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Accelerated gene

Department of Chemical and Materials En

Paul Anderson Tower, Lexington, KY 4050

+1-859-323-1929; Tel: +1-859-257-9844

Cite this: RSC Adv., 2015, 5, 18888

Received 31st October 2014Accepted 4th February 2015

DOI: 10.1039/c4ra13564d

www.rsc.org/advances

18888 | RSC Adv., 2015, 5, 18888–1889

ration of free radicals by ironoxide nanoparticles in the presence of analternating magnetic field

Robert J. Wydra, Catherine E. Oliver, Kimberly W. Anderson, Thomas D. Dziublaand J. Zach Hilt*

The surfaces of iron oxide nanoparticles are capable of catalytically generating reactive oxygen species

(ROS) through the Fenton and Haber–Weiss reactions. Fenton chemistry has been shown to be

temperature dependent with an increase in activity up to 40 �C and then a decrease above this

temperature as the hydrogen peroxide degrades into oxygen and water which limits the reaction. When

exposed to an alternating magnetic field (AMF), iron oxide nanoparticles absorb the energy from the

magnetic field and convert it into heat. In this study, we observed an increase in the degradation of

methylene blue when a suspension of magnetite nanoparticles (Fe3O4) was exposed to an AMF

indicating there was an increase in the ROS generation in response to the AMF. The increase in ROS

generation compared to the Arrhenius prediction was both time and concentration dependent; in which

we observed a decrease in ROS enhancement with increased time of exposure and concentration. We

postulate that the decrease is due to agglomeration in the presence of the field. As the nanoparticles

agglomerate, there is a decrease in surface area per mass limiting the reaction rate.

1. Introduction

Magnetic nanoparticles are of great interest for a wide range ofapplications due to their unique physical properties.1,2 Theseapplications include catalysis, biomedical imaging, anemiasupplement, drug delivery, thermal therapy, and environmentalremediation.3–9 One physical property that has presented itselfas a double-edged sword is the generation of free radicalsattributed to Fenton and Haber–Weiss chemistries. In the caseof biomedical applications, the generation of free radicals leadsto oxidative stress which is believed to be one of the keyunderlying mechanisms of concentration dependent cytotox-icity.10–16 At the same time, iron oxide nanoparticles arecombined with hydrogen peroxide and successfully used asadvanced oxidation processes for the removal organic contam-inates from wastewater.17 In both cases, iron oxide nano-particles act as a homogeneous and heterogeneous catalyst forthe degradation of hydrogen peroxide into free radicals. Thecatalytic mechanism is based on the environment. Underneutral conditions, Voinov et al. have demonstrated throughspin-trapping EPR that g-Fe2O3 nanoparticles produce hydroxylradicals primarily on the surface rather than dissolution of freeions.18 At lower pH, such as the microenvironment of a

gineering, University of Kentucky, 177 F.

6, USA. E-mail: hilt@engr.uky.edu; Fax:

3

lysosome, iron ions can be released from the nanoparticlesurface resulting in a greater extent of homogeneous catalysis.19

When exposed to an alternating magnetic eld (AMF),magnetic nanoparticles absorb the energy from the magneticeld and convert it into heat through primarily through Brow-nian relaxation (physical rotation of the particles) and Neelrelaxation (rotation of the magnetic moment).20 This heatgeneration has been extensively studied as magnetic uidthermal therapy for the treatment of cancer.21,22 Until recently,intracellular hyperthermia has been considered improbabledue to heat transport calculations by Rabin that demonstratedtheoretically the heat generated from a single nanoparticle orcluster of nanoparticles would be negligible to the cell.23

However, work by Creixell et al. has demonstrated that inter-nalized targeted nanoparticles can induce cellular death whenexposed to an alternating magnetic eld without a measurabletemperature rise.24 In follow-up work, the surface temperatureof the nanoparticles was shown to instantly increase and todrive a temperature responsive polymer beyond its lower criticalsolution temperature of 35 �C without an immediate increase insolution temperature.25 Thereby, they demonstrated that thelocalized heating from the nanoparticle surface is capable ofaltering surrounding chemistry and possibly a mechanism ofthe previously reported toxicity. The exact mechanism of toxicityis still under debate, however the evidence of a local heatingeffect provides interesting scenarios where the energy dissi-pated by the nanoparticles can be utilized to only alter the nearsurrounding volume.

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Paper RSC Advances

The kinetic behavior of the advanced oxidation processinvolving magnetic nanoparticles to mineralize pollutants hasbeen extensively studied.26–29 Temperature is one of the drivingfactors of the Fenton-like reaction up to about 40 �C.30 Abovethis temperature, hydrogen peroxide begins to degrade intooxygen and water limiting the reaction. To date, no one hasstudied the effect of AMF exposure on the generation of freeradicals from iron oxide nanoparticles.

The main objective of this study is to determine the inu-ence of AMF exposure on the degradation of methylene blue bymagnetite nanoparticles (Fe3O4) and hydrogen peroxide. Threeiron oxide concentrations were selected based on their ability toheat the surrounding media. The low concentration of ironoxide nanoparticles did not heat the solution above the radiantheat from the inductive source coil; whereas the high concen-tration actively heated the solution. The kinetic behavior wasrst determined using temperature controlled water baths. Thisdata was used to extrapolate an Arrhenius relationship whichprovided theoretical values based on the temperature achievedduring AMF exposure. The reported enhancement was denedas the ratio of the experimental degradation observed comparedto the theoretical value.

2. Materials and methods2.1 Materials

Iron(III) chloride hexahydrate (FeCl3$6H2O); iron(II) chloridetetrahydrate (FeCl2$4H2O); hydrogen peroxide (H2O2); andmethylene blue (MB) were obtained from Sigma Aldrich(St Louis, MO). Ammonium hydroxide (NH4OH) was obtainedfrom EMD Chemicals (Gibbstown, NJ). All materials were usedas received.

2.2 Iron oxide nanoparticle synthesis

A one-pot co-precipitation method was used to prepare the ironoxide nanoparticles as similarly reported previously.31 Briey,aqueous solutions of FeCl3$6H2O and FeCl2$4H2O werecombined in a 2 : 1 molar ratio in a sealed three-neck askunder vigorous stirring and an inert N2 environment. Once85 �C was reached, a 1.5 M solution of NH4OH was injected intothe vessel and the reaction was carried out for 1 hour. Thenanoparticles were initially decanted via magnetic decantationto remove majority of the impurities. Aer, they were resus-pended in water and transferred to a dialysis bag for 48 hours ofdialysis. Following the washing steps, the particles were storedin suspension.

2.3 Nanoparticle characterization

Dynamic light scattering (DLS). DLS measurements wereobtained using a Malvern Zetasizer, Nano ZS90 instrument.Nanoparticles were diluted in DI water to a concentration of200 mg ml�1 and dispersed using ultrasonication.

Alternating magnetic eld heating. The AMF heating prolewas observed using a custom made Taylor Wineld magneticinduction source and temperature wasmeasured with a LuxtronFOT Lab Kit. Nanoparticles were diluted in DI water to a

This journal is © The Royal Society of Chemistry 2015

concentration of 5 mg ml�1. One ml of solution was placed in a2 ml centrifuge tube and placed in the center of the coil. Thesolution was heated in a eld of approximately 60 kA m�1 instrength at 292 kHz frequency for 5 minutes.

2.4 Methylene blue degradation

The methylene blue degradation experiments were performed in2 ml microcentrifuge tubes in either temperature controlledwater baths or exposed to an AMF. Oneml samples were preparedby diluting stock concentrations of methylene blue to 5 mg ml�1

and iron oxide nanoparticles to 37.5, 75, and 150 mg ml�1. Thesamples were placed in the water bath for 10min to equilibrate tothe set temperature. The degradation was initiated by spiking thesamples with 25 ml of 30% H2O2 to a working concentration of245 mM. Aer given time intervals the samples were centrifugedfor 30 s using a Phenix Quickspin Centrifuge, magnetically dec-anted, and measured using UV-visible spectroscopy (maximumabsorbance at 665 nm) with a Varian Cary. To account fornanoparticle scattering from the nanoparticles that remain insuspension, samples containing only nanoparticles weremeasured and subtracted out from the sample absorbance.

Samples exposed to the AMF were prepared as describedabove. They were placed in water baths which corresponded tothe expected steady state temperature as a result of eld expo-sure. The samples were exposed to a eld of approximately51.0 kAm�1 in strength at 292 kHz frequency while temperaturewas measured with a Luxtron FOT Lab Kit.

2.5 Statistical analysis

Statistical analysis of the enhancement factor was determinedusing a one sample t-test where the hypothetical mean was setto 1. To indicate signicant enhancement a single, double, ortriple asterisk corresponding to P < 0.05, 0.01, and 0.001respectively were included in the gure.

3. Results and discussion3.1 Nanoparticle characterization

The hydrodynamic size of the particles was determined withDLS and reported as Z-average. Iron oxide nanoparticles were107 nm with a PDI of 0.176 when dispersed in water. Previouswork with TEM has determined the core nanoparticles to be onthe order of 10 nm. As these are uncoated nanoparticles with nostabilizing group, agglomerates are likely contributing the inthe increase in hydrodynamic size compared to the crystal sizeobserved in TEM.

The heating characteristics of the nanoparticle systems in anAMF were examined, and the heating proles can be seen belowin Fig. 1. The specic absorption ratio (SAR) was calculated:

SAR ¼ (P

iCimi)/m � (DT/Dt) (1)

where Ci is the heat capacity, mi is the individual mass of thecomponents heated (in this case water and iron oxide nano-particles),m is the mass of the component generating heat (ironoxide), and DT/Dt is the initial slope of the heating prole

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Fig. 1 DT heating profile uncoated iron oxide a nanoparticle wherestarting temperature was room temperature. The tangent line indi-cated the slope used in the SAR calculations.

Scheme 2 Diagram of potential Fenton/Haber–Weiss reactions initi-ated by iron oxide nanoparticles.

RSC Advances Paper

(the 25 and 35 second time points were used to calculate theslope). Calculations were based on specic heat capacities of0.65 and 4.18 J g�1 K�1 for iron oxide and water respectively. TheSAR value was determined to be 535.5� 25.8W g�1 at an AMF ofapproximately 60 kA m�1 in strength at 292 kHz frequency.

3.2 Methylene blue degradation

The rate of radical generation was determined by analyzing therate of methylene blue (Scheme 1) degradation. In a Fenton-likesystem, as illustrated in Scheme 2, iron ions react withhydrogen peroxide to generate highly reactive hydroxyl andsuperoxide radicals. These radicals attack bonds on methyleneblue fracturing the molecule into colorless intermediates.Possible splitting sites include the C–S+]C and the C–N]C ofthe central ring or hydroxylation of the aromatic side rings.32,33

It was determined that the degradation of methylene blue wascatalyst driven, as there were negligible effects of hydrogenperoxide alone or adsorption onto the nanoparticle surface onmeasured absorbance.

The kinetic data was initially t to a pseudo-rst-order andsecond-order models as explored previously in literature.34,35

Relating concentration as a function of relative absorbance, wehad the following rate models:

d(At/Ao)/dt ¼ �k1(At/Ao) (2)

d(At/Ao)/dt ¼ �k2(At/Ao)2 (3)

where k1 and k2 are the rst and second order rate constants,and At is the measured absorbance at a given time normalized

Scheme 1 Molecular structure of methylene blue.

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to the initial absorbance, Ao. Integrating eqn (2) and (3) resultsin the following:

ln(At/Ao) ¼ k1t (4)

1/(At/Ao) ¼ k2t + 1 (5)

The methylene blue degradation data was t using the aboveequations to determine the respective rate constants using alinear regression. The pseudo-rst-order model was rejectedbecause the y-intercept never approached 0 for any of theconcentrations or temperatures tested. The second-ordermodel, seen in Fig. 2, t the methylene blue degradation asobserved previously.35 The linear correlation coefficients (R2)were in the neighborhood of 0.95 again conrming the accuracyof the linear model.

By studying methylene blue degradation at various temper-atures, an Arrhenius type relationship was determined. Basedon the slopes observed in Fig. 2 the reaction was temperaturedependent where there was an increase in reaction withincreased temperature as expected by Fenton-like chemistry.Using the Arrhenius equation:

k ¼ A exp(�Ea/RT) (6)

where A is the pre-exponential factor, Ea is the activation energy,R is the ideal gas constant, and T is the reaction temperature.Converting eqn (6) into a linear relationship between ln(k) and1/T, the previously determined reaction rates were plotted intoFig. 3. The derived activation energy and pre-exponential factorare listed in Table 1 for the given concentration of iron oxidestudied.

Deriving an Arrhenius relationship was essential to provide acomparative basis for the AMF studies. The three iron oxideconcentrations studied were selected based on their ability toheat the surrounding media. When the AMF source is activated,the copper coil heats instantaneous and begins to radiate heatto the sample. At the AMF eld amplitude used, a solution ofmethylene blue would heat to 32–32.5 �C. At 37.5 mg ml�1 ironoxide concentration, the measured solution temperature neverexceeded this range with 15 minutes of heating. This impliesthat the amount of heat generated from the nanoparticle

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Fig. 2 Second-order kinetic plots of methylene blue degradation at37.5 mg ml�1 (a), 75 mg ml�1 (b), and 150 mg ml�1 (c) iron oxideconcentration.

Fig. 3 Arrhenius plots derived from second order kinetic model frommethylene blue degradation at 37.5 mg ml�1 (a), 75 mg ml�1 (b), and150 mg ml�1 (c) iron oxide concentration.

Table 1 Arrhenius constants of methylene blue degradation at variousconcentrations of iron oxide nanoparticles

Concentration �Ea (J mol�1) A (s�1)

37.5 mg ml�1 7.15 � 104 8.79 � 108

75 mg ml�1 9.91 � 104 1.05 � 1014

150 mg ml�1 8.48 � 104 7.27 � 1011

Paper RSC Advances

surface is insufficient to signicantly heat the surroundingvolume of water. At 75 mg ml�1 iron oxide, the measured solu-tion temperature was 33.5–34 �C, and at 150 mg ml�1, themeasured temperature was 36–36.5 �C resulting in a 1.5 and 4�C temperature rise respectively. At these concentrations theheat generated from the nanoparticle surface actively heatedthe solution temperature. From the measured steady statetemperature of each sample, a theoretical concentration wascalculated using the Arrhenius relationship. This value wascompared to the experimentally measured absorbance throughthe following enhancement factor (EF) equation:

EF ¼ (1 � (At/Ao)exp)/(1 � (At/Ao)theo) (7)

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where (At/Ao)exp is the experimental concentration and (At/Ao)theois the theoretical concentration of methylene blue. Theenhancement factor results are displayed in Fig. 4. The lowestconcentration, 37.5 mg ml�1, displayed very signicant ROSenhancement over the three time periods studied. At 5 minutes

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Fig. 4 Enhancement factor comparing extent of methylene bluedegradation based on experimental and theoretical values at differentconcentrations of iron oxide nanoparticles and length of AMFexposure.

RSC Advances Paper

of exposure, 75 mg ml�1 was signicant, but the enhancementdecreased to no measurable enhancement by 15 minutes. Thehighest concentration never displayed a signicantenhancement.

At the lower concentrations of nanoparticles, the localnanoscale heating effects would be more pronounced than atthe highest concentration. At these concentrations, energywould be dispersed to the local area driving the catalyticdegradation of methylene blue. This energy, however, is insuf-cient to heat the surrounding volume resulting in nomeasurable temperature rise. Thus, we would expect the kineticbehavior to be similar to the temperature elevated a few degreesresulting in the observed enhancement. At the highestconcentration the measured temperature corresponds closerthe local surface temperature and hence no enhancement.

When enhancement was observed, the degree of enhance-ment declined with length of AMF exposure. One possibleexplanation is the reversible agglomeration of the nanoparticlesinduced by the magnetic eld. Being paramagnetic, the nano-particles would be attracted to each other resulting in alignedchains or clusters.36,37 In this agglomerated state, the availablesurface area of the nanoparticles would be reduced affecting thecatalyst potential. This further conrms a recent observationmade by Sharma et al. where the degree of cellular oxidativestress was a function of iron oxide nanoparticle surface area asopposed to mass delivered.38 Nanoparticle concentration is oneof the hallmark factors of colloidal stability and could explainwhy no enhancement was observed at the highest concentra-tion. Elevated concentrations of magnetic nanoparticles displayobservable changes in colloidal stability when exposed to theAMF. While not signicant, the enhancement factor of the150 mg ml�1 system was below 1 at the 10 and 15 minuteexposure times. Future experiments investigating the role ofaggregation state on the magnetic eld heating enhancementfactor are warranted.

This study provides a direct example of utilizing the accel-erated surface reactivity with AMF exposure for the degradationof a model pollutant. When translating this material property toa biological system such as within the cellular environment,

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however, there is room for scholarly debate. For starters, theintracellular concentration ofH2O2 is typically around 0.5–0.7 mMbut can be as high as 1 mM during proliferation signaling.39

Cancer cells are known to have higher levels of H2O2 due toerratic signaling, so the intracellular level could conceivably beeven higher than 1 mM, but these levels are signicantly lowerthan the levels used in this study. With the decrease in relativeconcentrations of reactants, the reaction rates would predictablybe slower. Most cancer cells display an overexpression of Aktwhich causes them to be more susceptible to oxidative stressinduced apoptosis.40 Thus, even acute uctuations of ROS levelsfrom iron oxide catalyzed Fenton chemistry could have a detri-mental effect on cancer cells. Here in the best case, we havedemonstrated upwards of a two fold increase in ROS generationwith AMF exposure. Future research should investigate whetherthis ROS generation corresponds with an effective concentrationof iron oxide nanoparticles triggering a cytotoxic event.

4. Conclusions

This study reports the observed enhanced degradation ofmethylene blue by free radicals generated by iron oxide nano-particles heated in an alternating magnetic eld. The kineticbehavior of methylene blue degradation was modeled using asecond-order reaction, and an Arrhenius relationship wasextrapolated from the temperature dependence. When exposedto an alternating magnetic eld, the nanoparticles at lowerconcentrations and exposure time are capable of degradingmethylene blue at a greater extent than predicted by theArrhenius relationship. This observation has multiple futureapplications, such as improving intracellular hyperthermiaprocesses and reaction rates in advanced oxidation processes.

Acknowledgements

RW acknowledges the nancial support from a NCI-CNTC pre-doctoral traineeship and the project described was supportedby Grant Number R25CA153954 from the National CancerInstitute. The content is solely the responsibility of the authorsand does not necessarily represent the official views of theNational Cancer Institute or the National Institutes of Health.CO acknowledges the nancial support by the NSF REU GrantNumber EEC-1156667 from the National Science Foundation.

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