Chiral gold nanoparticles manipulate osteoimmune microenvironment via macrophage autophagy for bone regeneration

Abstract

The immune microenvironment orchestrates bone regeneration, which can be manipulated by macrophage autophagy. Herein, gold nanoparticles with L/D (left-handed or right-handed)-chirality (L/D-AuNPs) were synthesized with chiral glutathione ligands to stereoselectively regulate macrophage autophagy, and further manipulate immune microenvironment for bone regeneration. Notably, L-AuNPs exhibited superior macrophage uptake efficiency and higher autophagy level compared to D-AuNPs. Meanwhile, L-AuNPs improved the osteogenic microenvironment, further promoting bone regeneration. In addition, the in vivo results showed that the healing of skull defect was significantly enhanced by L-AuNPs. These findings demonstrated that chiral AuNPs can stereoselectively regulate macrophage autophagy and open an avenue for applications of chiral nanoparticles in osteoimmunity.

Keywords: Autophagy; Bone regeneration; Chirality; Gold nanoparticles; Macrophage.

Graphical abstract

Scheme 1

Schematic diagram of L/D-AuNPs. L-AuNPs exhibit enhanced macrophage uptake and increase autophagy levels, which further promotes M2 polarization and facilitates bone regeneration. In contrast, D-AuNPs suppress autophagy, tending to induce M1 polarization without significantly enhancing bone regeneration.

Fig. 1

Characterization of L/D-AuNPs. (A, B) TEM images of L/D-AuNPs, the inset exhibits size distribution histograms of L/D-AuNPs (n = 100 nanoparticles). (C, D) CD spectra (C) and g-factor (D) of L/D-AuNPs.

Fig. 2

Cellular uptake and autophagy of macrophage with L/D-AuNPs treatment. (A) Confocal images of RAW264.7 after treatment with L/D-AuNPs (5 μM) for 3 h (L/D-AuNPs: green; nuclear: blue). (B–E) Western blot analysis of Beclin1, p62 and LC3II/I expression in RAW264.7 after treatment with L/D-AuNPs. (F, G) Immunofluorescent images of RAW264.7 in each group (LC3/p62: green; nuclear: blue). All data were presented as the means ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig. 3

L/D-AuNPs regulated macrophage polarization. (A–D) Western blot analysis of CD206, Arg1 and iNOS expression in RAW264.7 after treatment with L/D-AuNPs for 48 h. (E) Immunofluorescent images of RAW264.7 cells treated by L/D-AuNPs for 48 h (iNOS: green; CD206: red; nuclear: blue). (F, G) Flow cytometry analysis of CD206 expression in RAW264.7 cells after L/D-AuNPs treatments for 48 h. All data were presented as the means ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig. 4

L/D-AuNPs regulated macrophage polarization via autophagy. RAW264.7 cells with or without 3-MA (2.5 μM) pretreatment for 6 h. (A) Immunofluorescent images of LC3/p62: green; nuclear: blue. (B, C) Western blot images and quantitative analysis of Arg-1 expression. (D) Immunofluorescent images of iNOS: green; CD206/Arg-1: red; nuclear: blue. All data were presented as the means ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig. 5

Chiral AuNPs regulated osteogenic differentiation via macrophage polarization in vitro. (A) Schematic representation of several macrophage-CMs for osteogenic induction of MC3T3-E1. (B–E) The relative mRNA levels of osteogenesis genes of MC3T3-E1 treated with CMs after 7 days. (F–I) Western blot analysis of OPN, OCN and RUNX2 expression in MC3T3-E1 after treatment with CMs after 7 days. (J) ALP staining photos of MC3T3-E1 treated with CMs after 7 days, followed by ARS staining after 21 days. (K) Quantitative assessment of ALP activity and mineralization in vitro. All data were presented as the means ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig. 6

Bone regeneration potential of chiral AuNPs in vivo. (A) Schematic diagram of detection bone regeneration potential of chiral AuNPs by the rat skull defect model. (B) Micro-CT and 3D reconstructed images for skull defects. (C–H) Quantitative analysis of bone-related parameters. (I) Representative HE, Masson and (J) osteogenic-relative protein immunofluorescent staining images of skull defects (OCN, OPN and RUNX2: green; nucleus: blue). All data were presented as the means ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.