Extracellular vesicles (EVs) are biologically derived nanovectors important for intercellular communication and trafficking, yet understanding of their underlying biological mechanisms remains poor. Advances have been hampered by both the complex biological origins of EVs and a lack of suitable imaging techniques. Here, we present a strategy for simultaneous in vitro imaging and molecular characterisation of EVs in 2D and 3D based on Raman spectroscopy and minimally-obstructive metabolic deuterium labelling. Metabolically-incorporated deuterium acts as a bio-orthogonal Raman-active tag for direct Raman identification of EVs and provides insights into their biocomposition and trafficking, with implications for their development as therapeutic delivery vectors.
Comprehensive single nanoparticle analysis of synthetic drug delivery systems, as well as natural occurring particles such as Extracellular Vesicles (EVs), is still a major challenge in the field, and is necessary to enhance their successful design, screening and study towards translational application. Investigating population heterogeneity is essential for nanoparticles, as their behaviour, characteristics and thus applicability are strongly affected by this, and cannot be resolved with conventional bulk analysis techniques. Here, we present a dedicated platform for comprehensive Single Particle Automated Raman Trapping Analysis (SPARTA). Nanoparticles ranging from synthetic polymer particles to liposomes or EVs can be integrally analysed by SPARTA without any modification, to obtain their size, determine functionalisation and composition, and monitor dynamic reactions occurring on their surface. The single nanoparticle nature of this approach allows highly detailed investigation in particle heterogeneity, resolving particle mixtures and tracking sequential functionalisations and dynamics on the particle surface. By using a Raman solution marker we demonstrated for the first time the capability to size single nanoparticles in a trap solely by Raman scattering, while simultaneously obtaining their compositional information, allowing novel insights in size-composition relationships. In addition, SPARTA can be applied to study in great detail the biochemical profiles of single EVs from cancerous and non-cancerous origin, towards the use of EVs as cancerous biomarkers for diagnosis, disease progression and evaluating therapeutic efficacy. SPARTA has great potential to critically impact fields from nano drug delivery system design to cancer biomarker identification and profiling.
Theranostic approaches to cancer management offer the possibility of tailoring treatments to individual patients or tumours. However, the development of theranostic nanoparticles optimally suitable for both diagnostic and therapeutic modalities to achieve this is challenging. Here we demonstrate an alternative nanoparticle-free theranostic approach that circumvents many of the difficulties currently hindering clinical translation. Through the use of a multimodal optical probe, we translate the theranostic burden from complex nanoparticles within the body to an external spectroscopic device, thus alleviating many of the constraints imposed on existing theranostic systems. Using this platform, we demonstrate the combination of Raman spectroscopic diagnosis with photodynamic therapy for optical cancer theranostics. Through selection of photosensitisers with suitable optical properties for combination with Raman spectroscopy, we achieve optical theranostics without the need for complex material systems. Sequential delivery of light for real-time Raman spectroscopic diagnosis followed by photosensitiser-specific illumination, enables cancer diagnosis and treatment during a single procedure. We demonstrate the feasibility of this theranostic approach using a panel of clinically-approved photosensitisers using in vitro cell assays across three cancerous cell lines, with in vivo demonstration using subcutaneous xenograft tumour models ongoing. Our results indicate that through careful instrument design, Raman spectroscopic diagnosis can be effectively performed on photosensitiser-containing cells and tissues without impaired diagnostic accuracy or undesired premature photosensitiser activation. Together, these results show that combination of Raman spectroscopy and photodynamic therapy for optical theranostics enables nanoparticle-free cancer detection, diagnosis, and treatment in real-time using a single optical system.
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