Investigation of nanoparticle-protein interactions with novel methods

Advancements in novel nanomaterials present promising avenue for therapeutics and diagnosis in nanomedicine. Importantly, in these applications, nanoparticles (NPs) are almost instantly coated with a complex layer of proteins in biological environment, termed as 'protein corona'€™. Understanding the interactions between the proteins and NPs has therefore been one of the key challenges in nanomedicine. The techniques established particularly for thermodynamic investigation of protein- NP interactions, however, suffer from spurious signals produced by aggregates in solution or require additional fluorescent labelling either of NPs or the proteins. In addition, there is a lack of careful systematic studies of the relationship between binding mechanism and the NP parameters such as size, surface chemistry and hydrophobicity. Presented within this thesis, a novel centrifugation-based methodology to investigate thermodynamic interaction parameters of NPs with proteins utilizing their hydrodynamic properties. With this technique, it is possible to monitor anisotropic shape evolution of NP-protein complex during the course of protein titration, especially for very small NPs. By exploiting Heteronuclear Single Quantum Coherence NMR spectroscopy, protein binding sites to NPs were carefully investigated with the help of diminishing cross peak signals of amino acids in the model protein, ubiquitin. Having developed high quality, monodisperse NPs with varying characteristics of size, hydrophobicity and surface chemistry, a systematic comparison study was carried out on the effects of such parameters on protein binding. The lack of correlation between the thermodynamic data and the mechanism of protein-NP interactions highlighted the importance of using multiple methods to fully describe these interactions. Finally, sub-10 nm gold NPs coated with amphiphilic ligand shell were proposed as efficient cargo delivery platforms for hydrophobic drug molecules with remarkable colloidal stability. The potential of these platforms was further corroborated áå îáíêç and áå îáîç with a variety of therapeutics. Overall, we believe the characterization techniques presented herein elucidate crucial aspects of NP-protein interactions and their relationship to structural parameters of the NPs. The implications of this work are anticipated to pave the way for better design of nanomedicine tools