The aqueous channel size of lipidic cubic phases can be a limiting factor for certain applications. For this reason, additives have been used to exquisitely control their nanostructure. In this study, two families of primary phosphoesters have been designed, synthesised and utilised to determine the effect of the positioning of the guest additive at the interface of the host mesophase, and to contrast the effect of headgroup ionisation and protonation. A general methodology has been developed to produce primary phosphoesters, and a unique use of P-31 NMR has been used in order to systematically investigate the influence of these additives on monoolein- and phytantriol-based bulk lipidic cubic phases and dispersed cubosomes. In general, di-phosphorylated additives exhibit a greater effect upon lipid packing than the mono- and tri-phosphorylated molecules due to their optimal positioning. In dispersion, the protonation state of the phosphate headgroups was manipulated by altering the pH, where shifts in pK(a) determined by P-31 NMR were used as a fluorescent label-free method to identify the location and ionisation state of the phosphate additives. This study systematically evaluates the influence of the positioning of the additive, headgroup size and charge of phosphorylated lipids on the behaviour of lipidic mesophases. (C) 2019 Elsevier Inc. All rights reserved.