Cell bystander effect induced by radiofrequency electromagnetic fields and magnetic nanoparticles

Induced effects by direct exposure to ionizing radiation (IR) are a central issue in many fields like radiation protection, clinic diagnosis and oncological therapies. Direct irradiation at certain doses induce cell death, but similar effects can also occur in cells no directly exposed to IR, a mechanism known as bystander effect. Non-IR (radiofrequency waves) can induce the death of cells loaded with MNPs in a focused oncological therapy known as magnetic hyperthermia. Indirect mechanisms are also able to induce the death of unloaded MNPs cells. Using in vitro cell models, we found that colocalization of the MNPs at the lysosomes and the non-increase of the temperature induces bystander effect under non-IR. Our results provide a landscape in which bystander effects are a more general mechanism, up to now only observed and clinically used in the field of radiotherapy.Comment: 16 pages, 4 figures, submitted to International Journal of Radiation Biolog

Protein adsorption onto Fe3O4 nanoparticles with opposite surface charge and its impact on cell uptake

Nanoparticles (NPs) engineered for biomedical applications are meant to be in contact with protein-rich physiological fluids. These proteins are usually adsorbed onto the NP surface, forming a swaddling layer called protein corona that influences cell internalization. We present a study on protein adsorption onto different magnetic NPs (MNPs) when immersed in cell culture medium, and how these changes affect the cellular uptake. Two colloids with magnetite cores of 25 nm, same hydrodynamic size and opposite surface charge were in situ coated with (a) positive polyethyleneimine (PEI-MNPs) and (b) negative poly(acrylic acid) (PAA-MNPs). After few minutes of incubation in cell culture medium the wrapping of the MNPs by protein adsorption resulted in a 5-fold size increase. After 24 h of incubation large MNP-protein aggregates with hydrodynamic sizes 1500 to 3000 nm (PAA-MNPs and PEI-MNPs respectively) were observed. Each cluster contained an estimated number of magnetic cores between 450 and 1000, indicating the formation of large aggregates with a "plum pudding" structure of MNPs embedded into a protein network of negative surface charge irrespective of the MNP_core charge. We demonstrated that PEI-MNPs are incorporated in much larger amounts than the PAA-MNPs units. Quantitative analysis showed that SH-SY5Y cells can incorporate 100 per cent of the added PEI-MNPs up to about 100 pg per cell, whereas for PAA-MNPs the uptake was less than 50 percent. The final cellular distribution showed also notable differences regarding partial attachment to the cell membrane. These results highlight the need to characterize the final properties of MNPs after protein adsorption in biological media, and demonstrate the impact of these properties on the internalization mechanisms in neural cells.Comment: 32 pages, 10 figure

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

Magnetic hyperthermia is currently an EU-approved clinical therapy against tumor cells that uses magnetic nanoparticles under a time varying magnetic field (TVMF). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, since therapeutic drugs available display severe side effects and 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 Fe_{3}O_{4} magnetic nanoparticles (MNPs) in order to remotely provoke cell death using TVMFs. The MNPs with average sizes of d approx. 30 nm were synthesized using a precipitation of FeSO_{4}4 in basic medium. The MNPs were added to Crithidia fasciculata choanomastigotes in exponential phase and incubated overnight. The amount of uploaded MNPs per cell was determined by magnetic measurements. Cell viability using the MTT colorimetric assay and flow cytometry showed that the MNPs were incorporated by the cells with no noticeable cell-toxicity effects. When a TVMF (f = 249 kHz, H = 13 kA/m) was applied to MNP-bearing cells, massive cell death was induced via a non-apoptotic mechanism. No effects were observed by applying a TVMF on control (without loaded MNPs) cells. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. Scanning Electron Microscopy showed morphological changes after TVMF experiments. These data indicate (as a proof of principle) that intracellular hyperthermia is a suitable technology to induce the specific death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies that combat parasitic infections.Comment: 9 pages, four supplementary video file

Field Dependence of Blocking Temperature in Magnetite Nanoparticles

Spherical magnetite nanoparticles having average particle size = 5 nm have been synthesized by coprecipitation of Fe(II) and Fe(III) salts in KOH with Polyvinylalcohol (PVA). The resulting dry powder displayed superparamgnetic (SPM) behaviour at room temperature, with a transition to a blocked state at TB ~ 45 K for applied field Happ = 500 Oe. The effect of dipolar interactions was investigated by measuring the dependence of TB on the applied field Hap and driven ac field in susceptibility data. A thermally activated model has been used to fit the dynamic data to obtain the single-particle energy barriers Ea = KeffV, allowing us to estimate the contributions of dipolar interactions to the single-particle effective magnetic anisotropy Keff. We have measured the dependence of TB with Hap in order to draw the transition contours of a H-T diagram. Two different regimes are found for the (TB-T0) ~H{\lambda} dependence at low and high fields, that can be understood within a pure SPM relaxation-time (N\'eel-Brown) landscape. The TB(H) data shows a crossover from {\lambda} = 2/3 to {\lambda} ~2 for applied magnetic fields of \approx 550 Oe.Comment: 6 pages, 4 figure

Cell death induced by the application of alternating magnetic fields to nanoparticle-loaded dendritic cells

In this work, the capability of primary, monocyte-derived dendritic cells (DCs) to uptake iron oxide magnetic nanoparticles (MNPs) is assessed and a strategy to induce selective cell death in these MNP-loaded DCs using external alternating magnetic fields (AMFs) is reported. No significant decrease in the cell viability of MNP-loaded DCs, compared to the control samples, was observed after five days of culture. The amount of MNPs incorporated into the cytoplasm was measured by magnetometry, which confirmed that 1 to 5 pg of the particles were uploaded per cell. The intracellular distribution of these MNPs, assessed by transmission electron microscopy, was found to be primarily inside the endosomic structures. These cells were then subjected to an AMF for 30 min, and the viability of the blank DCs (i.e., without MNPs), which were used as control samples, remained essentially unaffected. However, a remarkable decrease of viability from approximately 90% to 2-5% of DCs previously loaded with MNPs was observed after the same 30 min exposure to an AMF. The same results were obtained using MNPs having either positive (NH2+) or negative (COOH-) surface functional groups. In spite of the massive cell death induced by application of AMF to MNP-loaded DCs, the amount of incorporated magnetic particles did not raise the temperature of the cell culture. Clear morphological changes at the cell structure after magnetic field application were observed using scanning electron microscopy. Therefore, local damage produced by the MNPs could be the main mechanism for the selective cell death of MNP-loaded DCs under an AMF. Based on the ability of these cells to evade the reticuloendothelial system, these complexes combined with an AMF should be considered as a potentially powerful tool for tumour therapy.Comment: In Press. 33 pages, 11 figure

Validity of the N\'{e}el-Arrhenius model for highly anisotropic Co_xFe_{3-x}O_4 nanoparticles

We report a systematic study on the structural and magnetic properties of Co_{x}Fe_{3-x}O_{4} magnetic nanoparticles with sizes between $5$ to $25$ nm, prepared by thermal decomposition of Fe(acac)_{3} and Co(acac)_{2}. The large magneto-crystalline anisotropy of the synthesized particles resulted in high blocking temperatures ($42$ K \leqq $T_B$ $\leqq 345$ K for $5 \leqq$ d $\leqq 13$ nm ) and large coercive fields ($H_C \approxeq 1600$ kA/m for $T = 5$ K). The smallest particles ($=5$ nm) revealed the existence of a magnetically hard, spin-disordered surface. The thermal dependence of static and dynamic magnetic properties of the whole series of samples could be explained within the N\'{e}el-Arrhenius relaxation framework without the need of ad-hoc corrections, by including the thermal dependence of the magnetocrystalline anisotropy constant $K_1(T)$ through the empirical Br\"{u}khatov-Kirensky relation. This approach provided $K_1(0)$ values very similar to the bulk material from either static or dynamic magnetic measurements, as well as realistic values for the response times ($\tau_0 \simeq 10^{-10}$ s). Deviations from the bulk anisotropy values found for the smallest particles could be qualitatively explained based on Zener\'{}s relation between $K_1(T)$ and M(T)