The cytotoxicity of polycationic iron oxide nanoparticles: Common endpoint assays and alternative approaches for improved understanding of cellular response mechanism

Abstract

BackgroundIron oxide magnetic nanoparticles (MNP's) have an increasing number of biomedical applications. As such in vitro characterisation is essential to ensure the bio-safety of these particles. Little is known on the cellular interaction or effect on membrane integrity upon exposure to these MNPs. Here we synthesised Fe3O4 and surface coated with poly(ethylenimine) (PEI) and poly(ethylene glycol) (PEG) to achieve particles of varying surface positive charges and used them as model MNP's to evaluate the relative utility and limitations of cellular assays commonly applied for nanotoxicity assessment. An alternative approach, atomic force microscopy (AFM), was explored for the analysis of membrane structure and cell morphology upon interacting with the MNPs. The particles were tested in vitro on human SH-SY5Y, MCF-7 and U937 cell lines for reactive oxygen species (ROS) production and lipid peroxidation (LPO), LDH leakage and their overall cytotoxic effect. These results were compared with AFM topography imaging carried out on fixed cell lines.ResultsSuccessful particle synthesis and coating were characterised using FTIR, PCS, TEM and ICP. The particle size from TEM was 30 nm (−16.9 mV) which increased to 40 nm (+55.6 mV) upon coating with PEI and subsequently 50 nm (+31.2 mV) with PEG coating. Both particles showed excellent stability not only at neutral pH but also in acidic environment of pH 4.6 in the presence of sodium citrate. The higher surface charge MNP-PEI resulted in increased cytotoxic effect and ROS production on all cell lines compared with the MNP-PEI-PEG. In general the effect on the cell membrane integrity was observed only in SH-SY5Y and MCF-7 cells by MNP-PEI determined by LDH leakage and LPO production. AFM topography images showed consistently that both the highly charged MNP-PEI and the less charged MNP-PEI-PEG caused cell morphology changes possibly due to membrane disruption and cytoskeleton remodelling.ConclusionsOur findings indicate that common in vitro cell endpoint assays do not give detailed and complete information on cellular state and it is essential to explore novel approaches and carry out more in-depth studies to elucidate cellular response mechanism to magnetic nanoparticles.

Poly(allylamine) Magnetomicelles for Image Guided Drug Delivery

“…The subsequent stability problems in solution can be overcome by surface engineering of the particles with coatings such as silica and polymers [14,19]. However, in the last few years, increasing concern has arisen over the safety of polymer coated iron oxide nanoparticles [20][21][22]. This culminated in the withdrawal of Feridex ® from use in humans in the United Kingdom in 2012.…”

Rhamnose‐coated superparamagnetic iron‐oxide nanoparticles: an evaluation of their in vitro cytotoxicity, genotoxicity and carcinogenicity

“…The study of cell internalization of NPs opened a series of questions about the integrity of the cell membrane. With regards this point, the use of the LDH assay in many studies on nanoparticles (Naqvi et al, 2010;Singh et al, 2010;Hoskins et al, 2012b;) have been suggested. In our experiments, the LDH released is correlated with the increase in the nanoparticle's concentrations, confirming a trend previously observed in other studies (Naqvi et al, 2010;Hoskins et al, 2012b;).…”

Accumulation of Iron Oxide Nanoparticle and Conventional Iron Oxide in Rat Ovary and Oxidative Stress Caused by It

“…Moreover, Hoskins et al, showed that oxygen free radicals generated by iron nanoparticles increased, which ultimately led to cell death. It was expressed that iron oxide nanoparticles enter into lysosomes and by breaking down the membranes of lysosomes release from enzymes that damaged the cells (22). In addition to the changes listed for various oxidative stress factors, protein carbonyl index showed no statistically significant difference in any of the groups that was probably due to changes in the factors of chronic cases, or the other reasons associated with the creation of the intermediate elements such as Cu and Fe; and its impact on the circulatory system proteins and carbonyl group creation was very low.…”