We differentiated the subsarcolemmal and interfibrillar mitochondria by isolating them from the tissue using differential centrifugation (different speeds and duration isolate different fractions). For the interfibrillar mitochondria, we have to break the myofibrils using a protein. Once the two subpopulations of mitochondria are separated, we are able to measure the change in oxygen concentration, which reflects energy production, after the mitochondria have been fueled with glutamate, malate and ADP.
In terms of gene mapping, our initial investigations associated with proteomic data from heart tissue suggest that genes involved in cardiac fibrosis and enlargement may be affected by nanomaterial exposure.

Inert Gas Condensation (IGC) and Surfactant-Assisted Milling (SAM) each have advantages and disadvantages. IGC produces very uniform nanoparticles and gives us exceptional control over their growth; however, it requires ultra-high vacuum (making it more expensive and time consuming than SAM) and produces tens of milligrams of material per hour.
SAM is inexpensive and commonly used in industrial processes. We can produce gram-size quantities in the lab and industrial mills can produce kilogram and larger batches; however, milling produces a broad range of particle sizes. We thus have to use an additional centrifugation step to narrow the size distribution.
The computer algorithms that identify genetic pathways were developed and validated using data from the lung; however, initial pathway analysis on heart tissue indicates that the genetic pathways affected are involved in free radical scavenging, lipid metabolism, molecular transport and nucleic acid metabolism.

We used gold as an example in the video; however, the cardiac study used commercially available titanium dioxide nanoparticles, which have a mean aerodynamic diameter of 159 nm and a wide size distribution. Despite lack of control over the properties of commercially produced nanomaterials, a wide range of consumer products already use these materials, and we feel it is important to understand them.
Particle size is known to have important effects on biological function. One aspect of our approach is developing nanomaterials with very specific sizes, shapes and chemical functionalities so that we can understand the correlation between chemical and physical properties and biological impacts.

Our investigations to date have involved titanium dioxide and carbon nanotubes due to the wide array of potential applications and presence in commercially available products. A strength of our approach is that our methods are not nanoparticle specific, but can be applied to virtually any nanomaterial. We expect significant differences in biological response as a function of material, shape and size. One focus of our research is designing nanoparticles with very well controlled sizes and shapes so that we can investigate the specific dependence of the biological response on physical and chemical properties. We are currently focusing on fabricating iron and iron-based nanoparticles due to their medical applications.

We are particularly interested in two classes of nanomaterials: those that are already being used in consumer products (carbon nanotubes, silver, titanium dioxide) and model nanoparticles that will allow us to investigate the relationship between biological response and specific physical and chemical properties. Coupling our computer algorithms with micro-array data allows us to efficiently test and analyze any set of genes, but we are most interested in genes involved in cardiac fibrosis and enlargement. Whole-body exposure will be related to cellular mechanisms by analyzing blood and tissues for the expression of cytokines associated with inflammation or the presence of the nanoparticle.