Figure 5.

Uptake, lysosomal delivery and dissolution of synthetic AMCP nanoparticles.

PMA-differentiated THP-1 macrophages were incubated with calcein-labeled AMCP for 3 h and then washed to remove residual AMCP particles. Images were then acquired every 2 h by the Incucyte™. (A-C) Representative images from the green channel (right hand-side) and overlay images of phase contrast and green fluorescence (left hand-side) that were acquired one hour-post stimulation with (A) TCM, (B) AMCP and (C) 11 h-post stimulation with AMCP (10× magnification). (D) Particle count, as quantified by the IncucyteZOOM software, was normalized against cell confluence, and is expressed as percent of values obtained in the first scan. X-axis indicates monitoring time by live cell imaging post-stimulation, i.e., 0 h represents the first scan (i.e. straight after 3 h stimulation with calcein-labeled AMCP). Data are presented as mean ± SD (n = 5, obtained from two independent experiments performed in technical replicates). (E) Mean green fluorescence intensity of soluble calcein added at indicated concentrations to tissue culture medium (acellular environment) and measured by the Incucyte™. Dye quenching was induced by the addition of 1 mM iron as iron hydroxide adipate tartrate. Data are shown as connecting curves for the mean of technical triplicates from one experiment. (F) Image Stream summary of internalized synthetic calcein-labeled AMCP in peripheral blood monocytes (left column) that were concomitantly positive for the lysosomal marker CD107 (Right column; n = 4 ± SD).