A simple method to visualize and assess the integrity of lysosomal membrane in mammalian cells using a fluorescent dye.

Fluorescent dyes have been used as "nanosensors" for visualization and determination of various processes occurring inside a cell, or intracellular events, such as cell cycle progression and intracellular trafficking. Here, we describe a novel use of acridine orange to visualize lysosomes and discriminate cells with healthy lysosomes from cells with damaged lysosomes in two different types of mammalian cells: fibroblasts and macrophages. This method allows assessment of lysosomal membrane integrity upon exposure to various foreign particles, i.e., engineered nanoparticles. The uniqueness of this method enables investigators to acquire fluorescent images with a dye that is susceptible to photo-bleaching under UV light. These acquired images bolster the quantitative data, providing a visual representation of the cell morphology as well as assess its nucleus and lysosomes.

Nanomaterial cytotoxicity is composition, size, and cell type dependent.

BACKGROUND: Despite intensive research efforts, reports of cellular responses to nanomaterials are often inconsistent and even contradictory. Additionally, relationships between the responding cell type and nanomaterial properties are not well understood. Using three model cell lines representing different physiological compartments and nanomaterials of different compositions and sizes, we have systematically investigated the influence of nanomaterial properties on the degrees and pathways of cytotoxicity. In this study, we selected nanomaterials of different compositions (TiO2 and SiO2 nanoparticles, and multi-wall carbon nanotubes [MWCNTs]) with differing size (MWCNTs of different diameters < 8 nm, 20-30 nm, > 50 nm; but same length 0.5-2 microm) to analyze the effects of composition and size on toxicity to 3T3 fibroblasts, RAW 264.7 macrophages, and telomerase-immortalized (hT) bronchiolar epithelial cells. RESULTS: Following characterization of nanomaterial properties in PBS and serum containing solutions, cells were exposed to nanomaterials of differing compositions and sizes, with cytotoxicity monitored through reduction in mitochondrial activity. In addition to cytotoxicity, the cellular response to nanomaterials was characterized by quantifying generation of reactive oxygen species, lysosomal membrane destabilization and mitochondrial permeability. The effect of these responses on cellular fate - apoptosis or necrosis - was then analyzed. Nanomaterial toxicity was variable based on exposed cell type and dependent on nanomaterial composition and size. In addition, nanomaterial exposure led to cell type dependent intracellular responses resulting in unique breakdown of cellular functions for each nanomaterial: cell combination. CONCLUSIONS: Nanomaterials induce cell specific responses resulting in variable toxicity and subsequent cell fate based on the type of exposed cell. Our results indicate that the composition and size of nanomaterials as well as the target cell type are critical determinants of intracellular responses, degree of cytotoxicity and potential mechanisms of toxicity.