Fundamental understanding of the role of nanoparticle physicochemical properties (such as size, charge, hydrophilicity, etc.) on internalization into cells and intracellular localization is a requirement for the rational design and optimization of nanoparticles for imaging, sensing, drug or DNA delivery, cell sorting, and many other clinical applications. The long term objective of this application is to elucidate the relationship between nanoparticle physicochemical properties and internalization and fate of nanoparticles in cells. As a first specific aim, methods will be refined to use established pharmacological agents to systematically inhibit molecular internalization pathways of cells in vitro and demonstrate that such methods can be applied to elucidate the mechanism of internalization of carboxy-methyl dextran coated magnetite nanoparticles into the cancer cell lines of epithelial origin Caco-2 (human colon cancer derived) and MFC-7 (human breast cancer derived). The methods to be refined will permit discrimination between active and passive transport mechanisms, clathrin- dependent pathways (and the sub-divisions of receptor mediated endocytosis and fluid phase endocytosis), and clathrin-independent pathways (and the subdivisions of caveolar and lipid raft-dependent endocytosis). As a second specific aim, the role of surface charge on internalization and intracellular localization of carboxy- methyl dextran coated nanoparticles will be assessed by using magnetite nanoparticles of similar size, coated with carboxy-methyl dextran of the same molecular weight but varying degrees of carboxylic acid substitution. Nanoparticle internalization through each of the above-mentioned molecular pathways will be assessed qualitatively using confocal laser scanning microscopy and quantitatively using inductively coupled plasma mass spectrometry. The methods to be refined through the proposed work should find applicability in systematic study of nanoparticle internalization mechanisms for a wide array of inorganic core/organic shell nanoparticles and a wide array of target cells. The specific information to be obtained on mechanisms and effect of surface charge in internalization of carboxy-methyl dextran coated nanoparticles will be applicable to the rational design of magnetic nanoparticles with potentially improved efficacy as MRI contrast agents, magnetically targeted drug delivery vectors, and in magnetic fluid hyperthermia treatment of cancer. PUBLIC HEALTH RELEVANCE: The relationship between nanoparticle physicochemical properties and internalization in cells is a key step in developing medical applications of magnetic nanoparticles, such as MRI contrast agents, sensing, drug delivery, and magnetic fluid hyperthermia cancer treatment. The proposed work will systematically study internalization mechanisms of magnetic nanoparticles in cells and determine the effect of surface charge on rate of internalization and intracellular localization of nanoparticles. The methods refined in this work will also impact study and development of other types of polymer coated inorganic core nanoparticles for medical applications.