Liposomes: systematic screening and characterisation of lipid-membrane stability

Abstract

Liposomes are versatile nanoparticles for drug delivery, facilitated by surface modifications and controlled cargo release, making them a valuable tool in cancer treatment. However, despite their potential, the number of approved liposomal therapies is limited due to challenges in tumour targeting and drug release. To address this limitations, a liposome preparation technique published by Massing et al. in 2008, which uses dual centrifugation, was utilised. This method is particularly suitable for research due to its ability to produce multiple samples in parallel, its high encapsulation efficiency and the minimal amount of lipid required. The first project investigated how the chain length of dialkyl lipids affects their membrane anchoring and migration rate into other membranes. This is particularly important for surface-functionalised liposomes, as the loss of modified lipids can significantly reduce therapeutic efficacy. By covalently linking lipids with 16, 18 and 20 carbon atoms in their dialkyl chains with fluorescent dyes, a reduced anchoring stability of C16 lipids in liposome membranes compared to longer chain lipids was observed. In addition, a correlation between the anchoring stability and the proportion of phospholipids in the lipid composition, as well as the type of phospholipids used, was investigated. Overall, this study provided new insights into the development of stably modified liposomes. In the second project, dual centrifugation was utilised for parallel sample preparation to perform a screening aimed at identifying new pH-sensitive liposomes according to rational criteria. These liposomes were designed to release their cargo under acidic conditions, such as those found in the mature endosome. Initially, binary lipid compositions were evaluated for membrane stability, physicochemical properties and release characteristics. The selected and optimised candidates demonstrated improved intracellular release of the encapsulated cytostatic agent in cellulo compared to a liposomal control, while maintaining similar biodistribution over 24 h in a mouse model. Overall, this rational screening approach successfully identified three pH-sensitive liposomes, demonstrating the practical applicability of the method. In the third study, a novel method was developed to assess the biological activity of FTAD, a potential methyltransferase inhibitor, which was previously not possible due to its lack of membrane permeability. This was achieved by using the fusogenic liposome LFT1 as a transfection system for small molecules. Physicochemical characterisation revealed minimal effect of encapsulated cargos on the particle properties of LFT1. The successful intracellular release was demonstrated using a fluorescent phosphatase substrate as a model cargo. Finally, the activity of FTAD via liposomal transfection was evaluated in cellulo by analysing the methylation levels of total tRNA and comparing it with the effects of the methyltransferase inhibitor sinefungin. FTAD showed no inhibitory activity, whereas sinefungin effectively reduced six total tRNA methylation sites under comparable conditions. This work highlights the utility of LFT1 as a tool for early assessment of the biological activity of new small molecules in drug development.