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

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Journal of Colloid and Interface Science

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Doxorubicin-loaded magnetic gold nanoshells for a combination therapyof hyperthermia and drug delivery

http://dx.doi.org/10.1016/j.jcis.2014.07.0250021-9797/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Institute of Advanced Technology, Universiti PutraMalaysia, Serdang, Selangor 43400, Malaysia.

E-mail address: faruq_m@upm.edu.my (F. Mohammad).

Faruq Mohammad a,b,⇑, Nor Azah Yusof a

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

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 May 2014Accepted 18 July 2014Available online 2 August 2014

Keywords:Core-shell typeSuperparamagnetismSPIONs@AuHyperthermiaDrug deliveryDoxorubicin

In the present work, nanohybrid of an anticancer drug, doxorubicin (Dox) loaded gold-coated superpara-magnetic iron oxide nanoparticles (SPIONs@Au) were prepared for a combination therapy of cancer bymeans of both hyperthermia and drug delivery. The Dox molecules were conjugated to SPIONs@Au nano-particles with the help of cysteamine (Cyst) as a non-covalent space linker and the Dox loading efficiencywas 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 frequencyoscillatory magnetic fields. The hyperthermia studies investigated to be strongly influenced by theapplied frequency and the solvents used. The Dox delivery studies indicated that the drug release efficacyis strongly improved by maintaining the acidic pH conditions and the oscillatory magnetic fields, i.e. anenhancement in the Dox release was observed from the oscillation of particles due to the applied fre-quency, and is not effected by heating of the solution. Finally, the in vitro cell viability and proliferationstudies were conducted using two different immortalized cell lines containing a cancerous (MCF-7 breastcancer) and non-cancerous H9c2 cardiac cell type.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

In recent years, the involvement of nanotechnology-basedappliances have significantly advanced the traditional medical sec-tor due to the possibility of treating the degenerative diseases suchas cancer, diabetes, cardiovascular, and arthritis by means ofapplying the ‘‘combination therapy or polytherapy’’. The emergingapplications of nanotechnology with advanced drug delivery forexample, offer the possibilities for an early detection of disease,its diagnosis and treatment in addition to disease prevention bymaking use of the multifunctional nanomaterials [1]. The variouskinds of nanomaterials involved in the disease diagnosis and treat-ment purposes include the polymeric (liposomes, microspheres,micelles, etc), metallic particles (core–shell, onion type, composite,alloy, magnetic), quantum dots, carbon nanotubes, etc [2]. Thenanostructured magnetic materials in combination with ther-mally-sensitive polymers form magnetothermally-responsive sys-tems and the use of these systems for drug delivery applicationsallows for an outer controlled triggered release of encapsulatedor conjugated drug molecules to the site specific region of interest.

The use of magnetothermally-responsive systems (or magneti-cally-triggered, thermally-sensitive) significantly improves thetreatment efficiency especially for a disease such as cancer [3,4].With this approach, the diffusion and activated release of drugmechanism achieved through the incidence of a phase change inthe host material by means of alternating magnetic field triggersthe drug externally to the body, in addition to localized heatingin vivo. Since, most of the cancer therapeutic drugs are associatedwith several side effects and thus the benefits of applying the mag-netically-triggered formulations for treating cancerous diseases area reduction in the total amount of drug required to reach an effec-tive dose, less frequency of drug administration, targeted and outercontrolled delivery, possibility for an inclusion of other imagingagents and multiple modes of therapy (combination therapy orpolytherapy) [3,5]. The therapeutic heating can be easily achievedby the use of superparamagnetic particles (of typically less than20 nm size) and the applied field causes the magnetic particles torelease heat by means of two different mechanisms; Néel relaxa-tion (rotation of magnetizations within the particle) and Brownianrelaxation (rotation of particles against the dispersed medium). Incontrast to many other magnetic materials (Mn, Co, Cu, Gd, etc),the superparamagnetic iron oxide nanoparticles (SPIONs) are ofhighest priority due to their biocompatibility, biodegradability,magnetic behavior, and image enhancing (MRI) capabilities [3,6,7].

90 F. Mohammad, N.A. Yusof / Journal of Colloid and Interface Science 434 (2014) 89–97

One of the main advantages of employing magnetic fluid hyper-thermia technique to cancerous diseases is that the possibility ofdestroying only the cancer diseased cells through localized heatingof tumor while simultaneous protection of healthy normal cells.The recent developments such as the availability of various target-ing strategies (antibody, protein, etc), particle formation into nano-size (allowing the easy passage across biological membranes), easyadministration, effective and outer control of heat, more efficientand homogeneous heat release compared to macrosized particles,made the magnetic hyperthermia as one of the preferred ways oftreating the tumors which are located far inside the body (liver,brain, pancreas) [6,8]. Also, in our recent study, we observed thatvarious cell types, i.e. cancer and non-cancer cells respond differ-ently to the SPIONs-induced heat stress when applied using theoscillatory magnetic fields operating at low frequencies. The studyindicated that the normal GT1-7 neurons are more resistant to theheat induced stress than the LNCap prostate cancer cells by meansof expressing molecular chaperones in the form of heat shockprotein-70 only in GT1-7 neurons [9]. Similarly, some of theanticancer drugs which have been tested for external magneticfield-induced targeted drug release studies includes paclitaxel[10,11], doxorubicin (Dox) [10], hydroxycamptothecin [12],5-fluorouracil [13] and docetaxel [14]. It was observed from thesestudies that the presence of external magnetic field significantlyinfluenced the drug release kinetics, targeting ability, magnetichyperthermia and all these contributes finally to the efficiency ofcombination therapy.

In the majority of studies related to hyperthermia-based ther-apy and hyperthermia-based drug delivery, the main challenge inthe design of magnetic particles is that the high frequency of oscil-latory magnetic fields i.e. lies in the range of several kHz and MHz[15,16]. Further, to influence the specific power loss (SPL) which isdirectly correlated with the hyperthermia effect, the changes madein the composition of magnetic particles are negatively affectingtheir use for most of the biomedical applications. For example,even though the presence of Mn, Co, Pt, Ni in SPIONs compositionsignificantly enhancing the SPL, the oxidative instability and freeradical induced toxic mechanisms (apoptosis mostly) with theseelements are still a concern [6,17–19]. Similarly, during the devel-opment of magnetically influenced drug delivery systems, severalattempts have been made for the use of SPIONs while retainingtheir inherent magnetic and image enhancing properties. In thatview, the commonly used approach involves the conjugation orencapsulation of a drug of interest to the polymers such as chito-san, dextran, starch, poly(ethylene glycol), polylactide, andpoly(DL-lactide-co-glycolide). The presence of thermally respon-sive polymers in this approach requires the development of com-plex conjugation chemistry for drug loading (often results inlimited drug association with SPIONs) in addition to the formationof large-sized microparticles with limited drug encapsulation[10,20]. Finally, all these factors adversely influencing the SPIONsbiodistribution, imaging characteristics and drug targeting/releas-ing efficiency in vivo due to the occurrences of the changes in thephysical and/or surface characteristics of original SPIONs (zetapotential, hydrodynamic size, charge, stability and magnetization)[10]. In a similar approach, with the use of liposomal or emulsionformulations for the magnetic drug delivery and tumor imagingapplications, the observed drug loading efficiency is as low as only2–3% [21]. Therefore, construction of an ideal drug delivery systemfulfilling all the specifications such as superior magnetic behavior,no toxicity, and efficient drug loading capability is still remainingas a challenge.

Inspired by our recent review article based on the applicationsof magnetic nanomaterials for dual application sites of diagnosis aswell as therapy [22], the present work is aimed to develop a multi-ple application probe with improved drug loading efficiency and

possesses an externally controlled drug releasing system for can-cer. For that, we constructed a SPIONs probe with an ability tofunction as MRI contrast enhancing agent for the diagnosis andtherapy by two different means. In one way, the probe generateslocal hyperthermia in accordance with the external magnetic fieldwhile in the second way, and the outer controlled delivery of con-jugated drug can be achieved from the oscillation of particles withthe 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 tothe naked SPIONs. Also, the anticancer drug, Doxorubicin hydro-chloride (Dox�HCl) was selected for the study, as it already provedto be cancer therapeutic and its mechanism of action is via interca-lation with cell’s DNA and inhibition of macromolecular biosynthe-sis [10]. For the conjugation of Dox drug to the SPIONs@Auparticles, cysteamine (Cyst) biomolecule was used as a space linkerand the synthetic methodology is shown in the Fig. 1. The particleswere characterized at various stages using HRTEM studies, UV–Vis,FT-IR, magnetization and further used for testing the heat and drugrelease studies. In addition, we also performed the in vitro toxicitytest by using two different cell types which includes a cancerous(MCF-7 breast carcinoma) and the non-cancerous cell type (H9c2cardiomyoblast).

2. Materials and methods

2.1. Chemicals

Iron(III) acetylacetonate (Fe(C5H7O2)3, 99.9%), 1,2 hexadecane-diol (C16H34O2), 90%), oleylamine (C18H37N, 70%), oleic acid(C18H34O2, 99%), phenyl ether (C12H10O, 99%), Cysteamine hydro-chloride (C2H7NS�HCl, 98%), and Doxorubicin hydrochloride(C27H29NO11�HCl, 98%) were purchased from Sigma Aldrich(St. Louis, MO). Gold acetate (Au(C2H3O2)3) was purchased fromAlfa 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, wefirst synthesized SPIONs@Au (gold coated iron oxide NPs) asdescribed by Mohammad et al., i.e. the reduction of organometal-lics in the presence of oleic acid and oleylamine surfactants. Thebiocompatible Au coating on SPIONs expected to provide theoxidative stability for the iron oxide core and protects fromFe-induced free radical generation, in addition to allowing the easyconjugation with biomolecules [6]. The following step involvesthe stabilization of hydrophobic oleic acid/oleylamine coatedSPIONs@Au NPs with Cyst molecules to form a water-dispersiblesystem [23]. Thus obtained SPIONs@Au–Cyst NPs are stable, waterdispersible and further used for the loading of Dox molecules(of hydrophilic behavior) by using the method described by Jainet al. [10].

Briefly, 0.7 g of iron acetylacetonate was mixed with 20 mL ofphenyl ether, 2 mL of oleic acid, and 2 mL of oleylamine under inertatmosphere with vigorous stirring in a three necked round bot-tomed flask. To this, 2.6 g of 1,2-hexadecanediol was added andheated to 210 �C with reflux for 2 h in a nitrogen atmosphere. Fol-lowing the period, the reaction mixture was cooled, ethanol(degassed) was added and the precipitate of SPIONs (average diam-eter: 5.1 nm) was separated by centrifugation. In the second step,0.5 g of SPIONs was mixed with a solution containing 30 mL ofphenyl 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 atmo-sphere. The reaction mixture was heated to around 190 �C withreflux for about 1.5 h. Following the period, the contents were

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

F. Mohammad, N.A. Yusof / Journal of Colloid and Interface Science 434 (2014) 89–97 91

cooled to room temperature, ethanol was added, and the precipi-tate was separated out by centrifugation. The product was sus-pended in hexane, washed with ethanol 3–4 times to obtainSPIONs@Au NPs (hydrophobic nature). The third step involvesthe stabilization of formed SPIONs@Au so that they are waterdispersible and for that, a colloidal solution containing 30 mg ofSPIONs@Au in 10 mL of toluene was prepared. In another flask,10 mg of Cyst in 10 mL of distilled water was prepared. Both thesolutions were mixed together and stirred for about 10 min. Atfirst, one can see clearly two different phases with the bottom col-orless due to water and the top purple-colored toluene containingthe SPIONs@Au and after 2–4 h, the migration of SPIONs@Aucompletely into the Cyst water phase can be seen. This is a roomtemperature reaction and the driving force for this is provided bythe affinity of thiol (ASH) of Cyst with the gold surface to formSPIONs@Au–Cyst NPs. The SPIONs@Au–Cyst NPs were separatedwith the help of a magnet, washed 2–3 times with distilled water,and dried under inert atmosphere. In order to form Dox loadedSPIONs@Au–Cyst particles, an aqueous solution containingDox�HCl (at a concentration of 0.5 mg/mL) was added drop-wiseto a colloidal dispersion of SPIONs@Au–Cyst NPs (0.5 mg/mL in dis-tilled water) while stirring. The mixture was allowed to stir over-night (�16 h) to allow partitioning of drug into Cyst and oleicacid/oleylamine layer surrounding the SPIONs@Au core. Finally,the Dox-loaded SPIONs@Au–Cyst NPs were separated from the freedrug using a magnet, washed with distilled water, dried andstored. With the help of standard concentration curve of Dox (atthe absorption of 485 nm) generated from UV–Vis spectrophotom-eter using a series of Dox solutions of varying concentrations, theunbound Dox remained in the supernatant was analyzed [2]. Sim-ilarly by making use of the calibration curve, the samples from Doxrelease studies were also analyzed in order to estimate the amountof drug that got released into the solvent media due to a change inpH, 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 electronmicroscope operating at 200 kV and the samples were preparedby dispersing the particles in distilled water and then droppingonto the carbon coated copper grid. The EDAX were also simulta-neously recorded by focusing the electron beam onto the particles

using the same JEOL 4000EX electron microscope attached to anEDAX analyzer. The UV–Visible spectroscopic analysis were carriedout on SPECORD� PLUS double beam spectrophotometer andQE65000 spectrometer supplied by Ocean Optics limited and thesamples were prepared by using distilled water as solvent in a typ-ical wavelength range of 300–800 nm. The FT-IR spectrums wererecorded on Nexus 670 FT-IR spectrometer in the transmissionmode and the samples were prepared by grinding the materialwith dry powder of KBr and then made into a transparent pellet.The magnetic measurements were conducted with the help ofQuantum Design MPMS-5S superconducting quantum interferencedevice (SQUID) magnetometer using the dry powders of the sam-ple placed in a gel-capsule stuffed with kim-wipe paper which isthen placed inside a plastic straw. The magnetizations were mea-sured in two different modes; Zero-field-cooled (ZFC) and Field-cooled (FC). The sample was ZFC to 5 K, a field was applied andmagnetization was measured as a function of temperature, whilein FC, the sample was field-cooled from above 300 K to 5 K andthe magnetization was measured. The temperature at which thetwo curves ZFC and FC meet is considered to be the blocking tem-perature (TB). Hysteresis loops, in which the sample’s magnetiza-tion is measured as a function of applied field in the temperaturefrom of 5 K to 300 K.

2.4. In vitro cell viability and proliferation assay

The cell viability and proliferation were assessed based on met-abolic activity of cells using the CellTiter-Blue� reagent from Pro-mega (Madison, WI) and the assay involves the reduction ofresazurin, a non-fluorescent blue dye, into a pink colored, red-fluo-rescent resorufin. Briefly, the H9c2 rat embryonic cardiomyoblastswere 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 incuba-tor at 37 �C, 5% CO2, and in 95% humidity. Approximately 104

cells/well were seeded onto 96-well plates and allowed to growfor 24–36 h. When the cells reach to �80% confluence, the mediumwas replaced with fresh DMEM containing 2% (v/v) FBS and vary-ing concentrations (25–500 lg/mL) of SPIONs@Au–Cyst and incu-bated for additional 24 h. After the period, the CellTiter-Blue�

reagent (20 lL) was added and incubated for additional 3–4 h asper the manufacturer’s instructions. The amount of resorufinformed during this time was measured at excitation and emission

92 F. Mohammad, N.A. Yusof / Journal of Colloid and Interface Science 434 (2014) 89–97

wavelengths of 560 and 590 nm (respectively) using a SpectramaxEM Gemini spectrofluorimeter (Molecular Devices, Sunnyvale, CA).Background fluorescence from the wells that containedSPIONs@Au–Cyst in DMEM but no added cells was subtractedand phorbol myristate acetate (PMA; 10 lg/mL) used as a positivecontrol. The percentage cell viability was calculated with respect tothe untreated control (set at 100%) and the values shown aremean ± standard deviations (SDs) of three individual experiments[6,24].

The MCF-7 breast carcinoma cells were maintained in Eagle’sminimum essential medium (EMEM) containing 0.01 mg/mLbovine 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 viabilityfor MCF-7 cells was measured following the treatment with SPI-ONs@Au–cyst (25–500 lg/mL) in 2% FBS for 24 h, based on theconversion of resazurin to resorufin by viable cells.

2.5. Studies of hyperthermia

The apparatus used for applying the magnetic field and the tem-perature measuring conditions were discussed in our previousreports [6]. Briefly, the alternating magnetic field-induced heatreleasing experiments were carried out by using the custom-madesetup consisting of copper coil (Alpha-Core Inc.) and the magneticfield inside it is controlled with the P1351 Behlman AC powersource. The highest parameters that the setup can generate are of430 Hz field frequency at 123 V, and the lowest parameters of44 Hz field frequency at 15 V. In addition to the highest and lowestfrequencies, an intermediate frequency of 230 Hz at 67 V wasselected for the studies and the measurements were carried overa period of 60 min.

2.6. Release of Dox from SPIONs@Au–Cyst–Dox

The studies of drug release kinetics from the surfaces ofSPIONs@Au–Cyst–Dox NPs were conducted as against three differ-ent parameters of pH, oscillatory magnetic fields, and temperature.Each time, a colloidal solution was prepared by taking 5 mg ofSPIONs@Au–Cyst–Dox into 2.5 mL of distilled water and thechanges in the concentration of Dox in the dispersion mediumwere measured over a period of 3 h. With the help of the standardconcentration curve of Dox generated from the UV–Vis spectro-photometer, the release of Dox into distilled water was quantifiedduring the specified time intervals [2].

2.7. Statistical analysis

The hyperthermia data i.e. the recorded temperature change ofthe medium were arranged in multivariate repeated measures andanalyzed by using the PROC GLM in SAS software. However, thestatistical analysis of cell viability and drug release data is per-formed by using a one-way analysis of variance (ANOVA) andBonferroni’s method for multiple comparisons. All of the data inthis report are presented as mean ± SD of 3–5 separate experi-ments. The probability of p < 0.05 was considered as statisticallysignificant (represented by ⁄) and p < 0.01 as highly statistical sig-nificant (represented by ⁄⁄).

3. Results and discussion

In the present study, an anticancer drug, Dox-loadedSPIONs@Au–Cyst particles was prepared for the combined therapyof cancer by means of hyperthermia as well as chemotherapy. Theparticles are characterized thoroughly and were tested for the

hyperthermia and drug release efficacy by the influence of changesin pH, temperature and external magnetic field. In addition to thedrug release kinetics, the in vitro toxicity investigation was alsoperformed on two different immortalized cell lines of a cancerous(MCF-7 breast) and a non-cancerous (H9c2 cardiac). The reason forchoosing H9c2 cardiac cells is to see the effect of our synthesizedparticles toward cardiac cells, as it is well known for the observa-tion of cardiotoxicity in patients undergoing chemotherapeutictreatment involving Dox. Similarly, to see the response of cancercells with our particles, the MCF-7 breast cancer cells wereselected.

3.1. Physical characterization

Fig. 2(a)–(c) shows the HRTEM images, corresponding particlesize distributions and EDAX-based elemental analysis ofSPIONs@Au–Cyst NPs respectively. From the figure, one can seeclearly that the particles are spherical and uniform with an averageparticle size of around 6.8 nm. From the EDAX spectrum shown inFig. 2(c), the appearance of peaks resembles to the presence of cor-responding elements in the particle; the major peaks, S from ASHof cysteamine, Au and Fe from the gold coated iron oxide core. Itcan also be seen from the figure that the appearance of Cu peakhowever is generated from the carbon coated copper grid used inthe preparation of HRTEM samples.

The magnetic characterization of hysteresis curves and temper-ature-dependent magnetization curves (ZFC–FC) for theSPIONs@Au–Cyst is shown in the Fig. 3(a)–(b). From the hysteresisdata (Fig. 3a) recorded at 5 and 300 K, the observation of hysteresisloops at room temperature confirming the superparamagneticbehavior of SPIONs@Au–Cyst NPs. The saturation magnetization(Ms) values observed to be 84 and 78 emu/g at 5 and 300 K respec-tively, while the blocking temperature (TB) found to be 201 K(Fig. 3b). It was observed from our previous studies [6] that theSPIONs@Au NPs containing oleic acid/oleylamine surface ligandspossesses the Ms. value of 58 emu/g (at 5 K) and the same particleson their surface modification with cysteamine, the Ms. value sig-nificantly increased to 84 emu/g. This significant enhancement inthe magnetization value (at 5 K) are attributed to the intrinsicallydeveloped magnetism due to the modification of the local electronstructure of Au upon the formation of AuAS bond, i.e. by means ofsharing the 3d electron of Au with that of S of thiol in cysteamine[25]. In general, any system with high magnetization values is wellsuited 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 materialcan exhibit the corresponding magnetizations even at higher tem-peratures. This is particularly useful in hyperthermia applicationswhere the oscillating magnetic particles generally lose their mag-netic behavior with an increase of temperature of the mediumdue to its own heating property. But in our case, the observationof a high TB indicating that efficient, prolonged heat release canbe expected from the SPIONs@Au–Cyst particles under magneticfields due to the existence of high magnetizations at high temper-atures. The observation of superparamagnetic behavior, high Ms.and TB values is thus making the SPIONs@Au–Cyst NPs to be idealfor magnetic fluid hyperthermia and externally controlled mag-netic drug delivery applications.

The SPIONs@Au core contains oleic acid, oleylamine and cyste-amine molecules which possess the functional groups of unsatura-tion (C@C), carboxylic (ACOOH), amino (NH2) and thiol (ASH) andhence it can easily form p–p electronic, Van der Waals interactionand strong hydrogen bonding with the quinone portion of Doxmolecules containing the AOH, ANH2 groups. The UV–Vis spectro-scopic comparison of the SPIONs@Au–Cyst before and after loading

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 dependenceof magnetization.

F. Mohammad, N.A. Yusof / Journal of Colloid and Interface Science 434 (2014) 89–97 93

with Dox is shown in Fig. 4(i). From the figure, a strong absorptionmaximum for the SPIONs@Au–Cyst NPs is observed at 567 nm dueto the surface plasmon resonance property of gold and forpure Dox is at 485 nm. However, on loading of Dox drug withthe same SPIONs@Au–Cyst NPs, a broader peak with two absorp-tion maxima is observed; one being at 495 nm and the other at525 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

Fig. 4. Comparison of the (i) UV–Visible spectrum and (ii) FT-IR spectrums

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

of SPIONs@Au–Cyst with that of pure Dox and SPIONs@Au–Cyst–Dox.

94 F. Mohammad, N.A. Yusof / Journal of Colloid and Interface Science 434 (2014) 89–97

between the Dox groups and other ligand surface groups from themaintenance of neutral conditions while preparing the material.The FT-IR spectral comparison of the SPIONs@Au–Cyst andpure Dox drug was recorded and compared with that ofSPIONs@Au–Cyst–Dox to further confirm the bonding and isshown in Fig. 4(ii). From the figure, the sharp peak around1734 cm�1 in pure Dox (due to C@O) shifted to 1730 cm�1 andthe broad peak at 3415 cm�1 (due to –OH stretching) becomingsharp and shifted to 3428 cm�1 after forming the nanohybrid com-plex with SPIONs@Au–Cyst. Similarly, the peak in SPIONs@Au–Cystaround 780 cm�1 due to CASH bending changed to 760 cm�1 onbonding with Dox and the FeAO stretching in magnetite core ofSPIONs@Au–Cyst at 540 cm�1 changed to 530 cm�1 confirmingthe persistence of the SPIONs@Au even after the Dox loading.The similar results were observed when the Dox drug was usedfor conjugation with graphene oxide and during the formation ofthiol protected gold nanostructures [2,26]. The comparison of bothUV–Vis and FT-IR spectral data therefore, confirming the bondingof Dox molecules with SPIONs@Au–Cyst NPs.

3.2. In vitro cell viability and proliferation

The comparison of cell viability and proliferation studies of theSPIONs@Au–Cyst and SPIONs@Au–Cyst–Dox at concentrations inthe range of 25–500 lg/mL for an exposed period of 24 h towardthe H9c2 cardiomyoblasts and MCF-7 breast carcinoma cells isshown in Fig. 5(a)–(b). From the analysis of results with respectto positive and negative controls (Fig. 5a), it is clear that theSPIONs@Au–Cyst does not induce much change in the viabilityto either H9c2 cardiomyoblasts or MCF-7 carcinoma cells(under experimental conditions). However, the drug loadedSPIONs@Au–Cyst particles (Fig. 5b) under similar conditions exhib-ited significant decrease in the viability and proliferation towardboth the cell types over 100 lg/mL concentration. The reason forsuch a non-toxic activity by naked SPIONs@Au–Cyst is that thecore iron oxide particles are coated completely with the biocom-patible gold shell and their surfaces are well protected with bio-molecules such as oleic acid, oleylamine and Cyst. All thesesurface components are protecting the cell cultures from theFe-induced free radical reactions either by the direct oxidation ofintracellular components or indirect oxidation through the culturemedium. While the cell viability changes by SPIONs@Au–Cyst–Doxtreated cultures are attributed to the effects of the anticancer drug,Dox, since it already proved to be responsible for the pharmacolog-ical activity by the mechanisms of DNA strand breakage, mitochon-drial depletion, free radical-mediated doxorubicinol metaboliteformation, etc [27]. In addition, the reduction in the viability ofH9c2 cardiac cells also gives a hint that the targeted deliveryshould be strictly implemented especially when Dox is used duringcancer treatment so as to avoid the drug actions on non-target

Fig. 5. Comparison of the cell viability and proliferation studies of SPIONs@Au–Cyst NPsand H9c2 cardiomyoblast cells. From the figure, ⁄ represents significant and ⁄⁄ represen

tissues liver, lung, heart and kidneys. For any application in bio-medical sector, the selected probe should not possesses any kindof intrinsic toxicity and the absence of toxicity behavior inSPIONs@Au–Cyst primarily confirms that it can serve as a stableplatform for hyperthermia and drug delivery.

3.3. Effect of magnetic field and solvent viscosity toward hyperthermia

The studies of hyperthermia i.e. external magnetic field-induced temperature increase of medium with time at three differ-ent frequencies and solvents of varying viscosities are shown inFig. 6(a)–(d). The measurements were carried at the oscillation fre-quencies of 44, 230 and 430 Hz over a period of 60 min and the sol-vents include water, ethanol and toluene. From the comparison ofresults shown in Fig. 6(a)–(c), it can be observed that the heatrelease is both frequency and viscosity dependent, i.e. the heatingrate is increasing with an increase of applied frequency anddecrease of solvent viscosity. The temperature increase of mediumfollows the order of 44 Hz < 230 Hz < 430 Hz and toluene < etha-nol < water. One can also see from the figure that more tempera-ture raise is observed during the first 30 min of exposure period(significant increase being the first 15 min), followed by reductionof heat release on further exposure. This fall in the heat release canbe attributed to the possible quenching of the particles oscillationin accordance with the applied field, as gold is a better heat con-ductor. Also, under similar experimental conditions, the hydropho-bic magnetic particles (SPIONs@Au) tested in our previous studiesprovided the information that more heating is observed in case ofwater medium at the lowest frequency of 44 Hz [6]. However, withthe use of hydrophilic particles (SPIONs@Au–Cyst–Dox) in thepresent work, the more heating is observed in the same watermedium but at the highest frequency of 430 Hz. These results pro-vide the information that in magnetic fluid hyperthermia, theBrownian-rotation mechanism is also equally important to Néelrelaxation for heat release by the magnetic particles, as the heatingeffects are changing with respect to the frequency and solventmediums. Thus, while designing the magnetic particles for heat-related applications, it is necessary to consider the applied field,viscosity of the medium and the solvent dispersibility of the parti-cles. Further, to confirm whether the obtained data is statisticallysignificant or not, the data were arranged in multivariate repeatedmeasures and analyzed by using the PROC GLM in SAS software[28]. The results of multivariate tests showed that there were sig-nificant effects of TIME, TIME * FREQUENCY and FREQUENCY * SOL-VENT 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 rep-resented in terms of SPL, i.e. the ability to generate heat from the

(a) before and (b) after loading with Dox when tested against MCF-7 breast cancerts highly significant values against the untreated control measurements.

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.

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

Table 1SPL values of SPIONs@Au–Cyst–Dox measured at various frequencies and dispersionmediums.

Type of Medium SPL (W/g)

44 Hz 230 Hz 430 Hz

Water 697.5 460.3 1199.7Ethanol 203.3 227.7 256.2Toluene 67.8 87.7 210.9

F. Mohammad, N.A. Yusof / Journal of Colloid and Interface Science 434 (2014) 89–97 95

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

SPL ¼ CVsM

dTdt

where C is the specific heat of the sample (C = 4.185, 2.44, and1.13 J/g/�C for water, ethanol, and toluene, respectively), Vs is thevolume of the sample medium, M is the weight of the magneticmaterial in grams, and dT/dt is the raise in temperature per unittime.

The calculated SPL values for the SPIONs@Au–Cyst–Dox mate-rial tested in three different mediums of varying viscosities andapplied frequency are shown in Fig. 7 and are tabulated in Table 1.From the data, it is evident that the material is exhibiting high SPLfor water compared to other two mediums of ethanol and tolueneat all three frequencies and these results are in agreement with thespecific heat of the solvents. Also within the water medium, it isinteresting to note that the material is exhibiting high SPL(1199 W/g) at the highest applied frequency of 430 Hz and thelowest value (460 W/g) at the intermediate applied frequency of230 Hz. Surprisingly, the material exhibited the medium SPL(697 W/g) at the lowest applied frequency of 44 Hz in water med-ium. However, this effect is not observed in other two mediums ofethanol and toluene, i.e. the SPL values are increasing in accor-dance with the applied frequency. The similar effects of low SPLvalues at the same intermediate frequency (230 Hz) are observedwhen the hydrophobic SPIONs@Au NPs were tested for the heatrelease studies [6]. One probable reason for this can be due tothe fact that the applied frequency (at 230 Hz) is not completelytaken up by the particles so as to convert it into heat, it may beconverting into other forms or some times more possible quench-ing resulting in a loss of heat release.

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 SPI-ONs@Au–Cyst NPs, it may be used as a carrier material for drugdelivery. The studies of Dox drug release (%) from the SPI-ONs@Au–Cyst NPs as a function of solution pH, applied frequency

96 F. Mohammad, N.A. Yusof / Journal of Colloid and Interface Science 434 (2014) 89–97

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 highestdrug release efficiency of about 56% (over a 3 h period) in acidicconditions of pH 2.1, when compared against neutral pH of 7.4and basic pH of 10.3. The reason for the high efficacy at low pHcan be attributed to the effects of both the formation of ions andlack of hydrogen bonding between Dox and other ligand groups.Under acidic conditions, more number of H+ ions are availableand can associate easily with the –NH2 of Cyst or oleylamine (pres-ent as ligands on SPIONs@Au core) at which the Dox molecules arenon-covalently linked. The surface ligand molecules always try tolose the bulkier Dox molecules and facilitate for the formation ofammonium ions (NH3

+) by making use of the H+ ions. Similarly,under basic conditions (pH 10.3), the ACOOH of oleic acid existsas COO� and the ASH of cysteamine changes to AS� and is notavailable for participation in a strong hydrogen bonding with theDox groups, thereby better drug release are observed. Howeverin neutral conditions, due to the absence of H+ ions and the abilityof surface ligand groups to form a strong hydrogen bonding withthe Dox groups, a very low drug release was observed. Since themetabolic rate of cancer cells is very high compared to the healthynormal cells and due to this, the local tumor environment is acidicby the formation of lactic acid. In that view, the property of highdrug release rate under acidic conditions is especially suits for can-cer drug delivery, as more amount of Dox can be released into thetumor on localization [29]. These results of more efficient drugrelease under acidic conditions followed by basic environmentare supported by a similar study where the same Dox drug wasused in conjugation with graphene oxide [2].

The release of Dox under the influence of externally appliedmagnetic fields with increase of frequency in an acidic pH of 2.1is shown in the Fig. 8b. From the analysis of results, it is evidentthat the drug release rate increased in accordance with theincrease of applied frequency and is due to the coupling of oscillat-ing particle’s magnetic moment with the field. Under these condi-tions, the significant drug release (%) effects are observed during

Fig. 8. Studies of drug release kinetics as a function of (a) pH, (b)

the first 30–60 min of field exposure when recorded over a 3 h per-iod and the maximum drug released was investigated to be 72% at430 Hz, followed by 57% at 230 Hz and 49% at 44 Hz. These resultsare significant when compared with the pH effects where the max-imum release was seen at the highest period of 3 h; however,under the influence of magnetic field, a 72% release observed dur-ing the first 30–60 min period. Since the oscillation of magneticparticles also generates heat in addition to drug release, we there-fore tested the Dox release under the influence of temperature ofthe medium (without applying any magnetic field). These studieswere carried in order to make sure that the enhanced drug releaseeffects are only due to particles magnetic moment influenced bythe external field, but not from the heating of solution where thereis a possibility for the breakage of some heat sensitive bonds.Fig. 8c shows the Dox release studies under the influence of tem-perature of the medium measured at 28, 32 and 35 �C. The selec-tion of these parameters are based on the fact that, theapplication of a 430 Hz frequency causes the maximum tempera-ture raise of 35 �C (when started from room temperature of25 �C) in the medium and similarly, a 28 �C and 32 �C wereobserved for the oscillations at 230 Hz and 44 Hz respectively. Itwas already described that at 230 Hz (Fig. 7), the SPL generatedis low compared to other two frequencies due to some undefinedlosses i.e. the applied field at this frequency is not getting utilizedentirely for the conversion into heat and hence a lower tempera-ture of 28 �C assigned to 230 Hz frequency. From the analysis ofresults (Fig. 8c), a maximum Dox release of 57% was observed atthe highest time period of 3 h in a 35 �C containing temperaturemedium. This release was followed by the 32 �C containing med-ium with the total Dox release of 46%, and the temperature(32 �C) was corresponding to the 44 Hz frequency from the hyper-thermia measurements. However, the Dox release studies underthe influence of frequency giving the information that the drugrelease is increasing in accordance with the applied frequency(Fig. 8b), i.e. 430 Hz > 230 Hz > 44 Hz. The temperature depen-dence Dox release studies in the absence of applied field indicating

external magnetic field and (c) temperature of the medium.

Table 2Comparison 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

F. Mohammad, N.A. Yusof / Journal of Colloid and Interface Science 434 (2014) 89–97 97

that there is no effect of solution temperature on the drug releasebecause of the fact that the same amount of drug release (56%) wasobserved even at room temperature (Fig. 8a, Table 2). This meansthat although some activation energy available in the form of tem-perature, but is not getting utilized for the release of Dox from thesurface of SPIONs@Au–Cyst particles. The supplied energy in tem-perature form in turn may be getting absorbed by the metals likeAu and Fe due to their conductive behavior and is not allowingfor the enhancement of drug release rate by breaking the non-covalent interactions between the groups of cysteamine, oleic acid,oleylamine and Dox. All these analysis confirming that the mag-netic moment is the dominating factor for the observation ofenhanced drug release and the in situ generated temperature ofthe medium do not influence the Dox release rate.

4. Summary and conclusion

In the present report, we introduced novel magnetic gold nano-shells that can be useful in a ‘combination therapy’ for treatingcancer by multiple application modes. It was observed that themagnetic properties are greatly enhanced (Ms. of 84 emu/g at5 K) on stabilization with Cyst biomolecule and also found to beexhibiting the magnetic susceptibility at higher temperatures(201 K) compared to simple SPIONs@Au particles. With those mag-netic properties, the heat releasing efficiency was increased withincrease of applied frequency due to the possibility of effectivecoupling of particles magnetic moment with the field and withthat, water exhibited a very high SPL value (1199 W/g at 430 Hz)among other two frequencies and tested media. In addition, wealso proved that a simple amino acid derivative, Cyst can effec-tively functions to mask the hydrophobic character of oleic acid/oleylamine in addition to achieving the non-covalent conjugationof Dox molecules and with this system, the loading efficiencyobserved to be as high as 63%. The intrinsically non-toxic behaviorof Dox-unloaded SPIONs@Au–Cyst NPs primarily supporting thatthe particles can be suitable for biomedical applications asobserved from the cell viability and proliferation studies. Takingadvantage of the non-toxic behavior and high magnetic suscepti-bility which helps to generate enough heat and also induces Doxrelease by the coupling of magnetic moment with the applied fieldeven at low frequencies, the SPIONs@Au–Cyst particles can there-fore serves to be sophisticated for the application of simultaneoushyperthermia and outer controlled drug delivery. The magneticallycontrolled drug release phenomenon can especially be usefultoward cancerous diseases through the introduction of targeteddelivery of drug loaded magnetic particles and thereby achievingthe enhanced bioavailability for highly toxic anticancer drugs[30]. In addition, the frequency dependent drug and heat releasingproperty can be utilized for enhancing the cancer treatment effi-ciency by increasing the anticancer drug bioavailability and localtumor temperature respectively, all of which controlled by theexternal magnetic field. Moreover, the hyperthermia propertycan also be extended to operate for the decomposition ofthermo-responsive or heat sensitive polymers in order to release

encapsulated therapeutic drugs in controlled amounts at any spe-cific targeted site of interest controlled by external magnet. Fur-ther, this technology will empower the development of drugdelivery vehicles which are capable of delivering small or largemolecules with controlled pharmacokinetics. Future studies areunderway to understand the mechanism of cell death induced bythe use of targeted SPIONs@Au–Cyst–Dox particles when testedin combination study of hyperthermia with drug delivery on vari-ous cancer and non-cancer cell types.

Acknowledgments

FM would like to thank Nanofabrication and Nanomaterialsgroup of Centre for Advanced Microstructures and Devices(Louisiana State University, LA, USA) for providing some of theinstrumental facility. FM also acknowledges NIH-supported INBREprogram of NCRR (P20RR016456) and Universiti Putra Malaysiafor the funding.

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