Figure 7.

Cytotoxicity of PTX-loaded CLs against PC3 cells. (a) Viability of PC3 cells (relative to untreated cells) as a function of PTX concentration for cells treated with CLs of a molar composition of 50/50–x_PTX/x_PTX, DLinTAP/DLinPC/PTX, with x_PTX = 2, 4, 6, or 9 (black lines). Cell viability rapidly decreased with PTX concentration to a plateau. As a control, DLinTAP/DLinPC CLs without PTX were added to PC3 cells in amounts that match the lipid content of CLs with x_PTX = 2 (which have the highest lipid content) at the PTX concentrations tested (blue line). This CL-only control showed no appreciable cytotoxicity (≥ 90% viability) up to the lipid equivalent of 65 nM PTX delivered with a 2 mol% PTX formulation, demonstrating that PTX and not lipid drove cytotoxicity at the IC50 of every formulation. (b) Viability of PC3 cells (relative to untreated cells) as a function of PTX concentration for cells treated with control CLs of a molar composition of 50/50–x_PTX/x_PTX, DOTAP/DOPC/PTX, with x_PTX = 2, 4, 6, or 9 (black lines). Cell viability rapidly decreased with PTX concentration to a plateau. Previous work has shown that CLs alone do not contribute to cytotoxicity at the employed concentrations⁴⁸. (c) Plot of IC50 values for PTX cytotoxicity against PC3 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. The IC50 decreases (efficacy increases) with increasing PTX content in both DOTAP/DOPC/PTX and DLinTAP/DLinPC/PTX formulations. The efficacy of DLinTAP/DLinPC/PTX CLs is higher than that of DOTAP/DOPC/PTX CLs at all PTX contents except 2 mol%, but difference is not significant. This shows that replacing oleoyl with linoleoyl tails maintains cytotoxic efficacy against PC3 cells while increasing PTX membrane solubility (Fig. 4).