Figure 5.

Drug release characterization and therapeutic activity of minicell_DOX biohybrids. a) pH‐responsive cumulative release of DOX encapsulated in minicells for a two‐week period at pH 4.6, 6.5 and 7.4. b) Fluorescence microscopy images of in vitro drug uptake of SK‐BR‐3 cells incubated with minicell_DOX for 0, 24 and 48 h. DOX inside the cells was shown by the red color. Scale bar: 20 µm. c) Quantification of mean DOX fluorescence intensity on SK‐BR‐3 cells over time. The mean ± SD for n ≥10, ^*** P = 0.0010, ^**** P < 0.0001, t‐test. d) The percent viability of SK‐BR‐3 cells incubated with free DOX, minicell biohybrids and minicell_DOX biohybrids with and without magnetic actuation at pH 6.5 for 24 h. The error bars represent the SD of the mean for n = 3; ns: not significant, ^** P < 0.0018, ^**** P < 0.0001 ANOVA. In all experiments shown in this figure, drug loading into minicells was performed by overnight incubation of minicells with DOX at a concentration of 40 µg mL⁻¹.