Cytotoxicity

Cell viability of adherent cell lines can be assessed by a variety of methods.86 These methods fall broadly into four categories, assays that measure: (1) loss of membrane integrity; (2) loss of metabolic activity; (3) loss of monolayer adherence; and (4) cell cycle analysis. Data generated using these various viability assays can be used to identify cell lines susceptible to nanoparticle toxicity and potentially give clues as to the type (i.e., cytostatic/cytotoxic) and location of cellular injury. Many of the cytotoxicity assays discussed below are available as commercial kits. These kits should be used whenever feasible since they provide an extra level of quality control.

1. Membrane integrity assays are particularly important as a measure of cellular damage, since there is evidence that some cationic nanoparticles, such as amine terminated dendrimers, exhibit toxic effects by disrupting the cell membrane.87 Examples of assays that measure membrane integrity include the trypan blue exclusion assay and lactate dehydrogenase (LDH) leakage assay, which measures the presence of LDH released into the media through cell lysis.88,89 The LDH leakage assay was selected because of its sensitivity and suitability for the high-throughput, 96-well plate format.

2. Examples of assays which measure metabolic activity include tetrazolium dye reduction, ATP, and 3H-thymidine incorporation assays. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay was chosen for measurement of metabolic activity in the assay cascade, since it does not use radioactivity, and historically has been proven sensitive and reliable. MTT is a yellow water-soluble tetrazolium dye that is metabolized by live cells to water insoluble, purple formazan crystals. The formazan can be dissolved in DMSO and quantified by measuring the absorbance of the solution at 550 nm. Comparisons between the spectra of samples from nanoparticle treated and untreated cells can provide a relative estimate of cyto-

toxicity.90

The MTT assay requires a solubilization step that is not required for the newer generation of tetrazolium dyes that form water-soluble formazans (e.g., XTT). However, these analogs require an intermediate electron acceptor that is often unstable, adding to assay variability. Furthermore, the net negative charge of these newer analogs limits cellular uptake, resulting in extracellular reduction.91 MTT, with a net positive charge, readily crosses cell membranes and is reduced intracellularly, primarily in the mitochondria. Because nanoparticles have been shown to interact with cell membranes and could potentially interfere with the reduction of the newer generation analog via trans-plasma membrane electron transport, the traditional MTT assay would appear to be a better choice to assess cellular viability in nanoparticle cytotoxicity experiments. Analytes that are antioxidants, or are substrate/inhibitors of drug efflux pumps, have been shown to interfere with the MTT assay.92,93 Functionalized fullerenes, which have not identified as efflux pump inhibitors or substrates, but do possess potent antioxidant activity, have been observed in our laboratory to cause MTT assay interference, resulting in enhanced MTT reduction and overestimation of cell viability (unpublished data).

3. Loss of monolayer adherence to plating surfaces is often used as a marker of cytotoxicity. Monolayer adherence is commonly measured by staining for total protein, following fixation of adherent cells. This simple assay is often a very sensitive indicator of loss of cell viability.55 The sulforhodamine B total protein staining assay was selected for determination of monolayer adherence. Advantages of this assay include the ability to store the fixed, stained microplates for extended periods prior to measurement, making the assay especially suitable for high throughput.94

4. Cell cycle analysis is conducted using propidium iodide staining of DNA and flow cytometry.95 Flow cytometric can be used as a screening test for toxicity of chemicals. This method can determine the effect of nanoparticle treatment on cell cycle progression, as well as cell death. Cell cycle effects have been shown for a variety of nanoparticles. For instance, carbon nanotubes have been shown to cause G1 cell cycle arrest in human embryonic kidney cells, with a corresponding decrease in expression of G1-associated cdks and cyclins.96

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