Internally Self-Assembled Pickering Mesosomes Stabilized by Positively Charged Lactoferrin

Mesosomes as sub-micrometer-sized emulsions containing internally self-assembled lyotropic liquid crystalline (LLC) mesophases were engineered using lactoferrin (LF) as a Pickering stabilizer. LF was pretreated with ultrasonication to prevent agglomeration and applied under controlled pH conditions during processing, resulting in emulsions with a positive surface charge and high stability. Glycerol monooleate (GMO) in mixtures with oleic acid (OA) at varying concentration ratios were used to produce four different lyotropic mesophases: bicontinuous cubic (<i>Pn</i>3<i>m</i>), inverse hexagonal (H₂), inverse micellar cubic (<i>Fd</i>3<i>m</i>), and microemulsions (ME). At a preadjusted pH of 4, LF stabilized all four lyotropic mesophases via a Pickering-type mechanism, forming mesosomes with hydrodynamic diameters (<i>d</i>_H) ranging from ∼187 to ∼239 nm, as measured by dynamic light scattering (DLS). High-pressure ultrasonication was used to drive positively charged LF to adsorb at the LLC-water interface, confirmed via confocal microscopy. The sonication led to a distinct positive surface charge on the mesosomes, as evidenced by the ζ potential of ∼+30 mV, without compromising the mesosomes' size and stability. Time-resolved DLS experiments showed that all mesosomes had long-term stability against coalescence and aggregation for at least 1 month of storage at room temperature. Small-angle X-ray scattering was used to study the phase behavior of GMO/OA mixtures before and after their stabilization by LF. The results showed that all four bulk mesophases maintained their structural hierarchy after being homogenized into mesosomes. Notably, pure GMO stabilized by LF formed cubosomes of diamond <i>Pn</i>3<i>m</i>, the same morphology as GMO in the bulk. This behavior is different from what has been observed for the commonly used stabilizer for LLC systems, Pluronic F127, which induces a transition to a primitive <i>Im</i>3<i>m</i> cubic morphology upon stabilization.