ethanol (degassed) was added and the precipitate of SPIONs (average diameter: 5.1 nm) was separated by centrifugation. In the second step, 0.5 g of SPIONs was mixed with a solution containing 30 mL of phenyl ether, 3.1 g of 1,2-hexadecanediol, 0.5 mL of oleic acid, 0.5 mL of oleylamine, and 0.8 g of gold acetate under inert atmosphere. The reaction mixture was heated to around 190 °C with reflux for about 1.5 h. Following the period, the contents were

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Fig. 1. Schematic representation of the multiple steps involved in the formation of SPIONs@Au–Cyst–Dox.

cooled to room temperature, ethanol was added, and the precipitate was separated out by centrifugation. The product was suspended in hexane, washed with ethanol 3–4 times to obtain SPIONs@Au NPs (hydrophobic nature). The third step involves the stabilization of formed SPIONs@Au so that they are water dispersible and for that, a colloidal solution containing 30 mg of SPIONs@Au in 10 mL of toluene was prepared. In another flask, 10 mg of Cyst in 10 mL of distilled water was prepared. Both the solutions were mixed together and stirred for about 10 min. At first, one can see clearly two different phases with the bottom colorless due to water and the top purple-colored toluene containing the SPIONs@Au and after 2–4 h, the migration of SPIONs@Au completely into the Cyst water phase can be seen. This is a room temperature reaction and the driving force for this is provided by the affinity of thiol (ASH) of Cyst with the gold surface to form SPIONs@Au–Cyst NPs. The SPIONs@Au–Cyst NPs were separated with the help of a magnet, washed 2–3 times with distilled water, and dried under inert atmosphere. In order to form Dox loaded SPIONs@Au–Cyst particles, an aqueous solution containing DoxHCl (at a concentration of 0.5 mg/mL) was added drop-wise to a colloidal dispersion of SPIONs@Au–Cyst NPs (0.5 mg/mL in distilled water) while stirring. The mixture was allowed to stir overnight (16 h) to allow partitioning of drug into Cyst and oleic acid/oleylamine layer surrounding the SPIONs@Au core. Finally, the Dox-loaded SPIONs@Au–Cyst NPs were separated from the free drug using a magnet, washed with distilled water, dried and stored. With the help of standard concentration curve of Dox (at the absorption of 485 nm) generated from UV–Vis spectrophotometer using a series of Dox solutions of varying concentrations, the unbound Dox remained in the supernatant was analyzed [2]. Similarly by making use of the calibration curve, the samples from Dox release studies were also analyzed in order to estimate the amount of drug that got released into the solvent media due to a change in pH, temperature and exposure to magnetic field. 2.3. Characterization of SPIONs@Au–Cyst High-resolution transmission electron microscopy (HRTEM) images were collected on a JEOL 4000EX high-resolution electron microscope operating at 200 kV and the samples were prepared by dispersing the particles in distilled water and then dropping onto the carbon coated copper grid. The EDAX were also simultaneously recorded by focusing the electron beam onto the particles

using the same JEOL 4000EX electron microscope attached to an EDAX analyzer. The UV–Visible spectroscopic analysis were carried out on SPECORDÒ PLUS double beam spectrophotometer and QE65000 spectrometer supplied by Ocean Optics limited and the samples were prepared by using distilled water as solvent in a typical wavelength range of 300–800 nm. The FT-IR spectrums were recorded on Nexus 670 FT-IR spectrometer in the transmission mode and the samples were prepared by grinding the material with dry powder of KBr and then made into a transparent pellet. The magnetic measurements were conducted with the help of Quantum Design MPMS-5S superconducting quantum interference device (SQUID) magnetometer using the dry powders of the sample placed in a gel-capsule stuffed with kim-wipe paper which is then placed inside a plastic straw. The magnetizations were measured in two different modes; Zero-field-cooled (ZFC) and Fieldcooled (FC). The sample was ZFC to 5 K, a field was applied and magnetization was measured as a function of temperature, while in FC, the sample was field-cooled from above 300 K to 5 K and the magnetization was measured. The temperature at which the two curves ZFC and FC meet is considered to be the blocking temperature (TB). Hysteresis loops, in which the sample’s magnetization is measured as a function of applied field in the temperature from of 5 K to 300 K. 2.4. In vitro cell viability and proliferation assay The cell viability and proliferation were assessed based on metabolic activity of cells using the CellTiter-BlueÒ reagent from Promega (Madison, WI) and the assay involves the reduction of resazurin, a non-fluorescent blue dye, into a pink colored, red-fluorescent resorufin. Briefly, the H9c2 rat embryonic cardiomyoblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1% antibiotics (Penicillin, 100 units/mL; Streptomycin, 100 lg/mL) in an incubator at 37 °C, 5% CO2, and in 95% humidity. Approximately 104 cells/well were seeded onto 96-well plates and allowed to grow for 24–36 h. When the cells reach to 80% confluence, the medium was replaced with fresh DMEM containing 2% (v/v) FBS and varying concentrations (25–500 lg/mL) of SPIONs@Au–Cyst and incubated for additional 24 h. After the period, the CellTiter-BlueÒ reagent (20 lL) was added and incubated for additional 3–4 h as per the manufacturer’s instructions. The amount of resorufin formed during this time was measured at excitation and emission

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wavelengths of 560 and 590 nm (respectively) using a Spectramax EM Gemini spectrofluorimeter (Molecular Devices, Sunnyvale, CA). Background fluorescence from the wells that contained SPIONs@Au–Cyst in DMEM but no added cells was subtracted and phorbol myristate acetate (PMA; 10 lg/mL) used as a positive control. The percentage cell viability was calculated with respect to the untreated control (set at 100%) and the values shown are mean ± standard deviations (SDs) of three individual experiments [6,24]. The MCF-7 breast carcinoma cells were maintained in Eagle’s minimum essential medium (EMEM) containing 0.01 mg/mL bovine insulin, 10% (v/v) FBS, and 1% (v/v) antibiotics (Penicillin, 100 units/mL; Streptomycin, 100 lg/mL) and incubated at 37 °C, 5% CO2, and in 95% humidity. Similar to H9c2 cells, the cell viability for MCF-7 cells was measured following the treatment with SPIONs@Au–cyst (25–500 lg/mL) in 2% FBS for 24 h, based on the conversion of resazurin to resorufin by viable cells. 2.5. Studies of hyperthermia The apparatus used for applying the magnetic field and the temperature measuring conditions were discussed in our previous reports [6]. Briefly, the alternating magnetic field-induced heat releasing experiments were carried out by using the custom-made setup consisting of copper coil (Alpha-Core Inc.) and the magnetic field inside it is controlled with the P1351 Behlman AC power source. The highest parameters that the setup can generate are of 430 Hz field frequency at 123 V, and the lowest parameters of 44 Hz field frequency at 15 V. In addition to the highest and lowest frequencies, an intermediate frequency of 230 Hz at 67 V was selected for the studies and the measurements were carried over a period of 60 min. 2.6. Release of Dox from SPIONs@Au–Cyst–Dox The studies of drug release kinetics from the surfaces of SPIONs@Au–Cyst–Dox NPs were conducted as against three different parameters of pH, oscillatory magnetic fields, and temperature. Each time, a colloidal solution was prepared by taking 5 mg of SPIONs@Au–Cyst–Dox into 2.5 mL of distilled water and the changes in the concentration of Dox in the dispersion medium were measured over a period of 3 h. With the help of the standard concentration curve of Dox generated from the UV–Vis spectrophotometer, the release of Dox into distilled water was quantified during the specified time intervals [2]. 2.7. Statistical analysis The hyperthermia data i.e. the recorded temperature change of the medium were arranged in multivariate repeated measures and analyzed by using the PROC GLM in SAS software. However, the statistical analysis of cell viability and drug release data is performed by using a one-way analysis of variance (ANOVA) and Bonferroni’s method for multiple comparisons. All of the data in this report are presented as mean ± SD of 3–5 separate experiments. The probability of p < 0.05 was considered as statistically significant (represented by ⁄) and p < 0.01 as highly statistical significant (represented by ⁄⁄). 3. Results and discussion In the present study, an anticancer drug, Dox-loaded SPIONs@Au–Cyst particles was prepared for the combined therapy of cancer by means of hyperthermia as well as chemotherapy. The particles are characterized thoroughly and were tested for the

hyperthermia and drug release efficacy by the influence of changes in pH, temperature and external magnetic field. In addition to the drug release kinetics, the in vitro toxicity investigation was also performed on two different immortalized cell lines of a cancerous (MCF-7 breast) and a non-cancerous (H9c2 cardiac). The reason for choosing H9c2 cardiac cells is to see the effect of our synthesized particles toward cardiac cells, as it is well known for the observation of cardiotoxicity in patients undergoing chemotherapeutic treatment involving Dox. Similarly, to see the response of cancer cells with our particles, the MCF-7 breast cancer cells were selected. 3.1. Physical characterization Fig. 2(a)–(c) shows the HRTEM images, corresponding particle size distributions and EDAX-based elemental analysis of SPIONs@Au–Cyst NPs respectively. From the figure, one can see clearly that the particles are spherical and uniform with an average particle size of around 6.8 nm. From the EDAX spectrum shown in Fig. 2(c), the appearance of peaks resembles to the presence of corresponding elements in the particle; the major peaks, S from ASH of cysteamine, Au and Fe from the gold coated iron oxide core. It can also be seen from the figure that the appearance of Cu peak however is generated from the carbon coated copper grid used in the preparation of HRTEM samples. The magnetic characterization of hysteresis curves and temperature-dependent magnetization curves (ZFC–FC) for the SPIONs@Au–Cyst is shown in the Fig. 3(a)–(b). From the hysteresis data (Fig. 3a) recorded at 5 and 300 K, the observation of hysteresis loops at room temperature confirming the superparamagnetic behavior of SPIONs@Au–Cyst NPs. The saturation magnetization (Ms) values observed to be 84 and 78 emu/g at 5 and 300 K respectively, while the blocking temperature (TB) found to be 201 K (Fig. 3b). It was observed from our previous studies [6] that the SPIONs@Au NPs containing oleic acid/oleylamine surface ligands possesses the Ms. value of 58 emu/g (at 5 K) and the same particles on their surface modification with cysteamine, the Ms. value significantly increased to 84 emu/g. This significant enhancement in the magnetization value (at 5 K) are attributed to the intrinsically developed magnetism due to the modification of the local electron structure of Au upon the formation of AuAS bond, i.e. by means of sharing the 3d electron of Au with that of S of thiol in cysteamine [25]. In general, any system with high magnetization values is well suited for a majority of biomedical applications such as MRI, hyperthermia, drug delivery, magnetic separation, etc. In our case, the observation of a high TB value (201 K) in addition to high Ms. (78 emu/g at 300 K), implies that the SPIONs@Au–Cyst material can exhibit the corresponding magnetizations even at higher temperatures. This is particularly useful in hyperthermia applications where the oscillating magnetic particles generally lose their magnetic behavior with an increase of temperature of the medium due to its own heating property. But in our case, the observation of a high TB indicating that efficient, prolonged heat release can be expected from the SPIONs@Au–Cyst particles under magnetic fields due to the existence of high magnetizations at high temperatures. The observation of superparamagnetic behavior, high Ms. and TB values is thus making the SPIONs@Au–Cyst NPs to be ideal for magnetic fluid hyperthermia and externally controlled magnetic drug delivery applications. The SPIONs@Au core contains oleic acid, oleylamine and cysteamine molecules which possess the functional groups of unsaturation (C@C), carboxylic (ACOOH), amino (NH2) and thiol (ASH) and hence it can easily form p–p electronic, Van der Waals interaction and strong hydrogen bonding with the quinone portion of Dox molecules containing the AOH, ANH2 groups. The UV–Vis spectroscopic comparison of the SPIONs@Au–Cyst before and after loading

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Fig. 2. (a) HRTEM image (scale: 5 nm), (b) particles size distribution, and (c) EDAX of SPIONs@Au–Cyst.

Fig. 3. (a) Magnetic hysteresis curves of SPIONs@Au–Cyst NPs at two different temperatures of 5 K and 300 K and (b) FC and ZFC curves to show the temperature dependence of magnetization.

with Dox is shown in Fig. 4(i). From the figure, a strong absorption maximum for the SPIONs@Au–Cyst NPs is observed at 567 nm due to the surface plasmon resonance property of gold and for pure Dox is at 485 nm. However, on loading of Dox drug with the same SPIONs@Au–Cyst NPs, a broader peak with two absorption maxima is observed; one being at 495 nm and the other at 525 nm. The absorption maxima at 525 nm are attributed to gold, while the other absorption at 495 nm is due to the presence of Dox

drug and therefore, primarily confirming the bonding of Dox with SPIONs@Au–Cyst NPs [2,6]. The standard absorption peak for Dox at 485 nm was used for generating the calibration curve with changes in Dox concentration (figure not shown). From the standard curve, the amount of Dox drug that got loaded onto the SPIONs@Au–Cyst NPs to be 0.32 mg/mg i.e. about 63% of the initial concentration used for loading. The reason for such high loading efficiency may be the existence of a strong hydrogen bonding

Fig. 4. Comparison of the (i) UV–Visible spectrum and (ii) FT-IR spectrums of SPIONs@Au–Cyst with that of pure Dox and SPIONs@Au–Cyst–Dox.

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between the Dox groups and other ligand surface groups from the maintenance of neutral conditions while preparing the material. The FT-IR spectral comparison of the SPIONs@Au–Cyst and pure Dox drug was recorded and compared with that of SPIONs@Au–Cyst–Dox to further confirm the bonding and is shown in Fig. 4(ii). From the figure, the sharp peak around 1734 cm1 in pure Dox (due to C@O) shifted to 1730 cm1 and the broad peak at 3415 cm1 (due to –OH stretching) becoming sharp and shifted to 3428 cm1 after forming the nanohybrid complex with SPIONs@Au–Cyst. Similarly, the peak in SPIONs@Au–Cyst around 780 cm1 due to CASH bending changed to 760 cm1 on bonding with Dox and the FeAO stretching in magnetite core of SPIONs@Au–Cyst at 540 cm1 changed to 530 cm1 confirming the persistence of the SPIONs@Au even after the Dox loading. The similar results were observed when the Dox drug was used for conjugation with graphene oxide and during the formation of thiol protected gold nanostructures [2,26]. The comparison of both UV–Vis and FT-IR spectral data therefore, confirming the bonding of Dox molecules with SPIONs@Au–Cyst NPs. 3.2. In vitro cell viability and proliferation The comparison of cell viability and proliferation studies of the SPIONs@Au–Cyst and SPIONs@Au–Cyst–Dox at concentrations in the range of 25–500 lg/mL for an exposed period of 24 h toward the H9c2 cardiomyoblasts and MCF-7 breast carcinoma cells is shown in Fig. 5(a)–(b). From the analysis of results with respect to positive and negative controls (Fig. 5a), it is clear that the SPIONs@Au–Cyst does not induce much change in the viability to either H9c2 cardiomyoblasts or MCF-7 carcinoma cells (under experimental conditions). However, the drug loaded SPIONs@Au–Cyst particles (Fig. 5b) under similar conditions exhibited significant decrease in the viability and proliferation toward both the cell types over 100 lg/mL concentration. The reason for such a non-toxic activity by naked SPIONs@Au–Cyst is that the core iron oxide particles are coated completely with the biocompatible gold shell and their surfaces are well protected with biomolecules such as oleic acid, oleylamine and Cyst. All these surface components are protecting the cell cultures from the Fe-induced free radical reactions either by the direct oxidation of intracellular components or indirect oxidation through the culture medium. While the cell viability changes by SPIONs@Au–Cyst–Dox treated cultures are attributed to the effects of the anticancer drug, Dox, since it already proved to be responsible for the pharmacological activity by the mechanisms of DNA strand breakage, mitochondrial depletion, free radical-mediated doxorubicinol metabolite formation, etc [27]. In addition, the reduction in the viability of H9c2 cardiac cells also gives a hint that the targeted delivery should be strictly implemented especially when Dox is used during cancer treatment so as to avoid the drug actions on non-target

tissues liver, lung, heart and kidneys. For any application in biomedical sector, the selected probe should not possesses any kind of intrinsic toxicity and the absence of toxicity behavior in SPIONs@Au–Cyst primarily confirms that it can serve as a stable platform for hyperthermia and drug delivery. 3.3. Effect of magnetic field and solvent viscosity toward hyperthermia The studies of hyperthermia i.e. external magnetic fieldinduced temperature increase of medium with time at three different frequencies and solvents of varying viscosities are shown in Fig. 6(a)–(d). The measurements were carried at the oscillation frequencies of 44, 230 and 430 Hz over a period of 60 min and the solvents include water, ethanol and toluene. From the comparison of results shown in Fig. 6(a)–(c), it can be observed that the heat release is both frequency and viscosity dependent, i.e. the heating rate is increasing with an increase of applied frequency and decrease of solvent viscosity. The temperature increase of medium follows the order of 44 Hz < 230 Hz < 430 Hz and toluene < ethanol < water. One can also see from the figure that more temperature raise is observed during the first 30 min of exposure period (significant increase being the first 15 min), followed by reduction of heat release on further exposure. This fall in the heat release can be attributed to the possible quenching of the particles oscillation in accordance with the applied field, as gold is a better heat conductor. Also, under similar experimental conditions, the hydrophobic magnetic particles (SPIONs@Au) tested in our previous studies provided the information that more heating is observed in case of water medium at the lowest frequency of 44 Hz [6]. However, with the use of hydrophilic particles (SPIONs@Au–Cyst–Dox) in the present work, the more heating is observed in the same water medium but at the highest frequency of 430 Hz. These results provide the information that in magnetic fluid hyperthermia, the Brownian-rotation mechanism is also equally important to Néel relaxation for heat release by the magnetic particles, as the heating effects are changing with respect to the frequency and solvent mediums. Thus, while designing the magnetic particles for heatrelated applications, it is necessary to consider the applied field, viscosity of the medium and the solvent dispersibility of the particles. Further, to confirm whether the obtained data is statistically significant or not, the data were arranged in multivariate repeated measures and analyzed by using the PROC GLM in SAS software [28]. The results of multivariate tests showed that there were significant effects of TIME, TIME * FREQUENCY and FREQUENCY * SOLVENT observed for the SPIONs@Au–Cyst–Dox particles (Fig. 6d). 3.4. SPL and its dependency The heating efficiency of any magnetic material can also be represented in terms of SPL, i.e. the ability to generate heat from the

Fig. 5. Comparison of the cell viability and proliferation studies of SPIONs@Au–Cyst NPs (a) before and (b) after loading with Dox when tested against MCF-7 breast cancer and H9c2 cardiomyoblast cells. From the figure, ⁄ represents significant and ⁄⁄ represents highly significant values against the untreated control measurements.

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Fig. 6. Heat release studies of SPIONs@Au–Cyst–Dox at three different frequencies of (a) 44, (b) 230 (c) 430 Hz and (d) the average heat release at all three frequencies.

magnetic coupling between the NPs magnetic moment and the applied field. In general, the SPL is influenced by the Ms, TB, applied fields, viscosity and the amount of magnetic material. Therefore, we have determined the SPL of SPIONs@Au–Cyst–Dox by making use of the following equation,

SPL ¼

CVs dT M dt

where C is the specific heat of the sample (C = 4.185, 2.44, and 1.13 J/g/°C for water, ethanol, and toluene, respectively), Vs is the volume of the sample medium, M is the weight of the magnetic material in grams, and dT/dt is the raise in temperature per unit time. The calculated SPL values for the SPIONs@Au–Cyst–Dox material tested in three different mediums of varying viscosities and applied frequency are shown in Fig. 7 and are tabulated in Table 1. From the data, it is evident that the material is exhibiting high SPL for water compared to other two mediums of ethanol and toluene at all three frequencies and these results are in agreement with the specific heat of the solvents. Also within the water medium, it is interesting to note that the material is exhibiting high SPL (1199 W/g) at the highest applied frequency of 430 Hz and the lowest value (460 W/g) at the intermediate applied frequency of 230 Hz. Surprisingly, the material exhibited the medium SPL (697 W/g) at the lowest applied frequency of 44 Hz in water medium. However, this effect is not observed in other two mediums of ethanol and toluene, i.e. the SPL values are increasing in accordance with the applied frequency. The similar effects of low SPL values at the same intermediate frequency (230 Hz) are observed when the hydrophobic SPIONs@Au NPs were tested for the heat release studies [6]. One probable reason for this can be due to the fact that the applied frequency (at 230 Hz) is not completely taken up by the particles so as to convert it into heat, it may be converting into other forms or some times more possible quenching resulting in a loss of heat release.

Fig. 7. SPR values of SPIONs@Au–Cyst at three different frequencies and in varying dispersion mediums.

Table 1 SPL values of SPIONs@Au–Cyst–Dox measured at various frequencies and dispersion mediums. Type of Medium

Water Ethanol Toluene

SPL (W/g) 44 Hz

230 Hz

430 Hz

697.5 203.3 67.8

460.3 227.7 87.7

1199.7 256.2 210.9

3.5. Effect of pH, magnetic field and temperature toward Dox release In view of the considerable drug loading capacity (0.32 mg/mg) and intrinsically non-toxic (up to 500 lg/mL) behavior of SPIONs@Au–Cyst NPs, it may be used as a carrier material for drug delivery. The studies of Dox drug release (%) from the SPIONs@Au–Cyst NPs as a function of solution pH, applied frequency

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and temperature over a 3 h period is shown in the Fig. 8(a)–(c). From the Fig. 8a, it is shown that the material exhibited the highest drug release efficiency of about 56% (over a 3 h period) in acidic conditions of pH 2.1, when compared against neutral pH of 7.4 and basic pH of 10.3. The reason for the high efficacy at low pH can be attributed to the effects of both the formation of ions and lack of hydrogen bonding between Dox and other ligand groups. Under acidic conditions, more number of H+ ions are available and can associate easily with the –NH2 of Cyst or oleylamine (present as ligands on SPIONs@Au core) at which the Dox molecules are non-covalently linked. The surface ligand molecules always try to lose the bulkier Dox molecules and facilitate for the formation of ammonium ions (NH+3) by making use of the H+ ions. Similarly, under basic conditions (pH 10.3), the ACOOH of oleic acid exists as COO and the ASH of cysteamine changes to AS and is not available for participation in a strong hydrogen bonding with the Dox groups, thereby better drug release are observed. However in neutral conditions, due to the absence of H+ ions and the ability of surface ligand groups to form a strong hydrogen bonding with the Dox groups, a very low drug release was observed. Since the metabolic rate of cancer cells is very high compared to the healthy normal cells and due to this, the local tumor environment is acidic by the formation of lactic acid. In that view, the property of high drug release rate under acidic conditions is especially suits for cancer drug delivery, as more amount of Dox can be released into the tumor on localization [29]. These results of more efficient drug release under acidic conditions followed by basic environment are supported by a similar study where the same Dox drug was used in conjugation with graphene oxide [2]. The release of Dox under the influence of externally applied magnetic fields with increase of frequency in an acidic pH of 2.1 is shown in the Fig. 8b. From the analysis of results, it is evident that the drug release rate increased in accordance with the increase of applied frequency and is due to the coupling of oscillating particle’s magnetic moment with the field. Under these conditions, the significant drug release (%) effects are observed during

the first 30–60 min of field exposure when recorded over a 3 h period and the maximum drug released was investigated to be 72% at 430 Hz, followed by 57% at 230 Hz and 49% at 44 Hz. These results are significant when compared with the pH effects where the maximum release was seen at the highest period of 3 h; however, under the influence of magnetic field, a 72% release observed during the first 30–60 min period. Since the oscillation of magnetic particles also generates heat in addition to drug release, we therefore tested the Dox release under the influence of temperature of the medium (without applying any magnetic field). These studies were carried in order to make sure that the enhanced drug release effects are only due to particles magnetic moment influenced by the external field, but not from the heating of solution where there is a possibility for the breakage of some heat sensitive bonds. Fig. 8c shows the Dox release studies under the influence of temperature of the medium measured at 28, 32 and 35 °C. The selection of these parameters are based on the fact that, the application of a 430 Hz frequency causes the maximum temperature raise of 35 °C (when started from room temperature of 25 °C) in the medium and similarly, a 28 °C and 32 °C were observed for the oscillations at 230 Hz and 44 Hz respectively. It was already described that at 230 Hz (Fig. 7), the SPL generated is low compared to other two frequencies due to some undefined losses i.e. the applied field at this frequency is not getting utilized entirely for the conversion into heat and hence a lower temperature of 28 °C assigned to 230 Hz frequency. From the analysis of results (Fig. 8c), a maximum Dox release of 57% was observed at the highest time period of 3 h in a 35 °C containing temperature medium. This release was followed by the 32 °C containing medium with the total Dox release of 46%, and the temperature (32 °C) was corresponding to the 44 Hz frequency from the hyperthermia measurements. However, the Dox release studies under the influence of frequency giving the information that the drug release is increasing in accordance with the applied frequency (Fig. 8b), i.e. 430 Hz > 230 Hz > 44 Hz. The temperature dependence Dox release studies in the absence of applied field indicating

Fig. 8. Studies of drug release kinetics as a function of (a) pH, (b) external magnetic field and (c) temperature of the medium.

F. Mohammad, N.A. Yusof / Journal of Colloid and Interface Science 434 (2014) 89–97 Table 2 Comparison of Dox release (%) against pH, applied field and temperature. pH 2.1

44 Hz

230 Hz

430 Hz

35 °C

32 °C

28 °C

56

49

57

72

57

46

39

that there is no effect of solution temperature on the drug release because of the fact that the same amount of drug release (56%) was observed even at room temperature (Fig. 8a, Table 2). This means that although some activation energy available in the form of temperature, but is not getting utilized for the release of Dox from the surface of SPIONs@Au–Cyst particles. The supplied energy in temperature form in turn may be getting absorbed by the metals like Au and Fe due to their conductive behavior and is not allowing for the enhancement of drug release rate by breaking the noncovalent interactions between the groups of cysteamine, oleic acid, oleylamine and Dox. All these analysis confirming that the magnetic moment is the dominating factor for the observation of enhanced drug release and the in situ generated temperature of the medium do not influence the Dox release rate. 4. Summary and conclusion In the present report, we introduced novel magnetic gold nanoshells that can be useful in a ‘combination therapy’ for treating cancer by multiple application modes. It was observed that the magnetic properties are greatly enhanced (Ms. of 84 emu/g at 5 K) on stabilization with Cyst biomolecule and also found to be exhibiting the magnetic susceptibility at higher temperatures (201 K) compared to simple SPIONs@Au particles. With those magnetic properties, the heat releasing efficiency was increased with increase of applied frequency due to the possibility of effective coupling of particles magnetic moment with the field and with that, water exhibited a very high SPL value (1199 W/g at 430 Hz) among other two frequencies and tested media. In addition, we also proved that a simple amino acid derivative, Cyst can effectively functions to mask the hydrophobic character of oleic acid/ oleylamine in addition to achieving the non-covalent conjugation of Dox molecules and with this system, the loading efficiency observed to be as high as 63%. The intrinsically non-toxic behavior of Dox-unloaded SPIONs@Au–Cyst NPs primarily supporting that the particles can be suitable for biomedical applications as observed from the cell viability and proliferation studies. Taking advantage of the non-toxic behavior and high magnetic susceptibility which helps to generate enough heat and also induces Dox release by the coupling of magnetic moment with the applied field even at low frequencies, the SPIONs@Au–Cyst particles can therefore serves to be sophisticated for the application of simultaneous hyperthermia and outer controlled drug delivery. The magnetically controlled drug release phenomenon can especially be useful toward cancerous diseases through the introduction of targeted delivery of drug loaded magnetic particles and thereby achieving the enhanced bioavailability for highly toxic anticancer drugs [30]. In addition, the frequency dependent drug and heat releasing property can be utilized for enhancing the cancer treatment efficiency by increasing the anticancer drug bioavailability and local tumor temperature respectively, all of which controlled by the external magnetic field. Moreover, the hyperthermia property can also be extended to operate for the decomposition of thermo-responsive or heat sensitive polymers in order to release

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encapsulated therapeutic drugs in controlled amounts at any specific targeted site of interest controlled by external magnet. Further, this technology will empower the development of drug delivery vehicles which are capable of delivering small or large molecules with controlled pharmacokinetics. Future studies are underway to understand the mechanism of cell death induced by the use of targeted SPIONs@Au–Cyst–Dox particles when tested in combination study of hyperthermia with drug delivery on various cancer and non-cancer cell types. Acknowledgments FM would like to thank Nanofabrication and Nanomaterials group of Centre for Advanced Microstructures and Devices (Louisiana State University, LA, USA) for providing some of the instrumental facility. FM also acknowledges NIH-supported INBRE program of NCRR (P20RR016456) and Universiti Putra Malaysia for the funding. References [1] M. Patil, D.S. Mehta, S. Guvva, J. Indian Soc. Periodontol. 12 (2008) 34–40. [2] X. Yang, X. Zhang, Z. Liu, Y. Ma, Y. Huang, Y. Chen, J. Phys. Chem. C 112 (2008) 17554–17558. [3] C.S. Brazel, Pharm. Res. 26 (2009) 644–656. [4] S.R. Dave, X. Gao, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1 (2009) 583–609. [5] R.T. Branca, Z.I. Cleveland, B. Fubara, C.S.S.R. Kumar, R.R. Maronpot, C. Leuschner, W.S. Warren, B. Driehuys, Proc. Natl. Acad. Sci. USA 107 (2010) 3693–3697. [6] F. Mohammad, G. Balaji, A. Weber, R.M. Uppu, C.S.S.R. Kumar, J. Phys. Chem. C 114 (2010) 19194–19201. [7] A.M. Derfus, G.V. Maltzahn, T.J. Harris, T. Duza, K.S. Vecchio, E. Ruoslahti, S.N. Bhatia, Adv. Mater. 19 (2007) 3932–3936. [8] C. Leuchner, C.S.S.R. Kumar, W. Hansel, W. Soboyejo, J. Zhou, J. Hormes, Breast Cancer Res. Treat. 99 (2006) 163–176. [9] F. Mohammad, A.C. Raghavamenon, M.O. Claville, C.S.S.R. Kumar, R.M. Uppu, Nanotechnol. Rev. (2014), http://dx.doi.org/10.1515/ntrev-2013-0041. [10] T.K. Jain, J. Richey, M. Strand, D.L. Leslie-Pelecky, C.A. Flask, V. Labhasetwar, Biomaterials 29 (2008) 4012–4021. [11] F. Dilnawaz, A. Singh, S. Mewar, U. Sharma, N.R. Jagannathan, S.K. Sahoo, Biomaterials 33 (2012) 2936–2951. [12] A. Wang, S. Li, BMC Biotechnol. 8 (2008) 1–7. [13] S. Li, A. Wang, W. Jiang, Z. Guan, BMC Cancer 8 (2008) 1–9. [14] H.Y. Hwang, I.S. Kim, I.C. Kwon, Y.H. Kim, J. Control. Release 128 (2008) 23–31. [15] R. Sharma, C.J. Chen, J. Nanopart. Res. 11 (2009) 671–689. [16] M. Suto, Y. Hirota, H. Mamiya, A. Fujita, R. Kasuya, K. Tohji, B. Jeyadevan, J. Magn. Magn. Mater. 321 (2009) 1493–1496. [17] J.P. Fortin, F. Gazeau, C. Wilhelm, Eur. Biophys. J. 37 (2008) 223–228. [18] P. Pradhan, J. Giri, G. Samanta, H.D. Sarma, K.P. Mishra, J. Bellare, R. Banerjee, D. Bahadu, J. Biomed. Mater. Res. Part B: Appl. Biomater. 81B (2007) 12–22. [19] M. Zeisberger, S. Dutz, R. Müller, R. Hergt, N. Matoussevitch, H. Bönnemann, J. Magn. Magn. Mater. 311 (2007) 224–227. [20] C. Alexiou, W. Arnold, R.J. Klein, F.G. Parak, P. Hulin, C. Bergemann, W. Erhardt, S. Wagenpfeil, A.S. Lübbe, Cancer Res. 60 (2000) 6641–6648. [21] S. Dandamudi, R.B. Campbell, Biomaterials 28 (2007) 4673–4683. [22] C.S.S.R. Kumar, F. Mohammad, Adv. Drug Deliv. Rev. 63 (2011) 789–808. [23] C.S.S.R. Kumar, F. Mohammad, J. Phys. Chem. Lett. 1 (2010) 3141–3146. [24] C. Hoskins, L. Wang, W.P. Cheng, A. Cuschieri, Nanoscale Res. Lett. 7 (2012) 77. 12 pp. [25] J.S. Garitaonandia, M. Insausti, E. Goikolea, M. Suzuki, J.D. Cashion, N. Kawamura, H. Ohsawa, I. Gil de Muro, K. Suzuki, F. Plazaola, T. Rojo, Nano Lett. 8 (2008) 661–667. [26] K. Araki, E. Mizuguchi, H. Tanaka, T. Ogawa, J. Nanosci. Nanotechnol. 6 (2006) 708–712. [27] C.F. Thorn, C. Oshiro, S. Marsh, T. Hernandez-Boussard, H. McLeod, T.E. Klein, R.B. Altman, Pharmacogenet. Genomics 21 (2011) 440–446. [28] R.C. Littell, P.R. Henry, C.B. Ammerman, J. Anim. Sci. 76 (1998) 1216–1231. [29] S.Y. Choi, C.C. Collins, P.W. Gout, Y. Wang, J. Pathol. 230 (2013) 350–355. [30] N. Li, Y. Chen, Y.M. Zhang, Y. Yang, Y. Su, J.T. Chen, Y. Liu, Sci. Rep. (2014), http://dx.doi.org/10.1038/srep04164.