Advanced characterisation methods for the analysis of nanoformulations and extracellular vesicles

Abstract

Nanomedicine represents a challenging and highly multidisciplinary research field, concerned with the development and study of nanoformulations for diagnostic and/or therapeutic purposes. The nano-sized particles of interest are increasingly complex. Their translational potential has been hampered by difficulties in their thorough characterisation. On the nano scale, small variations in size and composition can have large implications for their pharmacodynamic and pharmacokinetic behaviour. More precise techniques are therefore required to address these challenges. This thesis describes novel, advanced characterisation methods designed for the detailed study of single nanoparticles and their interaction and uptake behaviour with cells. A platform technology for Single Particle Automated Raman Trapping Analysis – SPARTA - was developed, capable of non-destructive, label-free and automated comprehensive single particle analysis. With the SPARTA system, the composition, functionalisation, size and dynamic reactions on the surface can be investigated in detail, of a wide variety of nanoparticles, through their Raman spectra. A further improved, custom designed SPARTA 2.0 platform was built, optimised for the analysis of complex biological particles, such as EVs. EVs represent a high potential as biomarkers, studied here in the context of breast cancer. The SPARTA 2.0 platform was able to resolve compositional differences between non-cancerous and cancer cell-derived EVs with excellent sensitivity and specificity. This highlights the possibility for development of new minimally invasive diagnostic approaches. In addition, a new imaging strategy for investigation of the EV-cellular interaction is presented, based on 3D Focused Ion Beam – Scanning Electron Microscopy (FIB-SEM). FIB-SEM allows the generation of 3D models of the subcellular structure and visualisation of the cellular trafficking of nanoparticles. This represents a powerful new approach for investigating EV uptake. The methods developed in this thesis allow for the single particle-based analysis of a wide variety of nanoformulations and EVs, to aid in understanding their composition, applicability and cellular interactions.