Fig 1.
(a) Schematic illustration of complexation of microRNA-223 (miR-223) with hyaluronic acid-poly(ethyleneimine) (HA-PEI) conjugate. (b) Size distribution of HA-PEI/miR-223 (9:1) nanoparticles (NPs) in phosphate-buffered saline (pH 7.4) by dynamic light scattering. (c) Transmission electron microscopy (TEM) image of HA-PEI/miR-223 NPs in PBS (9:1). (d) Agarose electrophoretic analysis of miR-223 duplex encapsulation in HA-PEI NPs with different polymer-to-miRNA weight ratios (27:1 and 9:1 w/w).
Fig 2.
Assessment of cellular toxicity as measured by viability of J774A.1 macrophages incubated with hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles at different concentrations from 1 μg/mL to 1,000 μg/mL for 24 hours.
Fig 3.
Fluorescence microscopy analysis of uptake of miR-223-encapsulated in hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles in J774A.1 alveolar and primary peritoneal macrophages and macrophages at 2 h post-incubation.
Prior to cellular uptake study, HA-PEI was conjugated with a red fluorescence dye, Cy5.
Fig 4.
(a) Comparative transfection study of hyaluronic acid-poly(ethyleneimine) (HA-PEI) / miR-223 duplexes nanoparticles (NPs) and HA-PEI/plasmid DNA expressing miR-223 (pDNA-miR-223) NPs in J774A.1 at 24 h, 48 h, and 72 h post-transfection. (b) miR-223 expression in primary peritoneal macrophages at 24 h and 48 h post-transfection of HA-PEI/miR-223 and Lipofectamine®/miR-223. miR-223 expression was quantified by Taqman miRNA assay specific for miR-223; U6snRNA was used as a house keeping gene. *p<0.05 compared to untreated macrophages, n = 3.
Fig 5.
(a) iNOS-2 (M1 marker) and Arg-1 (M2 marker) in M1 J774A.1 macrophages treated with hyaluronic acid-poly(ethyleneimine) (HA-PEI)/miR-223 duplexes nanoparticles (NPs) or HA-PEI/plasmid DNA expressing miR-223 ((pDNA-miR-223) NPs for 48 h. (b) iNOS-2 (M1 marker) and Arg-1 (M2 marker) in M1 peritoneal macrophages treated with HA-PEI/miR-223 or Lipofectamine®/miR-223 for 48 h. *p<0.05 compared to M1 macrophages which were obtained by stimulating macrophages with lipopolysaccharide (LPS, 100 ng/mL) combined with interferon-gamma (100 ng/mL) for 16 h, n = 3.
Fig 6.
In vitro anti-inflammatory study in peritoneal macrophages: expression of pro-inflammatory cytokines (a) TNF-α, (b) IL-1β, and (c) IL-6 mRNA level at 48 h post-transfection of macrophages with hyaluronic acid-poly(ethyleneimine) (HA-PEI)/miR-223 duplexes nanoparticles (NPs) or Lipofectamine®/miR-223, followed by stimulation with LPS (100 ng/mL) for 6 h.
qPCR was used to quantify mRNA levels. *p<0.05 compared to LPS-treated group, n = 3.