Figure 4.

Internalization and cargo release by calcein-encapsulating PEG- and pMPC-functionalized vesicles incubated with P. aeruginosa cells and biofilms. Representative microscopy images of PA14 cells following 4 h (A) and 24 h (B) incubation, followed by thorough washing, with PEG- (upper panels) and pMPC- (lower panels) calcein-loaded liposomes. Cells were grown at 37 °C for 24 h prior to the incubation with liposomes. Images are presented as a fluorescent intensity of the calcein signal (green) and overlay between calcein-fluorescence and brightfield. Insert in lower right panel (A) shows a zoomed-in detail of bacteria cells displaying a weak but recognizable fluorescent intensity. Scale bars: main, 10 μm; insert, 1 μm. (C) Quantification of single cell microscopy images of the number of cells displaying luminal calcein signal following 24 h incubation of cells with either PEG-LUVs or pMPC-LUVs. Differences between groups shown in the box plot were tested with a one-way ANOVA. Boxes represent the 25–75 percentiles of the sample distribution, with black vertical lines representing the 1.5 × IQR (interquartile range). Black horizontal line represents the median. (D) and (E) Kinetic profile of calcein release from liposomes upon interaction with bacterial biofilms of strain PA14 (D) or LESB58 (E) at 37 °C incubated for 17 h. Liposomes were either nonfunctionalized (brown circles) or functionalized with 5% pMPC (red squares) and 5% PEG (green triangles) polymer. Calcein-loaded pMPC- LUVs were incubated with naive BM2G media as a negative control (blue circles). Results are an average of a minimum of three experiments.