(143c) Alterations in the Membrane Lipid Composition of Live Cells Affects the Ability of Cells to Internalize Nanomaterials

Over the past decades, engineered nanoparticles have been extensively used to deliver drugs or imaging agents to mammalian cells. Nanoparticles can be internalized into the cells through passive diffusion or a number of endocytic pathways. Previous studies on nanoparticle endocytosis have been primarily focused on elucidating the endocytic pathways, and the membrane receptors involved in each pathway. However, the role of the lipid microenvironment in regulating nanoparticle endocytosis has remained unexplored. In this study, live cell-lipid exchange was used to replace the lipids of the outer leaflet in A549, alveolar epithelial cells, with lipids of choice and examine their role in the uptake of silica nanoparticles (50 nm).

Methyl-alpha-cyclodextrin (MÉ‘CD) was used to alter the lipid composition of outer leaflet of cell plasma membrane. For the exchange studies, MÉ‘CD was pre-loaded with sphingomyelin (18:0, 18:1, and 24:0), 1,2-dioleoyl-sn-glycero-3-phosphocholine (36:2 PC, DOPC), and 2-((2,3-bis (oleoyloxy)propyl)dimethylammonio) ethyl hydrogen phosphate (36:2 inverse PC, DOCP). In each case, the exchange of cellular lipids with these synthetic lipids was confirmed by chromatography and mass spectrometry and cell viability after exchange was examined using the MTT assay. Control and lipid-exchanged cells were then exposed to fluorescent silica nanoparticle in serum-free medium or supplemented with fetal bovine serum (FBS) and incubated for one hour at 37 °C. The intensity of fluorescent nanoparticles, associated with or internalized into cell plasma membrane, was measured using flow cytometry. Lysosome and early endosome marker were used to study the intracellular fate of nanoparticles using confocal microscopy.

All exogenous lipids maintained cell viability; however, lipid-exchanged cells showed significant differences in their ability to endocytose nanoparticles in serum-free medium. DOCP and SM 18:0-exchanged cells showed significantly reduced nanoparticle uptake, SM 18:1-exchanged cells showed increased uptake, while DOPC-loaded cells showed a comparable level of uptake to control cells. Confocal microscopy confirmed the differences in observed in flow cytometry experiments, while showing differences in intracellular localization of nanoparticles in control and lipid-exchanged cells. No difference in nanoparticle uptake was observed in FBS-supplemented medium, likely due to the fact that serum proteins resulted in significant aggregation of nanoparticles, rendering them too large for endocytic uptake. These findings clearly indicate an important role for membrane lipid composition in nanoparticle uptake and are important in understanding the differences in uptake in cells with different membrane lipid compositions.