The influence of dioleoylphosphatidylcholine (DOPC) on Lipid Sponge phase system

Marshall Machingauta^1,2Aina McEvoy³Alma Karlsson³Justas Barauskas⁴Tommy Nylander^5,6,7,8*Jennifer Gilbert^1,3

• ¹Division of Physical Chemistry, Department of Chemistry, Lund University, Lund, Sweden
• ²NanoLund, Lund University, Lund, Sweden
• ³Chalmers University of Technology, Gothenburg, Sweden
• ⁴Camurus AB, Lund, Sweden
• ⁵Lund University, Lund, Sweden
• ⁶LINXS Institute of Advanced Neutron and X-Ray Science,, Lund, Sweden
• ⁷Sungkyunkwan University - Natural Sciences Campus, Suwon-si, Republic of Korea
• ⁸Lund University, NanoLund, Lund, Sweden

The use of lipid nanoparticles (LNPs) in pharmaceutical and food applications has gained momentum due to their capacity to encapsulate a wide range of biomolecules. Previous studies have demonstrated the effective entrapment of enzymes within lipid sponge nanoparticles, highlighting their potential as versatile delivery vehicles. Similar to inverse bicontinuous cubic phases, the sponge phase features a network of aqueous cavities separated by curved lipid bilayers, but with a more flexible structure and larger water cavities. The objective with this study is to reveal how the lipid composition affects the sponge phase properties. Based on food grade lipid mixtures of glycerol monooleate rich lipid mixture (GMO-50), diglycerol monooleate (DGMO), polysorbate 80 (P80) and water, which are known to form sponge phases, we have studied the incorporation of the zwitterionic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This is of particular interest due to its potential to increase biocompatibility of the formulation. Using Small Angle X-Ray Scattering (SAXS) and Cryogenic Transmission Electron Microscope cryoTEM, we show that DOPC generally promotes lamellar phases at 25 °C, but sponge phases can be preserved by adjusting GMO-50/DOPC ratios, adding P80, or increasing temperature to 40°C. Dispersions in excess water yielded mixtures of sponge nanoparticles and vesicles, while diluting the LNPs in ionic buffers (PBS and cell medium) induced multilamellar vesicles. These results demonstrate that DOPC provides a tuneable handle on lipid nanostructure, enabling temperature and medium responsive systems, and that the surrounding medium can restructure nanoparticles even after formation. This underscores the importance of considering both the conditions of nanoparticle assembly and their response to new environments, with direct implications for biopharmaceutical performance.