MicroRNA-223 Induced Repolarization of Peritoneal Macrophages Using CD44 Targeting Hyaluronic Acid Nanoparticles for Anti-Inflammatory Effects

Abstract

The aim of this study was to evaluate macrophages repolarization from pro-inflammatory M1 to anti-inflammatory M2 phenotype upon transfection with microRNA-223 (miR-223) duplexes and miR-223 expressing plasmid DNA encapsulated in CD44-targeting hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles (NPs). The HA-PEI/miR-223 NPs with spherical shape and an average diameter of 200 nm were efficiently internalized by J774A.1 alveolar and primary peritoneal macrophages and non-cytotoxic at HA-PEI concentration less than 200 μg/mL. Transfection of HA-PEI/miR-223 NPs in J774A.1 macrophages showed significantly higher miR-223 expression than that with HA-PEI/plasmid DNA expressing miR-223 (pDNA-miR-223). HA-PEI/miR-223 NPs mediated transfection increased miR-223 expression to 90 fold in primary peritoneal macrophages compared to untreated cells. The overexpression of miR-223 in both J774A.1 and peritoneal macrophages induced a phenotypic change from M1 to M2 state as indicated by a decrease in iNOS-2 (M1 marker) and an increase in Arg-1 (M2 marker) levels compared to those in lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)-stimulated macrophages (M1). The change in macrophage phenotype by HA-PEI/miR-223 NPs could suppress the inflammation in peritoneal macrophages induced by LPS as evidenced by a significant decrease in pro-inflammatory cytokine levels TNF-α, IL-1β and IL-6, compared to LPS-stimulated peritoneal macrophages without treatment. The results demonstrated that miR-223-encapsulated HA-PEI NPs modulated macrophage polarity toward an anti-inflammatory M2 phenotype, which has potential for the treatment of inflammatory diseases.

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. In vitro polarization study.

(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.