Figure 1.
PRINT Particle Characterization.
A) Dynamic light scattering (DLS) and zeta potential measurements of PLGA particles used in studies. Particle charge decreases with increasing size. B) Scanning electron microscope (SEM) images of PLGA particles. C) PEG particle composition and characterization. D) SEM of PEG particles. E) Confocal images of hydrogel particle uptake in MH-S alveolar macrophage cells after 4 hours of treatment. Scale bar is 50 µm.
Figure 2.
PRINT particles do not cause inflammation in bone marrow-derived macrophages from BALB/c or C57BL/6 mice.
A) Overnight stimulation with a panel of PRINT PLGA and hydrogel particles (PEG) at 100 µg/ml does not cause TNF-α, IL-6, or IL-1β release from bone marrow-derived macrophages from C57BL/6 mice as measured by ELISA. B) Both PLGA and hydrogel PRINT particles (PEG) tested negative for endotoxin contamination using a Limulus amebocyte lysate assay. C) PRINT particles are not cytotoxic in bone-marrow derived macrophages as determined by lactate dehydrogenase (LDH) release. D) 80×320 nm PLGA particles do not synergize with LPS to induce inflammasome activation as measured by IL-1β ELISA in BALB/c bone-marrow derived macrophages. E) Neither 80×320 nm nor 1 µm PLGA particles synergize with LPS to induce inflammasome activation as measured by IL-1β ELISA in C57BL/6 bone-marrow derived macrophages. MSU was dosed at 300 µg/ml. *** = p<0.001. Experiments were performed in triplicate. Data shown are representative of at least three independent experiments.
Figure 3.
80×320 nm PLGA particles do not cause lung inflammation in mice.
Mice were challenged with either 50 µg of 80×320 nm PLGA particles or 20 µg LPS i.t. and airway inflammation was assessed 48 hours post-challenge. A) Total cellularity of bronchoalveolar lavage fluid (BALF) in treated C57BL/6 mice is no different after 48 hours than PBS-treated mice and is significantly less than the inflammatory cell recruitment seen in LPS-treated mice. B) PLGA particle treatment does not induce any appreciable immune cell recruitment to the lungs of mice, as opposed to the heightened levels of monocytes and neutrophils seen in the lungs of LPS-treated mice. C) Histopathology revealed no significant differences in lung architecture between PBS- and 80×320 nm PLGA particle-treated mice. This is in stark contrast to the airway occlusion and significant innate immune cell recruitment seen in LPS-treated mice. D) Histopathology scoring confirmed that no significant differences were seen between the lungs of PBS and PLGA particle treated mice. E–F) The increased lung levels of pro-inflammatory IL-1β and IL-6 seen in LPS-treated mice is not found in PLGA-treated mice. PBS, n = 3; 80×320 nm PLGA particle-treated, n = 5; LPS-treated, n = 3. ND = Not Detected. * = p<0.05, *** = p<0.001. Experiments were performed using 3–5 mice per group. Data shown are representative of at least two independent experiments.
Figure 4.
Hydrogel particles do not cause lung inflammation in mice.
Mice were challenged with 50 µg of hydrogel particles (80×320 nm, 1.5 µm, or 6.0 µm donuts) i.t. and airway inflammation was assessed 48 hours and 7 days post-challenge. A) BALF analysis indicated no increased cellularity 48 hours after hydrogel particle treatment, whereas a significant cellular influx was seen in LPS-treated controls. B) At 48 hours, BALF cellular composition does not show any significant trend for immune cell recruitment in hydrogel particle-treated mice. C–D) BALF cellularity and composition was not significantly augmented seven days after hydrogel particle treatment. E) Histopathology analysis revealed no significant differences in lung architecture between PBS- and hydrogel particle-treated mice at either 2 or 7 days post-treatment. F) Histopathology scoring confirmed that no significant differences were seen between the lungs of PBS and hydrogel particle treated mice at any time points. *** = p<0.001. Experiments were performed using 2–5 mice per group. Data shown are representative of at least two independent experiments.
Figure 5.
Hydrogel particles remain in the lungs for multiple days without overt signs of inflammation.
A) 6 µm hydrogel particles (denoted by red arrows) are visible in the alveolar spaces 2 days after intratracheal installation. Lower insets are a magnified view of black bounding box. PBS treated mice are shown as control. B) Multiple 6 µm hydrogel particles (denoted by black bounding box and red arrows) are visible in the alveolar spaces 7 days after intratracheal installation. C) Two days after treatment with hydrogel particles, BALF cells were stained and visualized for particle uptake via epifluorescence microscopy. Particles (Dylight 650, red); nuclei (DAPI, blue); F-actin (Phalloidin 488, green). D) Magnified views of BALF cells taking up hydrogel particles as denoted by white bounding boxes in Figure C. E) Quantification of particle uptake indicates smaller particles are more readily taken up in BALF cells than larger particles. F) All types of hydrogel particles can still be seen in BALF cells seven days after treatment, though there is a marked decrease in the number of particles present as compared to the 2 day time point. G) Magnified views of BALF cells taking up hydrogel particles 7 days after treatment as denoted by white bounding boxes in Figure C. Scale bar is 20 µm. Data shown are representative of at least two independent experiments.