Abstract by Lasse Skjoldborg Krog

Lipid mesophases, which are self-assembled structures of amphiphiles and water, hold immense potential in drug delivery due to their ability to encapsulate and release drugs in a controlled manner. However, in physiological environments such as the gastrointestinal (GI) tract, lipid mesophases encounter destabilising factors like bile salts and enzymes, which disrupt their ordered structure and compromise drug release. Traditionally, small-angle X-ray scattering (SAXS) has been the primary method for studying mesophase structures, but laboratory instruments can be limited in their ability to record real-time transformations, while accessibility to synchrotron-based SAXS can be limited. Low-frequency Raman (LFR) spectroscopy emerges in this thesis as a novel technique for in situ probing of lipid mesophases, specifically through analysing the intermolecular vibrational density of states (VDOS) region of the LFR spectrum close to the laser line, which was hypothesised to provide information on phase transitions in lipid mesophases. This thesis suggests LFR spectroscopy as an accessible alternative technique to monitor mesophase behaviour in different simulated environments, aiming to enhance our understanding of phase transformations and stability, especially under conditions that mimic the GI tract.

The overarching goal was to examine the phase transitions and stability of lipid mesophases using LFR spectroscopy across different conditions of increasing complexity. Specifically, this thesis aims to: (1) investigate phase transitions in monoolein (MO) bulk mesophases with varying hydration levels, aiming to map out structural changes induced by the increases in water content; (2) explore the behaviour of dispersed mesophases containing ionisable components under different pH conditions to determine whether pH-induced transitions can be monitored effectively using changes in the VDOS; and (3) determine the utility of LFR to monitor the kinetics of mesophase transformations induced by lipid digestion and evaluate a strategy to inhibit detrimental changes to lipid mesophase structures during digestion in a bile-rich environment using cyclodextrins. The research builds on three main studies: Paper 1 focuses on MO bulk mesophases under increasing hydration, Paper 2 examines ionisable dispersed mesophases with automated pH adjustments, and Paper 3 assesses the behaviour of lipid mesophases during bile- and digestion-induced transformations.

LFR spectroscopy was utilised to analyse phase-specific changes in the VDOS of lipid mesophases, providing non-invasive real-time structural information about the sample. Experiments were conducted across three primary conditions: (1) hydration of MO bulk mesophases, where incremental water content was added either in-situ or ex situ to induce phase transitions detectable as changes in the VDOS; (2) pH-controlled studies on ionisable dispersed mesophases with ionisable components oleic acid (OA) or nicergoline (NG), where pH was altered to provoke phase shifts and assessed by LFR and SAXS; and (3) in a bile-rich environment where cyclodextrin was added to dispersed mesophases to evaluate its role in maintaining the V₂ cubic phase and phases formed during digestion. Complementary laboratory and synchrotron-based SAXS were used to validate findings from LFR spectroscopy.

In conclusion, the studies collectively demonstrated that LFR spectroscopy could effectively capture subtle changes in both bulk and dispersed lipid mesophase structures. Paper 1 showed that LFR detected distinct variations in the shape of the VDOS in the hydration-induced phase transitions of MO, from lamellar liquid crystalline phase (L_α) to two types of V₂ phases (the Ia3d space group followed by the Pn3m space group), highlighting the sensitivity of LFR to different hydration states. In Paper 2, ionisable dispersed mesophases exhibited changes in the intensity of the VDOS that aligned with pH adjustments, indicating that LFR could discern phase shifts. The protonation state was altered, inducing changes in the packing of ionisable OA or NG in the lipid mesophase structures. As a result, the apparent dissociation constant (pK_a^app) could also be determined using LFR. In Paper 3, the LFR spectroscopy, corroborated by SAXS, was showcased as a methodology for monitoring structural transformations of lipid mesophases during digestion. The predictability of the method for probing the transitions was better for simple systems without co-existing mesophase structures. Lastly, the addition of cyclodextrin in bile-rich settings successfully preserved the structural integrity of mesophase structures in MO dispersions upon digestion. The bile salts were shown to associate with various entities in the GI tract with an increasing affinity for mixing with proteins, lipids and exogenous cyclodextrin, respectively. These results collectively affirm the capacity of LFR to serve as a non-invasive method for observing dynamics of transitions between lipid mesophases, essential for optimising lipid-based drug delivery.