Hybrid assemblies of photoluminescent nanoparticles for biomolecular-specific cellular imaging

The last decade witnessed an exciting confluence of nanotechnology and biophotonics. The application of photoluminescent nanomaterials in biological systems represents one of the many manifestations of this confluence. A unique set of photoluminescent nanomaterial properties includes: (1) programmability of their physical and chemical characteristics that depend on the size, composition and production methods; (2) presence of reactive functional groups on the surface that enables adaption of the nanoparticles to a specific biological task via the process called biofunctionalisation; (3) optimal size compatible with cells and tissues; (4) high surface area that can carry a large amount of molecular cargo; (5) bright and stable photoluminescence distinguishable on the background of strong scattering and autofluorescence of biological tissues. The merits of photoluminescent nanoparticles are particularly recognised in the most dynamic and actively developing area of biomedicine – biomolecular-specific optical diagnostic imaging. For example, the use of photostable photoluminescent nanoparticles has enabled demonstration of the molecular trafficking in cells and high-sensitivity imaging in small animals, showing good promise for direct and minimally-invasive diagnostic optical imaging in hollow organs, skin and surgical fields. As compared to the state-of-the-art, this emerging imaging modality permits observation of single biomolecules in cells and in vivo imaging at depths up to several millimetres, with superb, unprecedented sensitivity. In this study, new-generation photoluminescent nanoparticles, including fluorescent nanodiamonds, upconversion nanoparticles (UCNPs) and nano-rubies, were introduced to the area of biomolecular-specific optical imaging. In order to realise their full potential, nanoparticles must be transformed to hybrid inorganic-biomolecular assemblies, which are capable of targeting and interacting with biological systems. A hybrid assembly represents an inorganic nanoparticle core grafted with functional moieties, such as targeting or therapeutic biomolecules. The grafting is performed by linking these subunits in an easy and flexible modular fashion. Demonstration of a bottom-up approach of the synthesis of these hybrid assemblies and their deployment to targeted delivery to cells, followed by biomolecular-specific imaging, represent the key objectives of this thesis. Development of ultrasensitive optical imaging modality suitable for photoluminescence nanoparticle-based biomolecular-specific imaging is another important thrust of this work. In order to push the optical contrast to the ultimate single-biomolecule sensitivity level, an emerging nanomaterial, UCNPs, were employed. The imaging sensitivity at the single-nanoparticle level was investigated in challenging scenarios of advanced biomedical imaging in skin and haemolysed blood. The single-nanoparticle imaging was also demonstrated on the example of nano-rubies developed by my colleagues and me, whose photoluminescence signal was clearly visualised on the crowded optical background of stained cells. One of the key results reported in this thesis is the development of a new approach for the integration of nanoparticles and biomolecules into hybrid assemblies via molecular adaptors. The feasibility of this approach was established via grafting a fluorescent nanodiamond core with fluorescent proteins through novel molecular adaptors, a high-affinity protein pair, barstar:barnase. The merits of barstar:barnase as the molecular adaptors in comparison with their highly acclaimed analogue [strept]avidin:biotin were demonstrated in a separate study. In order to realise biomolecular-specific optical imaging assisted by fully-functional hybrid photoluminescent assemblies, mini-antibodies specific to the human epidermal growth factor receptor type 2 (HER2/neu) were attached to UCNPs, via the barstar:barnase adaptors. These hybrid UCNP-mini-antibody assemblies were demonstrated to adhere preferentially to adenocarcinoma cells, which expressed HER2/neu in large quantities, and make these cells optically distinguishable. The demonstrated specific optical imaging of the photoluminescent nanoparticle-labelled adenocarcinoma cells lent itself to an extension to an experimentally and theoretically modelled scenario of optical diagnostic imaging of early-stage breast adenocarcinoma. The developed approaches of production and deployment of hybrid photoluminescent nanoparticle-biomolecular assemblies in ultrahigh-sensitivity imaging in cells and biological tissue models will provide new capabilities of single-biomolecule imaging in molecular biology and potential background-free imaging in challenging diagnostic imaging scenarios and applications in clinical practice.