In vitro characterization of LPS-NP and the associated pro-inflammatory properties. A) Scanning electron microscopic examination of the LPS-NP morphology (scale bar represents 1 µm). B) LPS release from the nanoparticles under sink conditions. C) LC50 values calculated based on the dose-response fitting of cell survival values following the overnight incubation of 4 × 105 RAW 264.7 macrophages or JAWS II DCs with different concentrations of LPS and LPS-NP. D) Co-localization of LPS and nanoparticles following overnight incubation of macrophages with LPS-NP (Pearson’s correlation for 50 cells: 0.8666 ± 0.03964, Mander’s overlap for 50 cells: 0.8693 ± 0.0396). Blue, red, green, and yellow colors represent the cell nucleus, nanoparticles, LPS, and the co-localization of the LPS and nanoparticles, respectively. Scale bars represent 10 µm. E) Induction of NF-κBp65 in 5 × 106 RAW 264.7 macrophages following 6 h incubation with LPS/LPS-NP. RAW 264.7 cells with TLR4 blocked signaling have been used as control to enable the determination of the TLR4-independent induction of NF-κBp65, potentially related to the PLGA matrix or impurities. F) Induction of pro-inflammatory cytokines following the overnight incubation of 4 × 105 RAW 264.7 macrophages or JAWS II DCs with LPS, LPS-NP, and blank PLGA nanoparticles with polymer concentrations corresponding to those of LPS-NP.