Revealing the Morphology of Small Multilamellar Lipid Nanoparticles (SMLPs) made by In-Vial Homogenization

Previous research shows that thermosensitive small multilamellar lipid nanoparticles (tSMLPs) offer promising features for temperature-triggered cytostatic drug delivery, remaining completely stable at body temperature (37°C) and releasing their payload under mild hyperthermia conditions (42°C). A distinguishing characteristic of tSMLPs is their unique particle morphology - multiple tightly packed bilayers with progressively decreasing intermembrane spacing toward the particle core. In this study, we shift the focus from their thermosensitivity to an in-depth exploration of the particle's morphology. Using in-vial homogenization by dual centrifugation (DC) at very high lipid concentrations (60%), we prepare SMLPs composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG₂). A systematic screening of DPPC/DPPG₂ 100-x/x (mol/mol) from x = 0 to 100 enabled the formation of SMLPs with sizes below 200 nm, narrow size distribution and well-distinguishable morphologies. These lipid nanoparticles also demonstrated the capacity to entrap hydrophilic compounds, despite their multilamellar structure and thus limited interlamellar aqueous space. We propose that specific headgroup interactions between DPPC and DPPG₂ underlie the observed water influx upon dilution of the initially formed vesicular phospholipid gels (VPGs) during in-vial homogenization by DC. With a combination of biophysical techniques (DLS, Time-resolved fluorescence, SAXS and WAXS) and morphological analysis (cryo-EM), we present a hypothesis to explain the evolving SMLP morphology as a function of increasing DPPG₂ content in the phospholipid blend.