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. 2021 Mar 31;11:7311. doi: 10.1038/s41598-021-86484-9

Figure 8.

Figure 8

Cytotoxicity of PTX-loaded CLs against M21 cells. (a) Viability of M21 cells (relative to untreated cells) as a function of PTX concentration for cells treated with CLs of a molar composition of 50/50–xPTX/xPTX, DLinTAP/DLinPC/PTX, with xPTX = 2, 4, 6, or 9 (black lines). Cell viability decreased with PTX concentration to a similar level as observed for PC3 cells, albeit more gradually. As a control, DLinTAP/DLinPC CLs without PTX were added to M21 cells in amounts matching the lipid content of CLs with xPTX = 2 (which have the highest lipid content) at the PTX concentrations tested (blue line). The control showed no cytotoxicity (≥ 90% viability) up to the lipid equivalent of 75 nM PTX delivered with xPTX = 2. This suggests that PTX, not lipid, drove cytotoxicity at the IC50 of every formulation because CL cytotoxicity manifests only at concentrations at least four times the IC50 (see part c). (b) Viability of M21 cells (relative to untreated cells) as a function of PTX concentration for cells treated with control CLs of a molar composition of 50/50–xPTX/xPTX, DOTAP/DOPC/PTX, with xPTX = 2, 4, 6, or 9 (black lines). The decrease in cell viability with PTX concentration is less steep than in part A, suggesting a lower efficacy. At the employed concentrations, CLs without PTX do not elicit cytotoxicity48. (c) Plot of IC50 values for PTX cytotoxicity against M21 cells for the CL formulations based on lipids with linoleoyl (“DLin”, red line) and oleoyl (“DO”, black line) tails. Each IC50 was determined by fitting the corresponding cell viability curve (from parts a and b) as described in the Methods section. Importantly, the efficacy of the DLin formulations was about two-fold higher (their IC50 values were two-fold lower) than that of the corresponding DO formulations. This effect amplifies the benefits of increased PTX membrane solubility (Fig. 4). In contrast to PC3 cells, the IC50 increases (efficacy decreases) with increasing PTX content for both DO and DLin formulations. This effect is less pronounced for the DLin formulations, and the IC50 of the DLin formulation at xPTX = 9 is lower than that of the DO formulation at xPTX = 2.