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Nano-Toxicity

   Although information about toxicity of SPION continues to increase, a significant knowledge gap still exists on a complete toxicological profile of these promising magnetic materials proposed for future use in many aspect of biomedical engineering. Without the data, risk assessment or regulation for safety of the materials shall suffer immeasurably. To fill the information gap, Mahmoudi Lab is centered on three primary goals including:

  • Cytotoxicity assessments of SPION
  • Introducing the new protocols in order to obtain reliable toxicity results
  • Effect of SPION on the cell-life cycle
  • Defining the main reason of toxicity

 

Cytotoxicity assessments of SPION

   To assess the biocompatibility of SPION, their interaction with two cell lines (adhesive and suspended) was investigated using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which is a non-radioactive, colorimetric assay. Primary mouse fibroblasts (L929, adhesive) and human leukemia cells (K562, suspended) from the National Cell Bank of Iran (NCBI), Pasteur Institute, were selected for this work. Since published reports confirm that use of the MTT assay for measuring the toxicity of magnetite nanoparticles has high variability and non-specificity, the outlier detection method was applied to minimize variability. Cell detachment upon exposure to SPIONs, also necessitated the development of customized protocols for the MTT assay; detachment increased with both increasing SPION concentration and contact time. In reducing the adhesive properties of L929 cells, SPION exposure may have increased error in the MTT assay through the elimination of crystals during removal of the supernatant. To accommodate for cell detachment, cells were examined by optical microscopy to ascertain the density of violet spots prior to detachment. All synthesized SPION samples demonstrated acceptable levels of cell viability following exposure, with none demonstrating toxic effects at the concentrations tested. In addition to the effects of exposure time and concentration, reductions in cell viability depended on the physical characteristics of the SPIONs. Different shapes and sizes, which are affected by the composition and reaction conditions during formation, impart different effects to the cells.

Introducing the new protocols in order to obtain reliable toxicity results

   The core hypothesis of this study is to make a direct correlation between in vitro and in vivo studies by understanding the effect of nanoparticles on the cell medium, in particular the interaction of nanoparticles with biomolecules. The effects of both PVA coated and uncoated superparamagnetic iron oxide nanoparticles on the cell medium were examined. It is shown that the conventional in vitro examination method may contain large errors as compared to the modified method. This may be partially attributed to the fact that SPION can cause significant changes in the cell medium, such as denaturation of proteins, which in turn can cause toxicity. The modified method is.

  • Introduction of the nanoparticles to the cell medium
  • Leaving the solution in contact for a period of 24 hours
  • Replacing the medium with a fresh one
  • Application of the surface saturated SPION to the assays
Using this approach, the toxicity of uncoated SPION is found to decrease significantly. It is also observed that the existence of chloride may increase the error magnitudes in the conventional in vitro method. Coated nanoparticles induce lower toxicity not only due to the presence of the biocompatible coating, but also due to the lower adsorption sites for proteins, ions and other components in medium.

IEffect of SPION on the cell-life cycle

   In this research, an MTT assay was used to investigate the cyto-compatibility of SPION at relatively high concentrations using L929 cells. Acceptable levels of cell viability were maintained after exposure to PVA-coated SPION for up to 72 h at 80 mM, whereas the cells exhibited toxic effects after exposure to bare SPION. The morphology of the treated cells differed in comparison with the controls, particularly in the case of bare SPION. Cell clustering was observed at high SPION concentrations likely due to the magnetostatic effect. Significant apoptosis was not observed in cells treated with PVA-coated SPION, although considerable levels of apoptosis were detected in cells exposed to the bare nanoparticles. An enhanced G2/M phase was observed in cells treated with both bare and coated SPION at various concentrations. DNA damage is believed to be the prime causative factor leading to the identified apoptosis with the bare SPION exposure. Since no significant traces of cell death were observed with coated SPION at low concentration, the DNA repair pathway appears to have been activated.

Defining the main reason of toxicity

   To visualize the morphology of cell damage due to the presence of the SPION, the cells were exposed to crystal violet. Gas vesicles were detected inside cells exposed to the SPION. Gas vesicles are likely used by archaea, bacteria and planktonic microorganisms to control vertical migration by regulating gas content and thereby buoyancy. Since neither significant apoptosis, nor necrosis occurred in cells exposed to the coated SPION, the formation of gas vesicles may be responsible for the SPION toxicity through local changes in the ionic equilibrium.