In vitro assessment of Nano Material Toxicity

Introduction
Nanomaterials are generally in the 1-100 nm range and can be composed of many different base materials (carbon, silicon, and metals such as gold, cadmium, and selenium). Nanomaterials also have different shapes: referred to by terms such as nanotubes, nanowires, crystalline structures such as quantum dots, and fullerenes.

Nanomaterials often exhibit very different properties from their respective bulk materials: greater strength, conductivity and fluorescence.

Major Advantages of In Vitro ASSESSMENT

Reduction in animal use Greater experimental control over chemical concentrations, environmental composition Uniform biological systems (cell lines) Ability to use human tissue/cells Potential for use in high through-out screening assays

Examples of Cell-based In Vitro Assays

Hepatocyte/liver slice cultures Fibroblast/cell line cultures 3-Dimensional Cell cultures Skin, eye irritation tests

What is the MTT assay?

Cultured cancer cells are grown in the presence of potential drug for a specific time period (e.g. 24,48,72, 96 h)

The amount of viable cells remaining can be determined spectrophotometrically

Living cells convert the yellow water soluble-tetrazolium salt into an insoluble purple formazan crystal Crystal is dissolved in a suitable solvent (e.g. DMSO, acid-IPA) and the absorbance at 570 nm is obtained A dose response curve is plotted and the concentration at 50% cell growth is obtained

N S N

BrN N N N

Living cancer cells Mitocondrial dehydrogenases

H N N

MTT

Formazan

MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Lactate dehydrogenase (LDH))

When a cell dies its LDH is released to the blood. LDH is one of the main components in blood tests. High level of LDH are indicative of heart attacks, cancer or anemia. If LDH levels are high, another test is performed to test the level of the different isozymes, so that the source of the problem can be more accurately inferred The enzyme catalyzes the following biochemical reaction:

Pyruvate + NADH

Lactate + NAD

Real-Time PCR mRNA quantification

DNAmRNAprotein

Reflect level of gene expression Information about cell response Protein production

Real time PCR

Real-time PCR Applications

Viral Quantitation Quantitation of Gene Expression Array Verification Drug Therapy Efficacy DNA Damage measurement Quality Control and Assay Validation Pathogen detection Genotyping

CASPASE 3 ACTIVATION ASSAY

ANNEXIN V / PI STAINING
Annexin V binds strongly to phosphatidyl serine which is normally excluded from the extra vascular side of plasma membrane but flips between the inner and outer side upon the onset of apoptosis. Annexin V is therefore be useful to detect apoptotic cell.

Stress response detection of ROS

Cellular stress response investigated with H2DCFDA (2-7-dichloro dihydroflouroscene diacetate). The acetylated non-flouroscence mole.is taken up by cell culture cells,is presumbly trapped in a cytosol by deacetylation and becomes flouroscent upon intracellular oxidation. A possible increase in flouoroscency of DCF.

Comet Assay Single Cell Gel Electrophoresis