Fig 2: Nano-formation-controlled attachment and polarization of macrophages include ROCK. a Fluorescent images of macrophages immunostained for ROCK2 (in green) and nuclei (in blue) in macrophages after 24 h after culture with basal medium, 36 h after M1-polarizing culture, or 36 h after M2-polarzing culture. b Fluorescent images of macrophages stained for actin (in red) and nuclei (in blue) under basal medium culture for 24 h, iNOS (in green), Arg-1 (in red), and nuclei (in blue) under M1-polarizing culture for 36 h, or Arg-1 (in green), iNOS (in red), and nuclei (in blue) under M2-polarzing culture for 36 h. The dimer formation with the Mg2+-BP NPs (MgBNP dimer) or the RGD-Mg2+-BP NPs (RGDBNP dimer) was directed after 0 h under culture. Cells in the “RGDBNP” dimer was also cultured with the ROCK-inhibiting drug, Y27632 (RGDBNP + Y27632). Scale bars are 50 µm. The representative areas and elongation factors of attached macrophages were determined after culture under basal medium. Data are means ± s.e.m. (n = 10). Expression levels of M2-type markers (Arg-1 and Ym2 genes) determined by RT-qPCR, after cell culture under M2-type-polarizing medium. Data are means ± s.e.m. (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA)

Fig 3: RGD-presenting dimer formation promotes M2-type polarization of macrophages. a Expression levels of M2-type markers (Arg-1 and Ym2 genes) determined by RT-qPCR, and b fluorescent images of macrophages immunostained for Arg-1 (in green), iNOS (in red), and nuclei (in blue) after 36 h under culture (the first 12 h under culture with basal medium, followed by 24 h under culture with M2-type-polarizing medium). The dimer formation with the Mg2+-BP NPs (MgBNP dimer) or the RGD-Mg2+-BP NPs (RGDBNP dimer) was directed after 0 h under culture. The time-controlled dimer formation was directed with the Mg2+-BP NP formation after 0 h, followed by the RGD-Mg2+-BP NP formation after 12 h (MgBNP-RGDBNP dimer). Data are means ± s.e.m. (n = 3). **P < 0.01, ***P < 0.001 (ANOVA). Scale bar is 50 µm

Fig 4: The dimer formation facilitates M2-type macrophages in vivo with M2 stimulators. a Schematic representation of the “BNP” substrate implanted into a subcutaneous pocket, which was then subjected to dimer formation by the administration of BP, Mg2+, or RGD-BP followed by M2-type-polarizing inducers (IL-4 and IL-13 cytokines). Five mice were used per group. b Fluorescent images of the attached host cells, immunostained for actin (green), Arg-1 (red), and nuclei (blue) at 24 h post implantation. Scale bar is 20 µm. c The representative densities, areas, and elongation factors of the attached macrophages were determined, which were randomly chosen, at 24 h post implantation. Data are means ± s.d. (n = 4 for densities). Data are means ± s.e.m. (n = 10 for areas and elongation factors). Expression levels of M2-type markers (Arg-1 and Ym2 genes) determined by RT-qPCR, for the attached host cells, which were randomly chosen, at 24 h post implantation. Data are means ± s.e.m. (n = 3). *P < 0.05, ***P < 0.001 (two-tailed Student’s t-test)