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

Authors Grazú V, Silber AM, Moros M, Asín L, Torres TE, Marquina C, Ibarra MR, Goya GF

Received 30 June 2012

Accepted for publication 25 July 2012

Published 8 October 2012 Volume 2012:7 Pages 5351—5360

DOI https://doi.org/10.2147/IJN.S35510

Checked for plagiarism Yes

Review by Single-blind

Peer reviewer comments 4

Supplementary video A (cells without magnetic nanoparticles not submitted to magnetic fields)

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V Grazú,1 AM Silber,2 M Moros,1 L Asín,1 TE Torres,1,3,5 C Marquina,3,4 MR Ibarra,1,3 GF Goya1,3

1
Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Zaragoza, Spain; 2Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil; 3Departamento de Física de la Materia Condensada, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; 4Instituto de Ciencia de Materiales de Aragón (ICMA), CSIC, Universidad de Zaragoza, Zaragoza, Spain; 5Laboratorio de Microscopías Avanzadas (LMA), Universidad de Zaragoza, Zaragoza, Spain

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

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