Structure, activity and dynamics of extra virgin olive oil-in-water nanoemulsions loaded with vitamin D3 and calcium citrate

Extra virgin olive oil-in-water (O/W) nanoemulsions loaded with vitamin D3 and calcium citrate were developed and studied in terms of structure, activity and dynamics. The nanoemulsions were formulated with a two-step emulsification procedure using water as the continuous phase, extra virgin olive oil (EVOO) as the dispersed phase and mixtures of food grade surfactants as stabilizers. The nanoemulsions were investigated for their particle size, polydispersity and stability over time by Dynamic light scattering (DLS) analysis. Among the different nanoemulsions studied, those based on polysorbate 20 in combination with lecithin produced systems owing EVOO droplets of 285 ± 5 nm diameter and 0.202 ± 0.01 PdI that were stable for 37 days. These stable nanoemulsions were loaded with calcium citrate and vitamin D3 to result formulations containing both water and oil soluble micronutrients. The presence of calcium ions in the aqueous phase strongly affected the stability of the nanoemulsions whereas vitamin D3 addition in the dispersed oil phase affected the size of the oil cores by several nanometers. Interfacial properties were investigated using Electron Paramagnetic Resonance (EPR) spectroscopy employing an amphiphilic spin probe. The nature of the surfactant and the presence of vitamin D3 and calcium citrate affected the properties of the surfactants' layer in terms of rigidity, local viscosity and polarity. More specifically, upon encapsulation vitamin D3 resulted in more ordered and viscous interfaces. Antioxidant potential of the proposed nanocarriers was investigated with an EPR procedure based on the scavenging the 4-hydroxy-TEMPOL. EPR signal inhibition was observed due to the scavenging activity of hydrogen donating moieties present both in empty and loaded nanoemulsions. Diffusion ordered NMR spectroscopy (DOSY NMR) successfully revealed the solubilization of the lipophilic vitamin D3 in the dispersed oil phase of the nanoemulsion formulation.