Abstract
Diether phospholipids are phospholipid molecules that contain glycero-ether instead of glycero-ester bonds in their molecular structure (Figure 1). Their presence is reported extensively in nature, however unlike diacyl phospholipids there has been limited effort put into the formulation and study of in vitro diether phospholipid systems. Both the assembly and liposomal formulations of diether phospholipids need to be further studied, to understand how they may support biological function, and any potential future application of these systems. Furthermore, the abundance of enantiomeric enrichment in biological phospholipid systems begs questions regarding the ability of racemic phospholipid systems to form vesicular aggregates, and the nature of chiral interactions between phospholipid membranes and optically active substrates.
Figure 1. Chemical structures of a generic diester phospholipid (A) and diether phospholipid (B)
In order to investigate the behaviour of diether phospholipids in vitro, racemic diether phosphatidylethanoline (PE), phosphatidylcholine (PC) lipids of C14 – C18 chain length (R’ and R’’, Figure 1) and a phosphatidylglycerol (PG) lipid of C16 chain length were produced in order for further use in in vitro application. Enantiomerically enriched diether PC lipids were also produced for use in analysis of the chiral phospholipid-substrate interactions.
The lipid assembly of the racemic diether PE and PC phospholipids of various chain lengths and the PG phospholipid was investigated through rehydration in both 1x Phosphate Buffered Saline (PBS) and 35% NH3/H2O, representing conventional and extremophilic environments respectively. The effect of PG inclusion on assembly of diether phospholipid systems primarily composed of PE and PC phospholipids was also investigated. This was completed to provide insight into the ability of these systems to form vesicular assemblies in these environments, a requirement for biological function. PG inclusion was analysed due to the role of PG lipids in eukaryotic respiration and bacterial plasma membrane maintenance.
In 1x PBS all PC, and the PG diether lipids were found to form aggregates measurable by Dynamic Light Scattering (DLS), and the inclusion of the PG lipid into the PC systems was found to induce faster equilibration of the observed particle size. In 1x PBS the PE systems analysed did not produce DLS measurable lipid aggregates. Short lived species were observed in the C14 chain length PE systems containing 10% and 20% PG lipid inclusion. In 35% NH3/H2O, all tested lipid formulations produced DLS observable lipid assemblies excluding the system containing only the PG lipid. Inclusion of PG into the primarily PC and PE systems here also resulted in universal acceleration of equilibration of particle size. In both 1x PBS and 35% NH3/H2O, the lipid nano assemblies were confirmed to be vesicular in nature through Transition Electron Microscopy (TEM) imaging.
The diether PC phospholipids were then used to produce 5(6)-carboxyfluorescien (CF) loaded liposomes to compare these systems with commercially available diacyl phospholipids, in terms of liposomal suspension properties and their ability to maintain a concentration gradient in 1x PBS and pH 12 environments. Overall, it was found that diether PC liposomes were slightly larger than their diacyl counterparts, however the Entrapment Efficiency (EE), liposomal concentration and TEM observed liposome morphology were similar.
In 1x PBS similar CF release was observed across diether and diacyl systems. A general trend of lower release with increased phospholipid tail length found at RT, regardless of diether or diacyl functionality. Chain length dependent CF retention was not observed in either system at 37 C, and a universal increase in CF release was observed. In pH 12 systems the diether PC liposomes were found to be universally stable in terms of CF release, whereas increased CF release was observed in diacyl PC systems. A trend of increased CF release was observed as the constituent diacyl phospholipid chain length decreased. At C18 tail lengths CF release was similar across both diether PC and diacyl PC systems.
Finally, the effect of lipid chirality on the liposomal loading and retention of the chiral Doxorubicin (DOX) molecule was investigated, using enantiomeric sets of diether and diacyl PC phospholipids of C18 chain length. Diether PC formulations were found to exhibit similar DOX loading regardless of their specific enantiomeric composition. In contrast the diacyl PC systems displayed discrete loading behaviours in every system analysed, with the racemic Distearoylphosphatidylcholine (DSPC) systems displaying an intermediate effective DOX concentration, in-between the two enantiomerically enriched DSPC systems. Furthermore, the release behaviour of DOX from the enantiospecific systems was nuanced, with diether and diacyl sets both containing a system that behaved different to the other two systems measured. In diether systems these were liposomes produced from the sn-1 enantiomer, and in diacyl systems from the racemic lipid.