Figure 4.

Lapatinib/fulvestrant colloids are cytotoxic after endocytosis and subsequent endosome disruption. Colloidal drug aggregates were formulated as described in Table S3, with the addition of 2 μM 7-aminoactinomycin D (7-AAD) to monitor endosome disruption in those experiments. Colloids composed of 50 μM lapatinib and 150 μM fulvestrant and stabilized with transferrin resulted in greater (A) cytotoxicity and (B) endosomal disruption (measured by amount of nuclear dye) than either co-colloids stabilized with PLAC-PEG or colloids of fulvestrant alone (150 μM fulvestrant plus stabilizer) (n = 9 biological replicates and separate colloid formulations for the toxicity experiment and n = 3 for the endosome disruption experiment, mean ± SD, two-way ANOVA with Tukey’s posthoc test, *p < 0.05, **p < 0.01, ****p < 0.0001). (C) Schematic describing how the membrane-impermeant nucleic acid stain 7-AAD was used to test for endosomal escape. (D) Nuclear 7-AAD fluorescence (red) is visible after treatment with lapatinib-fulvestrant colloids stabilized with transferrin, demonstrating its endosomal escape. (E) Permeabilized cells accumulate 7-AAD (red) in their nuclei as expected, whereas (F) live cells treated with 7-AAD alone do not. The presence of cells in each region of interest was verified using the transmission channel prior to capturing these images.