Label-free imaging for drug delivery across biophysical barriers

Abstract

Every year countless drug products fail to make it through the discovery and approval process required for commercialisation and widespread patient use. This failure is often due to issues with poor efficacy and side effects. Drug delivery systems based upon nanotechnology are currently at the forefront of drug discovery research and boast the ability to greatly improve treatment capabilities. However, a general lack of understanding of drug delivery and distribution at the cellular and subcellular levels, is ultimately making the widespread use of these systems an uphill battle. Current ‘go to’ techniques for in vitro and in vivo quantification primarily revolve around using fluorescent labels and/or processes which cause sample destruction. Both of these are less than ideal. Raman spectroscopy is a non-invasive and label-free technique that can provide molecular information on a wide variety of sample types, ranging from drug formulations to cell and tissue samples. When coupled with microscopy, Raman is able to generate vibrationally, and often chemically, specific images of samples. This thesis examines the use of microscopies utilising the process of Raman scattering, to gain insight into drug delivery systems. A novel methodology is presented using Raman spectral unmixing achieved through multivariate curve resolution alternating least squares analysis to quantify non-ionic surfactant vesicle formulations in terms of drug loading and vesicle component concentration. The method presented was able to accurately quantify a series of calibration samples before providing invaluable insight into vesicle component concentrations. Drug loading calculated using the technique showed similar results to those seen with fluorimetry. Investigation into the intracellular response of cell cultures treated with vesicular drug delivery systems was also completed. Analysis using microscopies based upon the multiphoton techniques of coherent anti-Stokes Raman scattering and two-photon excited fluorescence allowed for complementary label-free and label-based imaging. A cellular response was observed upon treatment. The source of this response was investigated further, quantified and a dose-response relationship identified. Extent of drug delivery from vesicles was also quantified and compared to that of delivery from drug free in solution. The research outlined in this thesis demonstrates the excellent versatility and quantitative power of Raman spectroscopy to further elucidate the role drug delivery systems can play in improving therapeutic treatments.

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Identifiers

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

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