Electron microscopy characterization of nanomaterials for biomedical applications [electronic resource]
Keywords: Materials Science and Engineering.

In the last fifteen years, the field of nanotechnology in medicine has grown tremendously. Applications using nanoparticles as optical and magnetic contrast agents have garnered consistent interest as promising means to detect diseases and monitor therapies. Also, advances in characterization techniques have enabled new investigations of naturally occurring nano-scale features in the body, in order to better understand biological functions associated with health and disease. This dissertation describes the application of electron microscopy to characterize a wide variety of nanomaterials of relevance to the field of medicine. A combination of imaging, diffraction and spectroscopy are employed for the evaluation of the morphological, crystallographic, chemical and optical properties of nanoparticles themselves, as well as their interactions with cells and tissues. In addition to engineered structures, naturally occurring biological nanoparticles are investigated. The first part of this work is focused on surface-enhanced Raman scattering (SERS) nanoparticles, which have been proposed as a tool for sensitive detection of tumors. Electron beam lithography was applied to generate model gold SERS structures. These were tested using electron energy loss spectroscopy in the scanning transmission electron microscope (STEM-EELS) to measure the plasmonic (optical) response of the structures with nm-scale resolution. Raman microscopy was also applied to compare relative signal enhancements. Cr and Ti layers, which are commonly used to promote the adhesion of gold onto various substrates, were identified as damping plasmon resonance and causing reduced SERS signal intensities. An alternative adhesion layer, (3-mercaptopropyl) trimethoxysilane, was evaluated and found to have no such negative effects. Additionally, imaging and spectroscopy in the scanning electron microscope (SEM) were used to confirm intravenously injected commercially available SERS nanoparticles cross the brain-blood barrier of naturally occurring tumors in dogs. This is an important result as prior testing has only been performed on mice with xenografted tumors. The successful application on real (though canine) patients is promising sign for potential clinical translation. In the second part, Ferumoxytol, a commercially available nanoparticle-based iron supplement, is characterized. The shape is not well controlled, with small aggregates forming with an average size of approximately 16 nm. They are found to consist of a magnetite (cubic Fe3O4) and/or maghemite (cubic Fe2O3) core, with a thin organic shell. Next, ferumoxytol was incubated with human cells to evaluate its performance as a magnetic resonance imaging (MRI) contrast agent for the monitoring of stem cell therapies. The incubation was performed in three different protein containing media, as well as two solutions containing transfection agents designed to increase the uptake of nanoparticles by cells. Transmission electron microscopy (TEM), combined with electron energy loss spectroscopy, established the internalized nanoparticles were primarily located within lysosomes in the cell regardless of the incubation method. Surprisingly, the greatest iron uptake and MRI contrast were found in cells treated with human serum (HS) media. Therefore, incubation with HS could provide a simpler and more effective means of uptake as compared to traditional transfection agent methods for MRI applications. The third and final section addresses characterization of two types of naturally occurring nanoparticles. First, a new filtration technique for the isolation of extracellular vesicles (EVs) was evaluated with transmission electron microscopy, and compared with the ``gold standard'' of ultracentrifugation. EVs can provide valuable information about the cells which generate them, and a simple method of isolation is desired for implementation in clinical applications. A negative staining technique was implemented to provide contrast in the TEM, and specific proteins of interest were labeled with antibodies conjugated to gold nanoparticles. Successful isolation by filtration was confirmed from patient urine, plasma, and lung fluids. Immunogold labeling indicated that CD9, CD63, and CD81 proteins were present on the surfaces of some EVs, consistent with previous studies. TEM images of samples produced by filtration and ultracentrifugation showed similar morphologies. This suggests the former method does not cause structural damage during processing, making it a viable low-cost alternative to the latter. The second half of this section focuses on naturally occurring iron oxide deposits in the brain. Misregulation of iron has been proposed as a potential cause of oxidative stress and damage to the surrounding tissue. SEM and TEM analyses were performed on iron deposits in the brains of humans who suffered from Alzheimer's disease. Correlative optical microscopy and SEM was applied to locate the deposits. Energy dispersive X-ray spectroscopy performed in the SEM found that some samples contain a combination of iron and zinc. One such deposit was extracted using a focused ion beam tool and thinned to electron transparency. A STEM-EELS chemical map determined that the two elements were unevenly distributed. The biological implications of these findings are not yet understood. Overall, scanning and transmission electron microscopy are demonstrated to be versatile and powerful tools for characterization of a wide variety of nanomaterials, as well as for the evaluation of their distribution in cells and tissues.