Luciferase-based protein denaturation assay for quantification of radiofrequency field-induced targeted hyperthermia: Developing an intracellular thermometer

Figures & data

Figure 1. Quantification of thermal dose in cells treated with RF without C225-AuNPs. (A) Loss of cell viability in SNU449 cells exposed to an RF field (13.56 MHz, 600 W) for varying durations with the corresponding bulk medium temperature (n = 3, symbols represent the mean ± SEM). (B) Loss of cell viability in cells exposed to water bath at varying temperatures for 30 min with corresponding calculated thermal dose. (n = 3, symbols represent mean ± SEM). (C) Loss of luciferase activity in SNU449 cells with increasing duration of RF exposure (n = 3, symbols represent the mean ± SEM). (D) Time- and temperature-dependent loss of luciferase activity after thermal exposure of SNU449 cells in a water bath (n = 3, symbols represent the mean ± SEM). (E) Thermal dose (CEM43) was calculated from data shown in Figure 1D and plotted against measured luciferase activity to generate a standard curve. (F) Using the standard curve (Figure 1E), intracellular thermal dose, CEM43 (Ti) was calculated for data shown in Figure 1C. Extracellular thermal dose (Te) due to bulk medium temperature was calculated from measured bulk temperatures and was found to be negligible (n = 3, symbols represent the mean ± 95%CI).

Figure 2. Quantification of thermal dose in cells treated with C225-AuNPs. (A) Preincubation of SNU449 cells with C225-AuNPs (100 µg/mL) for 4 h followed by RF exposure enhances thermal denaturation of luciferase. (B) Intracellular thermal dose (Ti) was quantified using the standard curve in Figure 1E. Extracellular thermal dose (Te) due to bulk medium temperature was calculated from measured bulk temperatures and was found to be negligible (n = 3, symbols represent the mean ± 95%CI). (All p values are from unpaired two-tailed t-test.)

Figure 3. Gold nanoparticle internalisation by SNU449 cells. Transmission electron microscopy images demonstrating uptake of C225-AuNPs (100 µg/mL) by SNU449 cells after 4 h of treatment.

Figure 4. Uptake of C225-AuNPs and their biological effects. (A) Intracellular amount of Au in each SNU449 cell as determined by ICP-MS increases with increasing duration of exposure to C225-AuNPs (100 µg/mL) (n = 3, bars represent the mean ± SEM). (B) Loss of luciferase activity in SNU449 cells incubated with or without C225-AuNPs (100 µg/mL) for varying duration followed by exposure to RF field for 3.6 min (n = 3, bars represent the mean ± SEM). (C) Relationship of duration of SNU449 cells incubation with C225-AuNPs (100 µg/mL) and intracellular thermal dose (same data as Figure 4B after intracellular thermal dose calculation using the standard curve in Figure 1E) (n = 3, symbols represent the mean ± 95%CI). (D) Heat shock proteins were analysed using western blot immediately after thermal exposure in a water bath (1) or radiofrequency field (2). (All p values are from unpaired two-tailed t-test.)