Fig 1.
Performance of our AMF system and results of examination of the heating efficiency of PSS-MNPs.
(a) Schematic device of high-frequency AMF-induced heating system. The system contains of three main parts—resonant RLC circuit machine, tank for cooling with circulated water and magnetic-field-induced sample tank. (b) Magnetic-field relative-frequency curve of our AMF device. (c) Time-dependent temperature (TDT) curve of PSS-MNPs (30 mg mL-1) under varied intensity of applied AMF (27, 71.5 and 143.5 G) for 20 min from initial temperature 23 oC. SAR and ILP values are labeled in the figure. (d) SAR vs PSS-MNP concentration (3.125, 6.25, 12.5, 25 and 50 mg mL-1) for varied intensity of applied AMF (27, 71.5 and 143.5 G) for 30 min. (e) SAR values vs H × f (product of generated AMF and operating frequency, generated AMF intensity s-1) with PSS-MNP concentrations 3.125, 6.25, 12.5, 25 and 50 mg mL−1 under hyperthermia operation for 30 min. (f) TDT curve of culture medium with added PSS-MNPs of concentration 2.5, 5, 10, 20, 40, 80 and 160 mg mL−1 in the case of a fixed AMF (43.7 kHz, 143.5 G) for 30 min.
Fig 2.
In vitro examination of a SK-Hep1 cell line to evaluate PSS-MNPs.
(a) SK-Hep1 cells (5×105 cell mL-1) were incubated with PSS-MNPs at 0, 0.05, 0.1, 0.5, 1, 5 and 10 mg mL-1, respectively. After incubation for 24 h, cell viability was tested with MTT analysis. (b) Examination of internalization efficiency of PSS-MNPs in a SK-Hep1 cell line through application of Prussian-blue staining. (c, d) Extracellular hyperthermia effects on a SK-Hep1 cell line induced by PSS-MNPs (concentrations 5 and 10 mg mL-1) after high-frequency AMF treatments (43.7 kHz, 143.5 G) for operating durations 0.5, 1, 2 and 3 h. Control groups in (c) and (d) mean the cells were untreated with PSS-MNPs. (e, f) Extracellular hyperthermia effects on a SK-Hep1 cell line induced by PSS-MNPs (concentrations 5 and 10 mg mL-1) after high-frequency AMF treatment (43.7 kHz, 143.5 G) for 0–3 cycles. The definition of cycle in hyperthermia-treated cell lines was set as the hyperthermia operation period 30 or 60 min with 30 min machine off; the definition of a cycle in a control group means that a MTT assay was implemented after the cells were co-cultured with PSS-MNPs for 1.5 h. Control groups in (e) and (f) mean the cells were treated with PSS-MNPs and cultured for varied duration of incubation, which was the duration of incubation with the hyperthermia examination and the product of duration of treatment operated.
Fig 3.
Cell morphologies and appearance of DAPI/PI-stained cells.
(a) Observations of SK-Hep1 cell line after incubation for 24 h with PSS-MNPs under optical and fluorescence microscopes (merged) and (b) photographs of SK-Hep1 cells treated with PSS-MNPs before and after 1 h of extracellular hyperthermia treatment. The scale bar is 30 μm.
Fig 4.
Images and distribution of cell size of DAPI-stained SK-Hep1 cells treated or untreated with MFH.
DAPI-stained MNP-treated SK-Hep1 cells were (a) untreated or (b) treated with MFH for 1 h; size comparison of SK-Hep1 cells before and after MFH operation (1 h) after co-culture 24 h with PSS-MNPs (10 mg mL-1). The SK-Hep1 cells were incubated with PSS-MNPs (i, iv) 0, (ii, v) 5 and (iii, vi) 10 mg mL-1 for 24 h, followed by PBS washing three times to remove the waste and excess PSS-MNPs from the medium. The cell solutions were then (a) observed under optical and fluorescence microscopes or (b) treated with hyperthermia for 1 h before microscope observation. Scale bars in figures are 30 μm. (c) Differences in size distribution of SK-Hep1 cells co-cultured with PSS-MNPs (10 mg mL-1) for 24 h before and after MFH (1 h). The inset figure presents an enlarged image of MFH-treated SK-Hep1 cells with a definition of measured cell size (yellow dashed line), which labels the maximum cell size of the calibrated individual single cells.
Fig 5.
Cell viability examination for intracellular MFH effects on SK-Hep1 cells with varied concentration and volume.
(a) Hyperthermia effect on HCC cells induced by intracellular MNPs (concentrations 0.05 and 0.5 mg mL-1) after high-frequency AMF hyperthermia for 1 h. The control groups indicate that the samples were treated with an AC magnetic field without added MNPs. (b) Intracellular hyperthermia magnetotherapy effects in a culture medium of varied volume. The cells were treated with PSS-MNPs (0.5 mg mL-1) for 24 h; untreated cell lines were incubated as control groups. Both MNP-treated and untreated cells were washed with PBS and filled with fresh culture medium (0.2, 0.4, 0.6, 0.8 and 1 mL) before AMF induction. Cells were treated for 2 h with AMF, at magnetic field 157 G and frequency 43.7 kHz. The degree of significance is given as * p<0.05; ** p<0.01 and *** p<0.001.
Fig 6.
Intermittent hyperthermia effect on PSS-MNP intracellular hyperthermia therapies with varied operating duration.
The duration was varied from (a) a few operations of MFH in one day (0–3 times) to (b) numerous MFH treatments operated within two days (3 times + incubation 24 h + 0–2 times next day). SK-HEP1 cells were treated with PSS-MNPs (0.5 mg mL-1) for cell endocytosis for 24 h, followed by washing with PBS to remove excess MNPs before AMF induction. The fresh culture medium volume was adjusted to 0.2 mL. (a) MNP-treated cells were treated for 2 h with an AMF of magnetic field 157 G and frequency 43.7 kHz, and then rested for 30 min before the next AMF operating cycle. (b) After AMF treatment three times of a first administration, the cells were treated with PSS-MNPs (0.5 mg mL-1) as a second administration for 24 h; intermittent hyperthermia steps were further treated and implemented for a few cycles. The degree of significance is given as * p<0.05; ** p<0.01 and *** p<0.001.