The role of ROS generation from magnetic nanoparticles in an alternating magnetic field on cytotoxicity

Abstract

Monosaccharide coated iron oxide nanoparticles were developed to selectively target colon cancer cell lines for magnetically mediated energy delivery therapy. The nanoparticles were prepared using a coupling reaction to attach the glucose functional group to the iron oxide core, and functionality was confirmed with physicochemical characterization techniques. The targeted nanoparticles were internalized into CT26 cells at a greater extent than non-targeted nanoparticles, and the nanoparticles were shown to be localized within lysosomes. Cells with internalized nanoparticles were exposed to an AMF to determine the potential to delivery therapy. Cellular ROS generation and apoptotic cell death was enhanced with field exposure. The nanoparticle coatings inhibit the Fenton-like surface generation of ROS suggesting a thermal or mechanical effect is more likely the source of the intracellular effect, unless the nanoparticle coating is unstable in the cellular environment.

Statement of significance: This is the first study to assess glucose coated MNPs for the delivery of MagMED therapy. With exposure of an AMF, the glucose-coated nanoparticles displayed a significant increase in cellular ROS and apoptotic cell death with no measurable increase in media temperature. To determine the mechanism of toxicity, we investigated the surface generation of ROS through Fenton-like chemistry. The coated systems displayed negligible ROS generation compared to uncoated nanoparticles. These observations suggest the cellular ROS measured is attributed to a thermal or mechanical effect of the internalized nanoparticles. In summary, this manuscript reports on some new insights as to the mechanism of MagMED therapies, which are of high interest to the biomaterials and cancer nanomedicine fields.

Keywords: Glyconanoparticles; Iron oxide; Magnetically mediated energy delivery; Reactive oxygen species.

Figure 1

FTIR spectra of citric acid coated iron oxide and glucose coated samples. The vertical line at 1088 cm⁻¹ indicates the location of a C-N vibration and at 1040 cm⁻¹ indicated the location of C-O stretch peak attributed to the D-glucosamine. The vertical lines 1560 cm⁻¹, 1360 cm⁻¹, and 1250 cm⁻¹ indicate the location of the C=O stretch, O-H bend, and C-O stretch bonds, respectively, typically attributed to citric acid coated particles.

Figure 2

Mass loss profile of citric acid and glucose coated iron oxide.

Figure 3

Iron content in CT26 cells when exposed to 200 μg/ml of nanoparticles over 0.5, 1, and 2 hours of incubation. Control group consists of cells never exposed to nanoparticles but underwent similar culturing conditions.

Figure 4

Representative localization images of control (a) and 50 μg/ml fluorescently tagged glucose coated nanoparticles (b) incubated with CT26 cells.

Figure 5

Measured ROS enhancement with field exposure determined by dividing the relative fluorescent means from the samples with field exposure by no field exposure. Significant differences between groups are indicated as *p < 0.05, **p < 0.01.

Figure 6

Ratio of Caspase 3/7 fluorescence of cells with and without field exposure when exposed to various nanoparticle systems. Significant differences between groups are indicated as *p < 0.05.

Figure 7

Fenton-like generation of ROS by nanoparticle systems measured by methylene blue dye degradation. 75 μg/ml nanoparticles were exposed to the AMF for 5 and 15 minutes in the presence of 0.75% H₂O₂. (a) Targeted magnetic nanoparticles bind to cell surface and are internalized into lysosomes. (b) AMF activation induces lysosomal permeability triggering cellular death. (c) Nanoparticle coating inhibits Fenton-like chemistry indicating a thermal or mechanical effect causes the lysosomal disruption.

Figure 7

Fenton-like generation of ROS by nanoparticle systems measured by methylene blue dye degradation. 75 μg/ml nanoparticles were exposed to the AMF for 5 and 15 minutes in the presence of 0.75% H₂O₂. (a) Targeted magnetic nanoparticles bind to cell surface and are internalized into lysosomes. (b) AMF activation induces lysosomal permeability triggering cellular death. (c) Nanoparticle coating inhibits Fenton-like chemistry indicating a thermal or mechanical effect causes the lysosomal disruption.

Scheme 1

Reaction schematic of monosaccharide coating of iron oxide nanoparticles displaying the attachment of the citric acid stabilizer and subsequent addition of D-glucosamine via amine-carboxyl coupling reaction