Establishing Three-Dimensional Super-Resolution Microscopy Methods For Quantifying Intracellular Nanoparticle Distribution

Abstract

The systemic administration of nanomedicine formulations has been described as a promising treatment option for solid tumors at both preclinical and clinical stages. However, these treatments are currently limited to improved safety over the administration of free drugs, while improvements to efficacy have been limited by a noted low accumulation of nanoparticles in cancer cells. The mechanisms for nanoparticle delivery across tumor blood vessels into the tumor microenvironment are not fully understood as a result of the physical limitations of the current standard methods of visualizing nanoparticle accumulation and intracellular transport in cancer cells. Most intracellular vesicles typically involved with the transport of nanoparticles across tumor blood vessels are sized smaller than the spatial resolution limit of light microscopy (~200 nm laterally), whereas electron microscopes, which provide sufficient lateral resolutions for visualizing these vesicles, are typically limited to thin biological samples, making it difficult to acquire three-dimensional (3D) visualizations of cells. To address these challenges, in this dissertation, quantitative 3D super-resolution light microscopy methods were applied to study the intracellular distribution of metallic and organic nanoparticle formulations in cultured cancer cells. We employed a method known as expansion microscopy, which involves embedding cell samples within swellable hydrogels to physically enlarge the sample >10X their original size for super-resolution imaging. Intracellular label-free metallic nanoparticles were visualized with light scattering imaging, while organic nanoparticles were visualized with internalized fluorescent tags. Since expansion microscopy is compatible with the labeling of intracellular features, this method enables the determination of the precise location of nanoparticles within cells, which can be used for studying intracellular nanoparticle trafficking with high spatial resolution in 3D. The successful application of this method will empower new research in nanomedicine for the development of safer and more effective treatments.