Multifunctional Nanoparticles for Bioimaging

Abstract

Bioimaging is a broad term that covers all processes in which biological tissues are imaged. It can range from single-cell visualization with fluorescence microscopy, to imaging of brain structures in living human beings with magnetic resonance imaging. As such, the term is not only interesting from a researcher’s point of view, but also to existing medical technologies. Imaging living organisms is mainly based on existing tissue densities between blood, brain, cartilage, and bone. However, in some cases these differences are not sufficient to separate e.g pathological tissue from healthy tissue. In these instances, a contrast agent may be administered to alter the contrast in the region of interest. Bioimaging of excised tissue usually involve staining with a dye that binds specifically to a cellular organelle or membrane, making it easier to separate intracellular structures from each other. Bioimaging of living and excised tissue both involves an external probe that enhances the available information in the image. Such probes must be synthesized from materials that are likely to improve the acquisition of the image, and must also have few toxic side-effects. Nanomaterials are in the size-range of molecules and proteins, and can potentially traverse cellular membranes as well as being systemically administered. Nanoparticles are described as multifunctional because they can show a range of optical responses, carry drugs and vibrate in response to heat. The nanomaterials in this thesis were chosen based on their plasmonic and magnetic properties, as well as their biocompatibility. The aim of this thesis is twofold. The first aim is to fully characterize and synthesize plasmonic nanomaterials of gold with biomineralization from proteins and from chemical synthesis. The second aim is synthesis and characterization of magnetic nanomaterials of iron and manganese. Characterization in this sense refers not only to physical description of the nanoparticles or nanoconstructs, but involves describing how these nanomaterials interact with biological tissues in vitro or in vivo, to assess their potential for bioimaging, hyperthermia and drug delivery. A plethora of methods have been used to characterize the nanomaterials synthesized in this thesis. The nanomaterials of gold synthesized from proteins were described with a range of optical techniques such as UV-visible and steady state spectroscopy, and time-correlated single-photon counting. Size and shape was assessed with dynamic light scattering, scanning transmission electron microscopy and high resolution transmission electron microscopy. The surface charge was determined with zeta potential measurements. The crystalinity and composition of nanoparticles were characterized with X-ray diffraction spectroscopy, and surface composition was described with X-ray photoelectron spectroscopy. Interactions between protein-stabilized nanomaterials and model membranes were described with Langmuir-trough studies and atomic force microscopy. Magnetic nanomaterials were characterized with magnetic resonance imaging and zero field cooling. Further, nanomaterials were co-incubated with human cancer and/or rodent cells to assess their in vitro uptake or cytotoxicity. The methods for in vitro assays involved fluorescence microscopy and a cytotoxicity assay. Finally, ultramicrotome sectioning and scanning transmission electron microscopy was used to describe cellular uptake as a function of time. The main findings in the first part of this thesis are that fluorescent protein-gold nanoconstructs show membrane interactions radically different from the native protein. We also describe how protein-directed self-assembly of fluorescent gold nanoclusters and plasmonic nanoparticles can be tuned to yield different optical gold nanostructures. In the second part of this thesis, the main findings are size, - and shape-dependent uptake of novel plasmonic and magnetic nanoparticles in vitro, as well as cell actuation in a magnetic field. The final part of this thesis culminates in synthesis of a nanoparticle system that can give dual contrast in magnetic resonance imaging while at the same time release a drug.