Physicochemical and molecular determinants of lipid migration through multicellular assemblies

Efficient movement of lipid molecules across multiple cell layers is a fundamental requirement for lipid-based drug delivery, but the physicochemical and molecular properties that enable this process remain poorly understood. To address this question, we generated a library of lipids with the fluorescent cyanine 3 and cyanine 5 dye headgroups, different tail lengths, and headgroup-tail linkers. We used multicellular 3D breast cancer spheroids to study the lipid penetration into the core (core/periphery ratio) with confocal microscopy. The parent non-lipid dye showed complete penetration into the core of spheroids. Short-chain (C12) lipids with stable linkers were distributed throughout the spheroids and reached the core, whereas long-chain (C18) lipids with stable linkers accumulated predominantly at the periphery. On the other hand, ester-based lipids underwent serum-mediated hydrolysis and the release of low-molecular-weight dye that showed deep spheroid penetration. Increasing the solubility and reducing the aggregation of C18 lipids via co-formulation with DSPE-PEG2000 dramatically enhanced migration and penetration into the core. The same principles are applicable to glioma spheroids, patient-derived glioma spheres, human midbrain organoids, and human patient glioma. Mechanistically, the migration and penetration of lipid molecules through multicellular layers is facilitated by increased lipid solubility and by decreased self-assembly. This work outlines key principles for designing lipid-based probes and therapeutics for effective tumor and tissue distribution.