Physicochemical characterization of liposomes coencapsulating curcumin and vitamin D3 produced using a mixture of purified and non-purified phospholipids

Currently the growing appeal of leading a healthy lifestyle has made its way to consumers, which includes the intake of higher nutritional foods. However, one of the main drawbacks found by the food industry is related to the incorporation of hydrophobic bioactives in aqueous-based food formulations. In this context, liposomes have been increasingly investigated for food applications as potential carriers for hydrophobic bioactives. In our study, liposomes coencapsulating curcumin (75 mg/100 g proliposome) and vitamin D3 (80,000 UI/100 g proliposome) were produced by proliposome hydration, a process which does not require expensive equipment or organic solvents other than ethanol. Different mass ratios of two phospholipids (Lipoid S40 and Phospholipon 90H) were used to produce the proliposomes: A (100% S40), B (70% S40) and C (50% S40). Phospholipon 90H is a purified phospholipid mainly composed by phosphatidylcholine (90% w/w) and lysophosphatidylcholine (maximum of 4% w/w). On the other hand, Lipoid S40 is non-hydrogenated and mainly composed by phosphatidylcholine (40% w/w) and phosphatidylethanolamine (12-15% w/w). The liposome dispersions were characterized in terms of hydrodynamic diameter, size distribution, span, zeta potential, and amount of bioactives retained over 15 days of refrigerated storage. In addition, the liposomes were analysed in terms of their morphology (using atomic force microscopy – AFM) and rheological properties. The liposomes presented average hydrodynamic diameters between 200 and 300 nm, characteristic of unilamellar vesicles. Interestingly, liposomes produced by proliposome hydration usually present higher values for hydrodynamic diameters, characteristic of multilamellar vesicles (MLV). This reduction in size was probably due to the use of sonication during the proliposome production step, due to the poor dispersibility of Lipoid S40 in ethanol. The large values of span obtained indicated multimodal vesicle size distribution. As reported in the literature, mixed vesicles formed by phosphatidylcholine and phosphatidylethanolamine usually present lower values of diameter than vesicles formed by only phosphatidylcholine. Zeta potential values obtained ranged from -149 ± 10 mV and -88 ± 8 mV which should be an indicative of an excellent colloidal stability. However, the dispersions remained stable for only 15 days, which reinforces the idea that zeta potential itself must be not considered an indication of stability. Curcumin and vitamin D3 contents were up to 96% in the formulations B and C, highlighting the good capacity of mixed liposomes to preserve both bioactives. The micrographs obtained by atomic force microscopy revealed that liposomes presented spherical or quasi-spherical shape. Besides, the size observed in micrographies agrees to the values of hydrodynamic diameters obtained from photon correlation spectroscopy. Rheologically, all the dispersions presented a slight non-Newtonian behavior and a decrease in viscosity when submitted to higher values of shear rate. The short stability period of the dispersions may be overcome by optimizing the parameters used in liposome production process or using techniques available in literature, as lyophilization. Besides, data revealed it was feasible to produce liposomes containing both curcumin and vitamin D3 stable for 15 days and the structures were highly capable to incorporate the bioactives in concentrations compatible with their recommended daily intake.

Financial support: FAPESP (São Paulo State Research Agency) – grant number 2017/10954-2