1. Analyzing ROS Generation from Magnetic Nanoparticles in an
Alternating Magnetic Field and its Role in Intracellular Hyperthermia
Catherine E. Oliver1, Robert J. Wydra2, David B. Cochran2, Kimberly W. Anderson2, Thomas D. Dziubla2, J. Zach Hilt2
1Department of Biomedical Engineering, University of Connecticut
2Department of Chemical and Materials Engineering, University of Kentucky
Research completed as part of the University of Kentucky REU program of Bioactive Interfaces and Devices
Kinetic Studies:
o The Arrhenius equation derived from the plot in Figure 5 can be used to calculate the theoretical
absorbance values at elevated temperatures to compare to the results obtained when using the
AMF.
Arrhenius Equation: 𝑘 = 𝐴𝑒
−𝐸 𝑎
𝑅𝑇 𝐴 = 4.46 × 10−2
𝑠−1
𝐸 𝑎 = 1.21 × 104
𝐽
Figure 5. Arrhenius plot of ln(k) (s-1) versus 1/T (K).
Figure 6. Plot of de-colorization comparing theoretical and experimental values of AMF exposure at various temperatures.
Conclusions:
o AMF heating does not fit Arrhenius trendline
o Experimental absorbance values are consistently lower than theoretical values indicating
improved kinetics due to AMF exposure
Viability-ROS in vitro assays:
o HT29 and CT26 colon cancer cells were seeded in 96 well plates
o Plate is doped with serial dilution of 800 to 50μg/ml Fe3O4 nanoparticles
o Calcein-AM and DCF-DA were used to measure viability and ROS production, respectively
o Fluorescent intensity was detected after 24 hours using spectrophotometer
Figure 6. Plot of percent fluorescent intensity versus magnetic nanoparticle (uncoated) concentration comparing cell viability and
ROS generation for CT26 colon cancer cells. Error reported as standard error.
Figure 7. Plot of percent fluorescent intensity versus magnetic nanoparticle (uncoated) concentration comparing cell viability and
ROS generation for HT29 colon cancer cells. Error reported as standard error.
Conclusions:
o As nanoparticle concentration increases, ROS generation is increased
o As nanoparticle concentration increases, cell viability is decreased
o Large error suggests variability among results
BACKGROUND
Hypothesis:
We hypothesize that the application of an alternating magnetic field (AMF) to magnetic
nanoparticles will increase production of reactive oxygen species (ROS) and that these ROS are a
key contributor to the toxicity demonstrated by intracellular hyperthermia.
Objectives:
o Demonstrate increase in ROS generation kinetics in the presence of an AMF
• Methylene blue dye-degradation assay
o Establish correlation between intracellular hyperthermia and ROS generation
• In vitro assays
Methylene blue dye degradation assay:
o With addition of hydrogen peroxide, iron oxide nanoparticles produce free (hydroxyl) radicals
• Fenton and Haber-Weiss reactions
o As radicals form, they oxidize methylene blue transforming the dye from blue to clear
o Color change monitored with UV-vis spectroscopy, specifically studying the peak at 665 nm
o At/Ao is the absorbance at time t divided by the absorbance of the sample without H2O2 at t = 0
AMF Studies:
Figure 1. Plot of de-colorization at room temperature, 37°C, and AMF exposure of 15 minutes (n=3) and 25 minutes (n=4) plus a 5
minute preheat at 37°C. Iron oxide nanoparticles concentration was 100μg/ml. Error reported as propagation of standard deviation.
Figure 2. Plot of average temperature versus time for AMF exposure of 15 and 25 minutes.
Figure 3. Plot of de-colorization at room temperature, 37°C, and AMF exposure of 15 minutes (n=5) and 25 minutes (n=3) plus a 5
minute preheat at 37°C. Iron oxide nanoparticles concentration was 75μg/ml. Error reported as propagation of standard deviation.
Figure 4. Plot of average temperature versus time for AMF exposure of 15 and 25 minutes.
Conclusions:
o Samples exposed to AMF experience increased ROS production
o Small error implies consistency with absorbance measurements
o Temperature heated to 37-38°C
Due to their unique physical properties, magnetic nanoparticles are being studied for a great
range of biomedical applications such as diagnostic imaging, drug delivery, and thermal therapy of
cancer [1-4]. Today, iron oxide is one of the most widely studied magnetic nanoparticle systems.
Furthermore, the ability of magnetic nanoparticles to be heated remotely by an alternating
magnetic field (AMF) is of particular interest because it can be utilized to overcome the barriers of
traditional hyperthermia and magnetic fluid hyperthermia methods [5-6]. Through Brownian
relaxation and Neel relaxation, magnetic nanoparticles absorb energy from the AMF and convert it
to heat [7]. Thus, via passive targeting, nanoparticles can collect at the tumor site and by the
application of the AMF provide localized heating throughout the tumor.
In magnetic fluid hyperthermia, treatment effectiveness is limited to solid tumors via direct
injection due to the high concentration of magnetic nanoparticles necessary to induce localized
heating of tissue. Recent work by Creixell et al. has demonstrated that internalized targeted
nanoparticles can induce cellular death when exposed to an AMF without a measurable
temperature rise, therefore stimulating great interest to develop targeted nanoparticles for the
treatment of metastatic cancer via intracellular hyperthermia [8]. Moreover, it has been shown
that when the solution temperature does not rise, the magnetic nanoparticle surface still
undergoes a significant increase in temperature when exposed to an AMF [9]. Currently, it is
unclear as to if this explains the toxicity of intracellular hyperthermia or if other phenomena are
contributing.
At the molecular level, it is thought that a significant contributing factor to the iron-oxide
toxicity is the generation of 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—beyond this temperature the hydrogen peroxide degrades into oxygen and
water limiting the reaction. [10] This research, in turn, focuses on the effects of an AMF on the
surface mediated production of ROS based on the hypothesis that these effects are a key
contributor to the toxicity demonstrated as intracellular hyperthermia.
[1] R.A. Frimpong, J.Z. Hilt, Nanomedicine, 5 (2010) 1401-1414.
[2] C.C. Berry, J. Phys. D-Appl. Phys., 42 (2009) 9.
[3] A. Ito, M. Shinkai, H. Honda, T. Kobayashi, J. Biosci. Bioeng., 100 (2005) 1-11.
[4] C. Sun, J.S.H. Lee, M.Q. Zhang, Adv. Drug Deliv. Rev., 60 (2008) 1252-1265.
[5] P. Moroz, S.K. Jones, B.N. Gray, Int. J. Hyperthermia, 18 (2002) 267-284.
[6] H.L. Rodriguez-Luccioni, M. Latorre-Esteves, J. Mendez-Vega, O. Soto, A.R. Rodriguez, C. Rinaldi, M. Torres-Lugo, Int. J.
Nanomed., 6 (2011) 373-380.
[7] R.E. Rosensweig, Journal of Magnetism and Magnetic Materials, 252 (2002) 370-374.
[8] M. Creixell, A.C. Bohorquez, M. Torres-Lugo, C. Rinaldi, ACS Nano, 5 (2011) 7124-7129.
[9] L. Polo-Corrales, C. Rinaldi, J. Appl. Phys. 111 (2012)
[10] M. A. Voinov, J.O. Sosa Pagán, E. Morrison, T. I. Smirnova, A. I. Smirnov, JACS Articles, 133 (2011) 35-37.
H2O2
·OH + OH-
AMF
·OH + OH-
H2O2
OH·, OOH·
H2O2
FeX+
HYPOTHESIS AND OBJECTIVES
NANOPARTICLE SYNTHESIS
Uncoated iron-oxide nanoparticles:
o 2:1 molar ratio of Fe2+ and Fe3+ is combined in a 3-neck flask
o At 85°C, 1.5 M solution of ammonium hydroxide is added to facilitate co-precipitation reaction
o After 1 hour, solution is removed from heat, cooled, and decanted once via magnetic decantation
o Nanoparticle suspension is transferred to a dialysis bag where it remains overnight
Fe3+ + H2O Fe(OH)X
3-X
Fe(OH)y
2-yFe2+ + H2O
Deprotonation
Oxidation
Dehydration
pH~9.0, 60°C
Magnetite
Fe3O4
RESULTS AND DISCUSSION
(colorless)
NN
N
S
H
Methylene Blue
(blue)
+
NN
N
S
reduction
oxidation
ACKNOWLEDGEMENTS
REFERENCES
o The financial support provided by the National Science Foundation REU Program #EEC-0851716
Metastatic Cell
Alternating
Magnetic Field Magnetic
Nanoparticles
Deprotonation
Haber Weiss Reactions
Fenton Reaction