Combining Bulk Temperature and Nanoheating Enables Advanced Magnetic Fluid Hyperthermia Efficacy on Pancreatic Tumor Cells

Abstract

Many efforts are made worldwide to establish magnetic fluid hyperthermia (MFH) as a treatment for organ-confined tumors. However, translation to clinical application hardly succeeds as it still lacks of understanding the mechanisms determining MFH cytotoxic effects. Here, we investigate the intracellular MFH efficacy with respect to different parameters and assess the intracellular cytotoxic effects in detail. For this, MiaPaCa-2 human pancreatic tumor cells and L929 murine fibroblasts were loaded with iron-oxide magnetic nanoparticles (MNP) and exposed to MFH for either 30 min or 90 min. The resulting cytotoxic effects were assessed via clonogenic assay. Our results demonstrate that cell damage depends not only on the obvious parameters bulk temperature and duration of treatment, but most importantly on cell type and thermal energy deposited per cell during MFH treatment. Tumor cell death of 95% was achieved by depositing an intracellular total thermal energy with about 50% margin to damage of healthy cells. This is attributed to combined intracellular nanoheating and extracellular bulk heating. Tumor cell damage of up to 86% was observed for MFH treatment without perceptible bulk temperature rise. Effective heating decreased by up to 65% after MNP were internalized inside cells.

Figure 1

Intracellular uptake of nanoparticles. (a) Exemplary TEM images of MiaPaCa-2 (left) and L929 cells (right) after incubation in with 225 µg(Fe) magnetoliposomes for 24 h. The uptake of nanoparticles (black dots) grouped into vesicles is clearly visible. (b) Magnetoliposomes (ML) internalized per cell after 24 h of incubation time as a function of initial incubation concentration of the ML in the cell culture medium. (c) Effect of initial incubation concentration of ML on cell survival after 24 h of incubation for MiaPaCa-2 cells. (d) Same as (c) for L929 cells. Control refers to untreated cells.

Figure 2

Effect of temperature on cell survival. Survival fraction and corresponding effective temperature of MiaPaCa-2 cells (a,b) and L929 cells (c,d) after magnetic fluid hyperthermia treatment for 30 min as well as 90 min and for different initial incubation concentration of iron in the cell culture medium (150 µg(Fe)/mL, 225 µg(Fe)/mL, 300 µg(Fe)/mL). All samples are compared to controls and ML-treated cells samples. Results marked with an asterisk (a,c) show statistically significant cell damage.

Figure 3

Comparing cytotoxicity of magnetic fluid hyperthermia and hotplate hyperthermia. (a) Effective temperatures for hotplate hyperthermia treatment of MiaPaCa-2 and L929 cells for either 30 min or 90 min. Dashed lines indicate body temperature and thermal damage threshold temperature. (b) Survival fraction for the MiaPaCa-2 and L929 treated at temperatures described in (a). Dashed lines indicate the 100% survival fraction line reached for the control sample.

Figure 4

Survival faction vs. cumulative equivalent minutes (CEM43). (a) MiaPaCa-2 cells and (b) L929 cells: Survival fraction substantially drops for MFH-treated cells at a CEM43 of approx. [1, 10] min. For hotplate hyperthermia treated cells the same substantial drop is observed at a CEM43 of approx. 50 min and above. Note that intracellular ML samples for the L929 cells are not shown here, as only samples showing significant damage caused by MFH were regarded (cf. Fig. 2d).

Figure 5

Specific loss power (SLP) of intracellular nanoparticles. Mean SLP values of ML internalized in MiaPaCa-2 (a) and L929 cell lines (b). The ordinate depicts the absolute amount of iron per sample, below which the relative amount of internalized ML is specified. Note that SLP values of intracellular ML for MiaPaCa-2 cells were below the detection limit and could thus not be quantified.

Figure 6

Total thermal energy deposited per cell. MiaPaCa-2 cells and L929 cells. Note that only samples showing significant damage by MFH treatment were considered (cf. Fig. 2b,d with p < 0.05). The inset displays the survival fraction on a logarithmic scale. The dotted line indicates the 100% survival fraction (control).