PhD Proposal: Morton Ehrenberg
The Physicochemical Basis of Nanoparticle Interactions with Cells: Application and Analysis
As technologies become ever more competent at producing objects at length scales comparable to the smallest levels of biological organization, opportunities are gained and challenges presented. This thesis contains research investigating the use of nanoparticles as probes for local mechanical responses of cells and interactions of nanoparticles with cells that underlie therapeutic uses and toxic mechanisms. Measurements of protein adsorption to particles were made following incubations in biological fluids of relevance to each context. Stably adsorbed proteins from particle surfaces were removed, quantified and identified, showing that particle composition and chemistry affects the spectrum of adsorbed protein. Particles were also monitored by fluorescent microscopy and other optical techniques in cell-free and cellular contexts. Binding interactions of proteins are found to be the most reliable predictors of the behavior of particles in biological systems.
High frequency and high resolution tracking of polystyrene nanoparticles within cells was developed and is covered first. This work shows that probe particles adsorbing disproportionate quantities of the cytoskeletal proteins actin and vimentin with surfaces exposed to the cytoplasm have restricted motions compared to particles only binding to one network. Motions of all injected particles are restricted in comparison to those that are endocytosed and thus in vesicles. Probe motions of actin adsorbing particles are also shown to measure weaker environments after specific poisoning of actin.
The other focus is on particle binding and downstream interactions at the endothelial surface; the layer of cells lining blood vessels that mediate transit of circulating materials and cells into tissue. Experiments with well characterized polystyrene nanoparticles separately addressed the binding capacity of particles for protein and associations of particles with cells measured by flow cytometry. Findings indicate a correlation between protein adsorption on particle surfaces and associations of particles with endothelial cells. Specific removal of highly abundant proteins from the system changes the profile of adsorbed proteins but has little effect on association, suggesting that this correlation is non-specific; that it does not depend on any receptor mediated pathways. Detailed analysis of the spatial distribution of particles within endothelial cells shows that they are internalized and trafficked to lysosomes in the perinuclear regions of cells, independent of surface chemistry. Particles are also grouped into clusters over time in a surface-dependent manner. Aliphatic and positive particles exhibit more clustering than acidic particles suggesting a role for decreased pH in this process. In addition, disruption of actin or microtubule networks with specific poisons are found to enhance clustering but have limited effect on the long time spatial distribution of clusters.