Application of magnetically induced hyperthermia in the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections

Abstract

Background

Magnetic hyperthermia is currently a clinical therapy approved in the European Union for treatment of tumor cells, and uses magnetic nanoparticles (MNPs) under time-varying magnetic fields (TVMFs). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, given that the therapeutic drugs available have severe side effects and that there are drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe₃O₄ MNPs in order to provoke cell death remotely using TVMFs.

Methods

Iron oxide MNPs with average diameters of approximately 30 nm were synthesized by precipitation of FeSO₄ in basic medium. The MNPs were added to C. fasciculata choanomastigotes in the exponential phase and incubated overnight, removing excess MNPs using a DEAE-cellulose resin column. The amount of MNPs uploaded per cell was determined by magnetic measurement. The cells bearing MNPs were submitted to TVMFs using a homemade AC field applicator (f = 249 kHz, H = 13 kA/m), and the temperature variation during the experiments was measured. Scanning electron microscopy was used to assess morphological changes after the TVMF experiments. Cell viability was analyzed using an MTT colorimetric assay and flow cytometry.

Results

MNPs were incorporated into the cells, with no noticeable cytotoxicity. When a TVMF was applied to cells bearing MNPs, massive cell death was induced via a nonapoptotic mechanism. No effects were observed by applying TVMF to control cells not loaded with MNPs. No macroscopic rise in temperature was observed in the extracellular medium during the experiments.

Conclusion

As a proof of principle, these data indicate that intracellular hyperthermia is a suitable technology to induce death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies combating parasitic infection.

Keywords:

• magnetic hyperthermia
• magnetic nanoparticles
• trypanosomatids
• Crithidia fasciculata

Acknowledgments

This work was supported by the Spanish Ministry Ministerio de Ciencia e Innovación (project MAT2010-19326 and Consolider NANOBIOMED CS-27 2006) and IBERCAJA. Partial support from Brazilian grants 08/57596-4 and 11/50631-1 from FAPESP and INBEQMeDI, respectively, is also acknowledged. We gratefully recognize Doctors M Vergés, MP Morales, and AG Roca for their kind donation of MNPs. We also thank J Godino from the Instituto Aragonés de Ciencias de la Salud, Zaragoza, for help with flow cytometer measurements, and I Echaniz for technical support, and L Casado for help with scanning electron microscopy imaging.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

The 2 × 2 design for evaluating the effect of magnetic nanoparticles and time-varying magnetic fields on Crithidia fasciculata

• File “a.mov”: Cells without magnetic nanoparticles not submitted to magnetic fields

• File “b.mov”: Cells with magnetic nanoparticles without magnetic field application

• File “c.mov”: Application of magnetic fields on unloaded cells

• File “d.mov”: Application of magnetic fields on magnetic nanoparticle-loaded cells

Figure S1 Magnetic response at T = 10 K from (A) unloaded cells, (B) response from Crithidia fasciculata cocultured with MNPs (sample incubated for 15 minutes), (C) difference between loaded and unloaded cells (B − A), and (D) pure magnetic colloid. Note that for the pure colloid (D), the curve was divided by 1.35 × 10⁴ to fit the same scale as the magnetic signal from loaded cells (C).

Notes: To calculate the amount of magnetic material m_mag incorporated by the cells, the M_S values from the pure colloids and from the magnetic nanoparticle-loaded cells were calculated as m_mag [g/cell]=M/MS× Number of cells. The number of magnetic nanoparticles per single cell was estimated from the known average particle diameter.