Figure 2.

In vitro cumulative release kinetics, cellular uptake, DC activation, and cytotoxicity of the empty and drug-loaded NPs. (A) NP release kinetics of encapsulated drugs simulated at 37°C in PBS and kept in a thermo-shaker at a constant shaking velocity. n = 3 from one representative experiment. (B) Uptake of NPs containing NIR dye (800 nm) by TC-1 cells (To-pro 3 iodide; 700 nm) over the times indicated. n = 3 from one representative experiment. (C) Uptake of NPs by TC-1 cells after 2 hours of incubation, shown by fluorescence microscopy. Red: cell membrane; purple: cell nucleus; green: NIR dye. (D) Activation of DCs measured by CD86 expression upon 48 hours incubation with NP(pIC+R848+MIP3α). NP(empty) and isotype controls are shown in red and grey, respectively. The cells were pooled from n = 3 from each condition, one representative out of three independent experiments. (E) Activation of DCs measured by the secretion of IL-12p40 upon 48 hours incubation with NP(pIC+R848+MIP3α). NP(empty) and pIC controls are shown in red and black, respectively. n = 3 from one representative out of three independent experiments. (F) Migration assessment using Boyden chamber assay. After 24 hours of pre-incubation of the lower chamber with either MIP3α (in solution) or NP(pIC+R848+MIP3α), RAW264.7 cells were added to the upper chamber and allowed to migrate for 24 hours. Medium was used as a negative control. n = 3 from one representative out of two independent experiments. (G) Cytotoxicity measurement of empty NPs on DCs incubated with increasing concentrations for 48 hours. The cytotoxic compound DMSO (black bar) was used as a positive control (100 percent of cell death). (H+I) Cell viability assessed by MTS cell proliferation assay upon 72 hours incubation with indicated compounds on TC-1 (H) or MC-38 (I) cells. n = 3 from one representative out of four independent experiments. All data are presented as mean ± SD.