Electron microscopy investigations of nanoparticles conjugated to biological systems [electronic resource]

Over the past decade, nanotechnology has shown great potential as a platform for cancer diagnosis and treatment. The use of novel imaging techniques including surface enhanced Raman scattering, SERS, to locate nanoparticles inside biological systems shows great promise as a way to detect cancer at an early stage before metastasis has a chance to occur. This dissertation describes the use of electron microscopy to characterize the interactions of SERS based nanoparticles with biological systems. As nanotechnology becomes increasingly employed in biology it is necessary to accurately characterize how these nanoparticles interact with the biological system. Due to the scale of these nanostructures, electron microscopy, with its sub nanometer spatial resolution, is essential to understanding these interactions. In the first section of this work, composite organic inorganic nanoparticles, COINs, composed of silver nanoparticles encapsulated in an organic binder and functionalized with antibodies were analyzed using both scanning and transmission electron microscopy. COINs were functionalized with anti-pEGFR, anti-pstat5 or anti-pstat1 and incubated with EGF stimulated A431 lung cancer cells. TEM analysis revealed that both the anti-pEGFR and anti-pstat5 COINs were internalized by the A431 cells while the anti-pstat1 COINs were not internalized. Anti-pEGFR COINs were located in the cytoplasm of the cell while anti-pstat5 COINs were able to translocate into the nucleus of the cell. In the second section of this work a novel SERS based nanoparticle called a Raman active gold core nanoparticle, R-AuNP, was characterized. The R-AuNP consists of a gold core surrounded by a silica shell. TEM analysis of the nanoparticles revealed that gold core has an average diameter of 65 nm with an overall nanoparticle diameter of 141 nm. In the third section of this work a STEM technique was developed to efficiently locate R-AuNPs in "large" volumes of mouse liver tissue as part of a large scale nanoparticle toxicity study. Mice were injected either intravenously or intrarectally with R-AuNPs and at set time points tissue samples were collected and the liver was analyzed using STEM for both the IV and IR injected mice. This analysis revealed that R-AuNPs were present in every liver sample from the mice injected intravenously with the R-AuNPs. No R-AuNPs were found in the liver samples from mice injected intrarectally with the nanoparticles. This indicates that the R-AuNPs were unable to pass through the colon wall and into the blood stream. With this method over 500,000 µm3 of mouse tissue has been examined to date. In the fourth and final section of this work electron microscopy was utilized to locate R-AuNPs in brain tumors and determine if the nanoparticles spread and accumulated across the entirety of the tumor. A correlative light microscope / scanning electron microscope technique was established utilizing light microscopy to provide information about the overall structure and location of the brain tumor through the use of histological staining protocols and utilizing scanning electron microscopy to accurately locate R-AuNPs within the tissue sample. By mapping the R-AuNP locations onto the light microscopy images it was possible to determine the location of the R-AuNPs with respect to the tumor and show that the R-AuNPs were located at the peripheries of the tumor at multiple locations. This indicates that the R-AuNPs infiltrated and spread across the entirety of the tumor. The utility of the electron microscopy approach is confirmed by these various examples.

Keywords: Materials Science and Engineering
Advisor: Sinclair Robert (Materials scientist) primary advisor. Thesis advisor
Gambhir Sanjiv Sam
Wang Shan