Identification of a cell subpopulation with different response to lipid nanoparticles and effect of protein corona on uptake and transfection.

Lipid nanoparticles (LNPs) have enabled the approval of the first RNA-based drugs. However, there are still many aspects of their cellular behavior that remain unclear. Here, we used LNPs of composition comparable to Onpattro and we analyzed their uptake, transfection efficiency and effects in thousands individual HeLa cells by flow cytometry. This allowed us to determine the variability in outcomes in individual cells within the cell population. We found that upon incubation with the LNPs, a sub-population of cells with comparable uptake but much lower transfection, as well as signs of cellular stress and toxicity could be distinguished. This sub-population was observed with both empty LNPs and LNPs carrying RNA. Comparable outcomes were observed with a different LNP formulation and in other cell types. Moreover, when increasing LNP concentration, uptake, endosomal leakiness and transfection first increased, but then they decreased, and the higher uptake levels were associated with loss of cell viability. Characterization of the dispersions formed at different LNP concentrations showed that the amount of proteins adsorbed on the LNPs in medium with serum decreased at higher LNP concentrations. Similarly, uptake and transfection varied greatly when increasing serum concentration towards more physiological conditions. Overall, these results indicate that the response to LNPs is highly heterogeneous, and some cells within the cell population are more sensitive to the LNPs. Furthermore, LNP uptake and transfection are strongly affected by the modifications LNPs encounter upon dispersion in biological environments.