Cell damage produced by magnetic fluid hyperthermia on microglial BV2 cells

Abstract

We present evidence on the effects of exogenous heating by water bath (WB) and magnetic hyperthermia (MHT) on a glial micro-tumor phantom. To this, magnetic nanoparticles (MNPs) of 30-40 nm were designed to obtain particle sizes for maximum heating efficiency. The specific power absorption (SPA) values (f = 560 kHz, H = 23.9 kA/m) for as prepared colloids (533-605 W/g) dropped to 98-279 W/g in culture medium. The analysis of the intracellular MNPs distribution showed vesicle-trapped MNPs agglomerates spread along the cytoplasm, as well as large (~0.5-0.9 μm) clusters attached to the cell membrane. Immediately after WB and MHT (T = 46 °C for 30 min) the cell viability was ≈70% and, after 4.5 h, decreased to 20-25%, demonstrating that metabolic processes are involved in cell killing. The analysis of the cell structures after MHT revealed a significant damage of the cell membrane that is correlated to the location of MNPs clusters, while local cell damage were less noticeable after WB without MNPs. In spite of the similar thermal effects of WB and MHT on the cell viability, our results suggest that there is an additional mechanism of cell damage related to the presence of MNPs at the intracellular space.

Figure 1

TEM images (left) of PAA-MNPs (A) and LA-MNPs (B), their histograms (right, C,D) and hydrodynamic diameter (E,F).

Figure 2

SPA values (H = 23.9 kA/m, f = 560 kHz) of both PAA-MNPs and LA-MNPs, as prepared and after dispersing in DMEM cell culture medium.

Figure 3

MNPs per cell detected after 24 hours of incubation in BV2 cells as function of MNPs added.

Figure 4

TEM images of BV2 cells with MNPs: (A) Control cells (B) BV2 incubated with PAA-MNPs 10 and 50 µg/ml; (C) BV2 incubated with LA-MNPs at 10 µg/ml and magnified image of the clusters; (D) BV2 incubated with LA-MNPs at 75 µg/ml and magnified image of clusters. (E–G) STEM images of BV2 cells incubated with 50 μg/ml of PAA-MNPs; H-K) STEM images of BV2 cells incubated with 50 μg/ml of LA-MNPs. The last column shows the Energy-dispersive X-ray spectroscopy EDS-HAADF spectra of PAA-MNPs (upper) and LA-MNPs (lower) inside the cell.

Figure 5

SEM images of BV2 cells incubated at different concentrations of PAA-MNPs and LA-MNPs during 12 h. The short projections from the cell membrane are visible, as well as the major axon-like structures extending from the two opposite ends of the cell poles.

Figure 6

SEM-FIB (Dual–Beam) images of BV2 cells incubated with PAA-MNPs (50 μg/ml, ≈12 pg/cell) showing several clusters pinned at different points of the cell membrane (A,B and C). Cross-sectioning of one cell at one cluster location (D and E) showed clearly the insertion of the agglomerates through the cell membrane and penetration into the cytoplasm (C and D).

Figure 7

BV2 viability results (n = 3) after water bath and magnetic hyperthermia. Viability was measured 5 min (filled bars) and 4.5 h (dashed bars) after hyperthermia at 46 °C for 30 min. Magnetic hyperthermia experiments were performed at f = 560 kHz and H₀ = 23.9 kA/m.

Figure 8

TEM images of BV2 cells containing 100 µg/ml of PAA-MNPs after 30 min of MHT at 46 °C. (A) vesicle-containing MNPs, ER: endoplasmatic reticulum, NE: nuclear envelope, MC: marginal chromatine, (D) dictyosome and M: mitochondrie. (B) MNPs cluster wrapped in lysosomal membrane (LM) and close to a residual body (RB). (C) Disrupted cell membrane near a large PAA-MNPs cluster after MHT. (D) Typical image of a completely damaged cell due to AMF and PAA-MNPs. (E) Cell with lysosome containing PAA-MNPs. Apoptotic cells (F and I) and dead cells (G and H) were found to coexist after MHT with (seemingly) alive cells containing high variable amounts of PAA-MNPs incorporated.

Figure 9

TEM images of BV2 cells after 30 min of water bath hyperthermia at 46 °C. TEM images of BV2 cells containing 100 µg/ml of PAA-MNPs after 30 min of water bath hyperthermia at 46 °C.

Figure 10

Scanning electron microscopic (SEM) images of BV2 cells after 30 min of water bath hyperthermia at 46 °C. SEM-FIB (Dual-Beam) images of BV2 cells containing 100 μg/ml of PAA-MNPs after 30 min of water bath hyperthermia at 46 °C. Dual Beam images of BV2 + PAA − MNPs + AMF, final Tª = 46 °C; (100 μg/ml PAA-MNPs, 24 k/m, f = 560 kA/m kHz and 30 min).