Date of Award

12-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Environmental Toxicology

Advisor

Klaine, Stephen J

Committee Member

Baldwin, William S

Committee Member

Lee, Cindy M

Committee Member

Mount, Andrew S

Committee Member

Roberts, Aaron P

Abstract

Over the past decade, there has been an exponential growth in products and applications that utilize nanotechnology, as these materials present exciting opportunities for the development of new products, enhancement of existing products, and the evolution of innovative procedures. However, this recent growth has occurred in the absence of detailed knowledge concerning the interactions of nanoparticles with biological systems. Nanoparticles must usually be bioavailable in order to elicit a biological response, and to reach an active site they must usually cross a membrane. As a result, cell culture studies have proven to be beneficial and are generally one of the first tests conducted upon the synthesis of a new particle type. Furthermore, the development of in vitro assays to predict biological interactions with nanoparticles may facilitate future endeavors towards accurate assessments of the risk that nanoparticles may pose on the environment. The goal of my dissertation was to characterize the uptake of gold nanoparticles by the mammalian cell line A549 through determining the roles of particle characteristics and culture medium components, and by describing potential modes by which particles are endocytosed. To assess the influence of the physical and chemical characteristics of gold nanospheres on cell uptake, a semi-factorial design was followed, examining three different particle sizes (roughly 5nm, 15nm and 50nm) modified by three different surface charges (anionic, cationic, and nonionic). The influence of particle concentration on cellular uptake was analyzed for 5nm and 50nm citrate-capped spheres over 24 h, and particle uptake increased as exposure concentration increased. The influence of particle size and surface charge on uptake was analyzed for each particle type over 2 h, and with the exception of 50nm nonionic particles, uptake was charge dependent; as cationic particles were taken up to a greater extent than anionic particles, which were taken up more than nonionic particles. To assess the influence of the culture medium components on particle uptake, cells were exposed to 30nm citrate-capped particles in exposure media supplemented with differing concentrations of fetal bovine serum (FBS) and bovine serum albumin (BSA). The uptake of particles exposed in FBS was inversely related to protein exposure concentration, which was not observed with particles in media supplemented with BSA, suggesting that certain components of FBS deter particle uptake. When cells were exposed to particles in media supplemented charcoal/dextran treated FBS, particle uptake increased significantly suggesting that growth factors and steroids may inhibit particle uptake. When cells were exposed to particles in media supplemented with BSA in addition to fetuin, uptake decreased significantly suggesting that fetuin may deter particle uptake. Also, particle uptake decreased when calcium was removed from the exposure media, suggesting that uptake occurs through a calcium dependent process. To assess potential modes of endocytosis utilized in the cellular trafficking of nanoparticles, cells were co-exposed to 30nm citrate-capped nanospheres and known inhibitors of specific endocytic pathways. Filipin and nystatin were used to inhibit caveolae-dependent endocytosis, chlorpromazine and phenylarsine oxide to inhibit clathrin-dependent endocytosis, and cytochalasin D and 5-(N-ethyl-N-Isopropyl) amiloride (EIPA) were utilized to inhibit macropinocytosis. No statistical decrease in particle uptake, in comparison to controls, was observed following exposure to any of the inhibitors, suggesting that the uptake of nanoparticles was not dependent upon any one specific mode of endocytosis. Overall, the results of my dissertation indicate that uptake is dependent upon both characteristics of the particle and exposure medium, and that the uptake of particles is a complex process which cannot be described solely by pharmacological inhibition.

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