A cell-based assay originally designed to test whether magnetic particles can repair damaged nerve cells has instead found that these nanoparticles, widely assumed to be safe, are actually neurotoxic.
The study, conducted by researchers at the University of California, San Diego, and published in the June issue of Biomaterials, raises questions about the continued use of these particles in biomedicine, as well as broader questions about research priorities in the field of nanobiotechnology, according to one of the paper’s authors.
“People have to pay more attention to the study of toxicity for nanomaterials,” especially in early-stage nanobiotechnology studies, Sungho Jin, a professor of materials science at UCSD and senior author on the paper, told Cell-Based Assay News this week. Funding agencies “have poured in billions and billions of dollars for nanotechnology and nanobiotechnology, but compared to that big chunk of money, the portion spent on [studying] toxic effects — side effects — is extremely small.”
The molecules used in the study, iron oxide nanoparticles, are currently used in a broad range of in vitro and in vivo biomedical applications, including cell sorting, targeted delivery of drugs or genes, magnetic hyperthermia for cancer therapy, and magnetic resonance imaging.
“There are thousands of groups in the world working with iron oxide magnetic nanoparticles,” Jin said.
Assuming that the molecules were safe, Jin and his colleagues thought they might be useful in a new application: nerve cell repair. “What we were hoping to do was put some magnetic particles in the two broken ends of the neuron, and see if we can apply a magnetic field so that they are magnetically attracted to each other and eventually reconnect,” Jin said.
However, he said, “in the course of the study, we found that the magnetic iron oxide particles did something strange: They deteriorated the functionality of the neurons.”
Deciding to look deeper into that finding, Jin and his colleagues designed an assay to measure the toxic effects that increasing concentrations of nanoparticles had on the neurons — one of the first quantitative studies to be performed in the field, according to the researchers.
“Although a few studies have been performed investigating the acute cytotoxicity of [magnetic nanoparticles] and their qualitative effects upon cellular morphology, little work has focused on quantifying the effects that [iron oxide] internalization has upon cell behavior and, in particular, the ability of cells to appropriately respond to biological cues,” the authors write in the Biomaterials paper.
The UCSD researchers used PC12 pheochromocytoma clonal cells for the study, which tested the response of treated and control neurons to nerve growth factor. Untreated PC12 cells have a “rapid and reversible response to NGF,” the authors write, “resulting in the extension of neurite-like processes up to 1 cm in length.”
Their findings showed that neurons treated with nanoparticles sprouted far fewer neurites than the control cells. While normal cells produced an average of 2.79 neurites per cell, the cells exposed to 0.15, 1.5, and 15 mM concentrations of nanoparticles produced an average of 2.67, 1.9, and 0.97 neurites per cell, respectively.
Funding agencies “have poured in billions and billions of dollars for nanotechnology and nanobiotechnology, but compared to that big chunk of money, the portion spent on [studying] toxic effects — side effects — is extremely small.”
In addition, the length of those neurites that were produced in the treated cells were “dramatically affected,” according to the authors.
Overall, PC12 cells exposed to the nanoparticles “show reduced viabilities, increased cytoskeletal disruption, and a diminished ability to form mature neurites in response to NGF exposure as compared to control cells,” the authors write. “This may have significant implications for in vivo and phenotypic-dependent in vitro uses” of the nanoparticles, they added.
One interesting finding was that dimercaptosuccinic acid, or DMSA, a material used to coat the iron oxide particles to prevent them from clumping, appeared to increase the toxicity of the nanoparticles despite being proven to have no toxic effect on its own.
The authors suggest three explanations for this behavior: the combination of the iron oxide and coating somehow “magnifies” the positive and negative interactions with cellular components; the coatings may make the nanostructures more likely to take part in adverse interactions; or a combination of those effects.
The primary finding of the study, Jin said, is that far more research needs to be done in this area, including studies around alternative coatings that could minimize the toxic effects of nanoparticles.
Specifically, he noted, the study underscores the importance of incorporating toxicity testing into early-stage nanobiotechnology studies. “It has to be done in the research stage, so people have a clear understanding of what they are doing, what the consequences are, and how to prevent [toxic effects],” he said.
Jin noted that toxicity issues are often overlooked in nanobiotechnology research due to the pressure to publish promising results. “When people study the effects of nanotechnology, they tend to report positive things — this is good for curing disease and so on — and people rarely talk about how bad it could be. There should be some balance in the future,” he said.
“People are beginning to study these effects, but what we’re saying is that there should be more,” he said.
In particular, funding agencies “need to pay more attention in terms of stimulating and supporting research of this nature instead of concentrating on breakthrough therapeutics and breakthrough imaging and those aspects” of nanobiotechnology, Jin said.