Among different lipid nanoparticle systems, sponge-phase nanoparticles (SPNPs) have recently attracted interest due to their ability to encapsulate large macromolecules along with demonstrated high interfacial activity. The potential application of SPNPs calls for investigations into how buffer conditions affect SPNP structure and interfacial activity. Herein, we systematically investigated how different buffer conditions affect SPNP preparation by characterizing solution-phase colloidal properties and interfacial adsorption behavior on oxide surfaces. Dynamic light scattering, electrophoretic mobility, and small-angle X-ray scattering (SAXS) measurements showed that SPNPs prepared by the same dispersion method had similar size, charge, and internal structure largely independent of the buffer condition. Interestingly, however, the interfacial activity of the different SPNP samples depended strongly on the buffer condition used for nanoparticle preparation. Quartz crystal microbalance-dissipation (QCM-D) experiments revealed that certain buffer preparation conditions increased attractive SPNP-SiO