Photoluminescence-based characterization of bioengineered nanovesicles and erbium emitters

Abstract: In recent decades, photoluminescence properties of single molecules and ions have opened new possibilities for studies on smaller size scales, below the light-diffraction limit. In this thesis, the advantages of such single photon emitters were harnessed and studied mainly in the field of biophysics, but also, in investigations within solid state photonics. The first aspect encompassed studies on extracellular vesicles (EVs) and nanovesicles derived from red blood cell (RBC) membranes which were bioengineered for drug delivery applications. RBC-derived vesicles demonstrated high biocompatibility and low immunogenicity, offering superior production scalability over physiological EVs. However, thorough physical characterizations of such nanovesicles are yet to be developed. Such investigations are essential for their clinical deployment, for therapeutic and diagnostic purposes. Initially, the morphology and size-stability of vesicles were investigated by applying atomic force microscopy and dynamic light scattering. These studies demonstrated the size heterogeneity and agglomeration tendencies of the vesicles. Comparative studies on physiological EVs revealed a higher size stability, while RBC-produced vesicles showed about 50 % reversible agglomerations. Secondly, a dual-colour coincident fluorescent burst (DC-CFB) experimental analysis technique was developed. DC-CFB was then used to characterize and profile the cargo-loading yields of bioengineered nanovesicles, overcoming challenges related to their small size (below the diffraction limit), their inherent heterogeneity, and the presence of free, non-encapsulated cargoes. The developed methodology was then applied to explore the loading with relatively small single nucleotides (dUTP) as well as larger antibody (Ab) molecules, motivated by the prospective role of such EVs and EV-mimetic bioengineered vesicles as nanocarriers of therapeutic drugs. The studies demonstrated consistent average loading yields of around 14-20 % for both cargo types (dUTP and Ab) into both vesicle categories, i.e., EVs and RBC-derived vesicles. Additionally, the analysis capability of the DC-CFB technique at single-vesicle and single-molecule levels, afforded analyses of the number of loaded molecules inside each vesicle, and how this number varied with the vesicle size. On average, this number was found to be greater than two, for both cargo types. Overall, the developed techniques based on fluorescence single photon counting provided a comprehensive assessment of the drug loading properties of nanovesicles. Such bioengineered nanocarriers have a disruptive potential for pharmaceutical applications. The last part of the thesis investigates the realm of solid-state on-chip photon emitters. Specifically, it considers the integration of erbium ions, exhibiting photoluminescent emission in the telecommunication C-band, into thin film lithium niobate (TFLN) waveguides. An Er-ion implantation process compatible with integrated optical circuits in x-cut TFLN was developed and the erbium photoluminescence properties were investigated versus temperature. These preliminary studies provide a foundation for future integration of Er single photon emitters into TFLN-based photonic components.