The effect of cell cluster size on intracellular nanoparticle-mediated hyperthermia: is it possible to treat microscopic tumors?

Abstract

Aim: To compare the measured surface temperature of variable size ensembles of cells heated by intracellular magnetic fluid hyperthermia with heat diffusion model predictions.

Materials & methods: Starch-coated Bionized NanoFerrite (Micromod Partikeltechnologie GmbH, Rostock, Germany) iron oxide magnetic nanoparticles were loaded into cultured DU145 prostate cancer cells. Cell pellets of variable size were treated with alternating magnetic fields. The surface temperature of the pellets was measured in situ and the associated cytotoxicity was determined by clonogenic survival assay.

Results & conclusion: For a given intracellular nanoparticle concentration, a critical minimum number of cells was required for cytotoxic hyperthermia. Above this threshold, cytotoxicity increased with increasing cell number. The measured surface temperatures were consistent with those predicted by a heat diffusion model that ignores intercellular thermal barriers. These results suggest a minimum tumor volume threshold of approximately 1 mm(3), below which nanoparticle-mediated heating is unlikely to be effective as the sole cytotoxic agent.

Figure 1. Experimental design

Schematic showing the experimental design: from left to right, DU145 cells are loaded in culture flasks with starch-coated BNF nanoparticles and PDL. Cell pellets are formed by centrifugation. The pellets are treated by AMF. AMF-treated and untreated cells are plated and fractional survival determined by clonogenic assay. AMF: Alternating magnetic field; BNF: Bionized NanoFerrite; PDL: Poly-D-Lysine.

Figure 2. Particle characteristics

(A) Particle size. Photon correlation spectrograph (PCS) showing mean hydrodynamic diameter of starch-coated BNF particles in phosphate-buffered saline (PBS) and PBS with PDL. (B) Particle transmission electron microscopy. Magnetic core of a particle comprising multiple crystals. (C) Particle ζ-potential. The ζ-potential of starch-coated BNF particles either in PBS or PBS with PDL is shown as a function of pH. (D) Particle SLP. Vertical dashed line indicates field amplitude used in study. BNF: Bionized NanoFerrite; PDL: Poly-D-lysine; SLP: Specific loss power.

Figure 3. Microscopy images of DU145 cells containing nanoparticles

(A–C) Light micrographs (color, 40×). Cell samples were treated under conditions (listed in Table 1) corresponding to (A) 70 pg Fe/cell, (B) 105 pg Fe/ cell and (C) 200 pg Fe/cell. They were fixed and stained with Prussian Blue to highlight iron deposits. (D) Transmission electron micrographs under similar conditions as (A). Magnifications are: 4000×, inset (i) 12,000×, and inset (ii) 40,000×. Note that the intracellular nanoparticle distribution is inhomogeneous, with cells appearing to phagocytize particle clusters (i) that are further concentrated within intracellular compartments or endosomes (ii).

Figure 4. Dynamic temperature profile of cell pellet

Measured surface temperature profile during alternating magnetic field treatment for a 500,000 cell cluster with an iron content of 199 pg/cell (cells were allowed to equilibrate to 37°C in sample chamber for 10 min before the power was turned on; the arrow indicates when the power was turned off).

Figure 5. Thermometry and cell survival results

(A) Comparison of the measured maximum change of cell pellet surface temperature (markers, M = Measured) versus the square of the diameter, D², with calculated maximum cell pellet surface temperature using Equation 4 in the text (solid lines, C = Calculated) for cell pellets with different intracellular iron concentrations. The dashed lines indicate linear least square fits of measured data with R² values of 0.99, 0.96 and 0.99 corresponding to 70, 105 and 199 pg of Fe/cell, respectively. (B) Fractional survival of cells from alternating magnetic field-treated pellets as a function of their measured pellet surface temperature. (C) Surviving fraction of DU145 containing Bionized NanoFerrite nanoparticles versus calculated cell-pellet core temperature. (D) Surviving fraction of DU145 containing Bionized NanoFerrite nanoparticles versus cell number separated by level of intracellular iron concentration. Error bars refer to standard error of the mean of measured values.