Investigation of intracellular compound dynamics by enhanced Raman scattering technologies

Abstract

The highly attritional nature of drug discovery research is underpinned by a fundamental lack of knowledge regarding drug compound behaviour inside cells. At present, no single technique is capable of providing measurement of the localisation, concentration and metabolism of exogenous compounds at the subcellular level. Current platforms rely on the use of fluorescent tags, destruction of samples or are limited by poor spatial resolution. Raman spectroscopy interrogates the full vibrational fingerprint of biological samples in a label-free and non-invasive manner but is limited by inherently weak scattering intensity for intracellular studies. Signal enhancement can be achieved utilising unique optical properties possessed by plasmonic nanostructures or coherent wave mixing properties, exploited by surface-enhanced Raman scattering (SERS) and coherent Raman imaging (CRI) experimental platforms respectively. Research presented within this thesis demonstrates the ability of these technologies to perform highly sensitive intracellular measurements of exogenous molecule dynamics and actions within human cancer cell lines. The voluntary uptake of spherical gold nanoparticles (AuNPs) for use as intracellular SERS probes is established. Resulting SERS analysis subsequently evaluates molecular-level changes induced to cells following excessive AuNP internalisation with greater sensitivity than traditional biological assays. An optimised intracellular SERS methodology then performs intracellular mapping of an exogenous small molecule inside the endolysosomal pathway that is improved with application of multivariate technique principal component analysis (PCA). CRI technique coherent anti-Stokes Raman scattering (CARS) microscopy was developed for quantifiable monitoring of cytosolic lipid droplet (LD) responses to both repeated laser exposure and application of a chemotherapeutic agent in live cells. The chemical selectivity of CARS was compared with that of the second predominant CRI platform, stimulated Raman scattering (SRS) microscopy, by comparative hyperspectral imaging and statistical analysis. The research evidences capability of enhanced-Raman scattering techniques to provide valuable new pharmacological insights for drug discovery research, informing future development of label-free and industry-viable assays that would further existing knowledge and improve clinical outcomes.

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Identifiers

ORCID for Sumeet Mahajan: orcid.org/0000-0001-8923-6666

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