Investigations of the Role of Triglycerides in Synthetic Colloidal Systems

Abstract

The base unit of all life on earth, the cell, is delimited by the biological membrane. Biological membranes, broadly speaking, are composed of phospholipid (PL) bilayers and a variety of membrane-associated proteins that assist in the maintenance of cells and their organisms. Since the 1990's, the components of synthetic biological membranes have been employed in the design drug-delivery vehicles, referred to as lipid nanoparticles (LNPs). The structure and components of LNPs vary based on the application, and they have been deployed clinically for a variety of treatments, including chemotherapeutics, pain management, antimicrobials, and vaccines. This work focuses on the physics of these lipid systems in an effort to develop new tools and understanding for use in their design. The primary focus of this work has been to investigate the behavior of triglycerides (TGs) in colloidal systems. TGs are glycerolipids with three strongly hydrophobic tails, generally thought of as oils. The tails are connected via a glycerol backbone, however, which is somewhat polar. This structure them unique properties compared to PLs, which have two strongly hydrophobic tails and a charged, strongly hydrophilic headgroup; while PLs are fixed to oil/water interfaces, TGs can occupy a variety of positions, and have been shown to impact membrane mechanics in interesting ways. In a PL bilayer, they can occupy interfacial positions, with the glycerol backbone acting as a headgroup (the "m" conformation), isotropic oil phases between leaflets, swelling the membrane (referred to as "blisters"), as well as intermediate positions, where their tails interdigitate into the leaflets (the "h" conformation). Inclusion of TGs has been shown to be critical to the success of liposomal encapsulation via water/oil/water double emulsions, which involve a drying process to remove the volatile intermediate organic component, and result in high encapsulation efficiencies. The mechanism of their action, however, was previously unexplained. In this work, using C13-Nuclear Magnetic Resonance (C-NMR) to assess the hydration level of TG's glycerol backbone, the position of TGs throughout the double-emulsion's drying process was tracked. It was determined that, at early stages of the drying process while the organic solvent is present and the oil/water interfaces are large, TGs sit at an interfacial position. As the solvent is removed via evaporation, TGs recede from the interfaces, occupying an oil phase once the solvent is completely removed. This indicates that TGs stabilize the interfaces as they shrink by smoothly receding into an oil phase, preserving the integrity of the emulsion and yielding high encapsulation efficiency. This property of TGs, referred to as "conformational flexibility", is also present in diacylglycerides. Both of these lipids have been found to be critical to biological processes involving membrane fusion and budding, and it is likely that their conformational flexibility, as well as inducement of negative membrane curvature, is critical to their roles in these processes as well. TGs were then applied to surfactant-coated microbubbles, which are clinically employed as ultrasound-contrast agents. Theoretically, TGs may be able to flexibly coat the expanded microbubble interface during rarefaction. Preliminary acoustic experimentation indicates that microbubbles produced with triglycerides have longer lifetimes under ultrasonication, and further experimentation is proposed to investigate this mechanism. A secondary focus of this work was to use multiparametric analysis of the fluorescence behavior of Laurdan to investigate PL membrane phase. Ternary systems of ordering PLs, disordering PLs, and cholesterol exhibit complex lateral phase behavior; as a function of composition, these systems can exhibit three distinct phases, as well as lateral phase coexistence between any pair or all three. Triggered LNP drug delivery systems in development rely on inducement of a phase-change via an environmental or applied stimulus, resulting in the release of the LNP's contents. As a result, it is of interest to locate phase-boundaries. Existing methods of phase-boundary detection are relatively laborious and/or expensive. Laurdan is a fluorescent probe commonly employed to investigate membrane fluidity and hydration, largely due to its solvent-induced spectral shift. Its use in the detection of boundaries of phase coexistence, however, have not previously been reported. It was found that by assessing Laurdan's fluorescence emission, anisotropy, and lifetime in membranes of varying composition, several key features of the ternary phase-map can be determined. By combining emission and anisotropy assessed at different emission wavelengths, the boundaries of the region of phase coexistence can be determined in binary PL/PL systems. Additionally, by combining anisotropy and lifetime to calculate rotational correlation time (a measure of membrane fluidity) and comparing this with the spectral shift (a measure of membrane hydration), the supercritical region beyond the region of liquid-liquid phase coexistence in ternary systems can be located, proximal to the critical point. These results are typical of a three-dimensional supercritical fluid; the supercritical liquid phase of membranes is characterized by the hydration of the more ordered phase with the fluidity of the disordered one.