Understanding Interactions of Nanomaterials with Biological Environments

Kelly L McNear, Purdue University

Abstract

Nanomaterials with sizes comparable to that of features of mammalian cells have a variety of applications in the clinical setting. In this thesis, I focused on functionalized silicon nanowires (SiNWs) as well as graphene for both biological and biomedical applications. First, I demonstrated the potential of using graphene as a protective coating in biological environments. As a preliminary test, I carried out a chemical experiment was conducted in a 37 °C oven for several days in order to mimic in vitro and in vivo conditions. This test was used to confirm that the presence of a graphene layer on copper substrates effectively inhibited the corrosion of the underlying metal substrate by ensuring that the Cu2+ ion concentration was low in aqueous biological media. For the in vitro studies, osteosarcoma cells were incubated in the presences of the samples and the viability of the cells was measured daily using the metabolic activity assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). The MTT dye undergoes a color change when exposed to metabolic activity, so the solution was purple when exposed to living cells and yellow when the cells had died. Absorbance measurements were obtained for the samples as well as the control (cells without the presence of samples) and the measurements were converted to viability. The results of this cell viability test suggested that the graphene coating effectively protected the metal substrate from corrosion and thus, protected the cells from exposure to Cu2+ ions. Additionally, to boost the protective properties of graphene, a thiol coating was assembled on the copper surface to fill in any defects in the graphene coating. This was also tested with transferred graphene, which showed acceptable protection when the thiol coating was utilized. Importantly, since metal ions are known to cause inflammatory reactions in many patients, a lymphocyte transformation test was carried out to confirm that the graphene layer present on the copper substrate inhibited an immune response. Finally, an in vivo experiment was carried out by surgically inserting either bare copper foil or copper foil with a coating of single layer graphene next to the spine of a live rat. This was used to confirm that graphene exhibits protective properties under typical physiological conditions. Lastly, I utilized functionalized silicon nanowires to show, for the first time, that the uptake mechanism of these high aspect ratio one-dimensional (1D) nanomaterials occurs via a physically-driven membrane wrapping mechanism. Experiments were carried out at two different incubation temperatures, 37 °C and 4 °C, in order to confirm that the process is physically driven rather than receptor-mediated. We chose 4 °C because it is well understood that many endocytic pathways are temperature dependent and that these pathways are limited to high temperatures, so uptake at a lower temperature suggest that the uptake is physically driven. For all experiments, the SiNWs with length of 5 µm were co-cultured with CHO-β cells and imaged at various time points. First, optical microscopy was used to confirm that the wires were binding and interacting at both incubation temperatures. These results showed that the interaction of the wires was insensitive to temperature. Next, to capture snapshots of the key features of the uptake process, I fixed the cells after culture with the wires and utilized cross-sectional TEM to visualize the cell membrane. From the samples incubated at both 37 °C and 4 °C, I was able to visualize the wires binding tangent to the membrane, the membrane changing shape to accommodate the wires, and a pocket of membrane surrounding the internalized wires. This confirmed that the membrane wrapped around the wire in order to internalize it. Finally, a fluorescent confocal experiment was carried out to further understand the membrane interactions as well as gather three-dimensional (3D) information through a z-axis scan. To visualize the cell membrane, the cells were stained with DiI (red) after the removal of the wires and images were taken at steps along the z-axis. Notably, when the SiNWs were incubated with DiI without the presence of cells, they did not fluoresce. However, after co-culture with the cells, the wires that were internalized fluoresced. These results further confirmed that the internalization pathway occurred through membrane wrapping.

Degree

Ph.D.

Advisors

Yang, Purdue University.

Subject Area

Chemistry

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