Engineering of L-Plastin Peptide-Loaded Biodegradable Nanoparticles for Sustained Delivery and Suppression of Osteoclast Function In Vitro

Figure 6

Confocal microscopy analyses of osteoclasts stained for filamentous (F-) actin with phalloidin. (A) Actin staining of osteoclasts with rhodamine phalloidin: osteoclasts were transduced with TAT-fused sNT-LPL peptides (P1—panels a, b, & I; P3—panels c, d, & j) or treated with nanoparticles loaded with peptides (NP1—panels e, f, & k; NP3—panels g, h, & l). After treatment cells were stained for actin (red) to determine the formation of nascent sealing zones (NSZs) and sealing rings (SR). Arrowheads point to actin punctate stainings; arrows point to NSZs; wavy arrows point to mature sealing rings. Osteoclasts plated on coverslips and treated as indicated above with peptides, and bone particles were stained with rhodamine phalloidin. An asterisk indicates podosome localization at the cell periphery. Scale bar 150μm. These results represent one of the three experiments performed with similar results. (B)-(F) Statistical analyses of the number of NSZs and sealing rings (SRs). The number of NSZs and SRs was counted in 75-80 osteoclasts from three experiments and presented as graphs (B=E) and table  (F). The treatments are shown below each graph. The number of NSZs and SRs is presented per osteoclasts in graphs shown in (B) and (D). Data are also given as scatterplots for the indicated number of osteoclasts ((C) and (E)). The total number of NSZs and SRs is also provided for the indicated peptides and the number of osteoclasts in Table  (F). The effect of P1 or NP1 is significant in the inhibition of NSZs and SRs as compared with respective control groups (P3 or NP3). p<0.01; p<0.001 versus respective control groups (P3 or NP3).