(442i) Triglycerides Stabilize Organic/Water Interfaces of Changing Area Via Conformational Flexibility

Background: The role of Triglycerides (TGs) in both synthetic and biological membranes has long been the subject of study and debate, involving metabolism, disease, and colloidal synthesis. TGs have been found to be critical components for successful liposomal encapsulation via double emulsion, which this work attempts to explain.

It has been shown that TGs can occupy multiple positions in a biological membranes. The glycerol backbone can sit at the membrane surface, adjacent to phospholipid headgroups, in what is termed the M Conformation; TGs in this confirmation have relatively low (~3%) solubility in membranes. TGs can also occupy several “oil” phases, where the glycerol backbone is isolated from water, in either mid-membrane positions or lipoprotein-style phospholipid-coated TG droplets. ¹³C-Nuclear Magnetic Resonance Spectroscopy (NMR) can be used to locate the backbone, as the carbonyl peaks are shifted downfield when exposed to water.

Methods: Liposomes were prepared using a 2-step Water/Organic/Water emulsion (W/O/W), where the organic phase consists of membrane constituents dissolved in chloroform, which is subsequently removed via a 24-hour drying process. Membrane constituents were Glycerophospholipids (PCs), Cholesterol, and TGs. NMR was performed for samples prepared using a triglyceride, Triolein (TO) with a ¹³C-enriched glycerol backbone. Samples were taken at different time points in the drying cycle to determine backbone hydration throughout the process. Membrane fluidity was assessed during drying via Fluorescence Anisotropy (FA) using Laurdan and Diphenylhexatriene (DPH) as fluorescent membrane probes. Finished colloidal suspensions were fractionated via Centrifugation at 50,000g in order to examine the different structures produced, resulting in a pellet, infranatant, and supernatant containing lipids. NMR and FA were repeated for these fractions. Encapsulation, composition, and size of structures were characterized using Flow Cytometry (FCM). Encapsulation efficiency was investigated as a function of TG chain length and saturation using a Calcein Leakage Assay.

Results: NMR revealed a transition in TG backbone location from surface to oil phase that begins with the disappearance of the bulk chloroform and ends with the disappearance of partially hydrated chloroform. A new, transitional TG backbone position has been identified, with a level of hydration between surface and oil. FA shows that membrane order increases as chloroform is removed, interfaces shrink, and TO exits the surface monolayers. Supernatant fraction is shown to be rich in TGs and significantly less ordered, while pellet and infranatant are shown to contain fewer TGs, more order, and to contain more encapsulated material. These results suggest that TGs are able to temporarily coat and stabilize the large water/organic interfaces present just after emulsification. As the chloroform evaporates and interfaces shrink, the TGs are able to recede into a mid-membrane space or to bud off into separate coated oil droplets, which can be removed via centrifugation. Finally, encapsulation efficiency is found to be negatively related to both chain saturation and length, implying that membrane fluidization is a key property arising from the presence of TGs.

Implications: Beyond clarification of a mechanism for high-efficiency and throughput liposomal encapsulation, these results implicate TGs as components that are able to stabilize biological membrane transitions involving changing interfacial area. This role for TGs may be of use in designing drug delivery systems and investigation of membrane transitions in life sciences.