Special Considerations

Many of the standard methods used to evaluate biocompatibility of new molecular and chemical entities are fully applicable to nanoparticles. However, existing test protocols may require further development and laboratory validation before they become available for routine testing. Careful attention must be paid to potential sources of interference with analytical endpoints that may lead to false-positive or false-negative results. Nanoparticle interference could result from: interference with assay spectral measurements; inhibition/enhancement of enzymatic reactions61,62; and absorption of reagents to nanoparticle surfaces. In the event of nanoparticle interference, additional sample preparation steps or alternative methods may be required.

When evaluating the results of in vitro assays, it is important to recognize that dose-response relationships will not always follow a classical linear pattern. These atypical dose-response relationships have previously been attributed to shifts between the different mechanisms underlying the measured response.63 In the case of nanomaterials, it is also important to bear in mind that concentration-dependent changes in the physical state (e.g., aggregation state, degree of protein binding) may also result in apparent nonlinearity.

Another key consideration when evaluating the results of nanoparticle research is the impact of dose metric (e.g., mass, particle number, surface area), sample preparation (e.g., sonication), and experimental conditions (e.g., exposure to light) on the interpretation of results. For example, surface area or particle number may be a more appropriate metric than mass when comparing data generated for different sized particles. This has been shown to be the case for 20- and 250-nm titanium dioxide nanoparticles, in which lung inflammation in rats, as assessed by percentage of neutrophils in lung lavage fluid, correlated with total surface area rather than mass.64 The importance of experimental conditions in study design is highlighted by an investigation of functionalized fullerenes, demonstrating that the cytotoxicity of dendritic and malonic acid functionalized full-erenes to human T-lymphocytes in vitro is enhanced by photoexcitation.41 The standardization of these experimental variables should limit inter-laboratory variability and make data generated more comparable.

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