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

Abstract

We have recently demonstrated that a small molecular weight amino-terminal peptide of L-plastin (10 amino acids; "MARGSVSDEE") suppressed the phosphorylation of endogenous L-plastin. Therefore, the formation of nascent sealing zones (NSZs) and bone resorption are reduced. The aim of this study was to develop a biodegradable and biocompatible PLGA nanocarrier that could be loaded with the L-plastin peptide of interest and determine the efficacy in vitro in osteoclast cultures. L-plastin MARGSVSDEE (P1) and scrambled control (P3) peptide-loaded PLGA-PEG nanoparticles (NP1 and NP3, respectively) were synthesized by double emulsion technique. The biological effect of nanoparticles on osteoclasts was evaluated by immunoprecipitation, immunoblotting, rhodamine-phalloidin staining of actin filaments, and pit forming assays. Physical characterization of well-dispersed NP1 and NP3 demonstrated ~130-150 nm size, < 0.07 polydispersity index, ~-3 mV ζ-potential, and a sustained release of the peptide for three weeks. Biological characterization in osteoclast cultures demonstrated the following: NP1 significantly reduced (a) endogenous L-plastin phosphorylation; (b) formation of NSZs and sealing rings; (c) resorption. However, the assembly of podosomes which are critical for cell adhesion was not affected. L-plastin peptide-loaded PLGA-PEG nanocarriers have promising potential for the treatment of diseases associated with bone loss. Future studies will use this sustained release of peptide strategy to systematically suppress osteoclast bone resorption activity in vivo in mouse models demonstrating bone loss.

Figure 1

Immunoblotting analyses with an L-plastin (LPL) and a p-Serine antibody. (a) Analysis of time-dependent expression of LPL in osteoclasts treated with (+; lanes 2-5) and without (-; lane 1) bone particles in the presence of TNF-α. (a) Immunoblotting analysis with an antibody to LPL (top panel) and GAPDH (bottom panel). (b) Amino acid sequences of TAT (11aa) -fused sNT-LPL (10aa) and control TAT alone (11aa) peptides are shown: P1) unsubstituted (S5S7); P2) double substituted (S5S7 to A5A7); P3) scrambled; P4) control TAT sequences only. (c). Immunoprecipitation and immunoblotting analyses. The effect of indicated sNT-LPL peptides (P1 to P4) on the phosphorylation of endogenous LPL is shown. An equal amount of osteoclast lysates was immunoprecipitated with an antibody to LPL and subjected to immunoblotting (IB) with a p-Serine antibody (top). This blot was stripped and blotted with an LPL antibody (panel (c); middle). Immunoprecipitation of lysates made from P2-transduced cells with a species-specific nonimmune serum was used as a control for immunoprecipitation (lane 5). An equal amount of total protein (Input) used for immunoprecipitation was assessed by direct immunoblotting of lysates with a GAPDH antibody. The experiment was repeated thrice and obtained comparable inhibitory effect with P1.

Figure 2

Nanoparticle size distribution and morphology. A narrow size distribution of PLGA-PEG_P1 (a) and PLGA-PEG_P3 (b) nanoparticles measured by dynamic light scattering is shown. (c) and (d) Transmission electron microscopy (TEM) images show well-dispersed round shaped PLGA-PEG_P1 (c) and PLGA-PEG_P3 nanoparticles (d). Scale bars = 200 nm.

Figure 3

In vitro release profile of peptide from PLGA-PEG nanoparticles. Graphical representation of in vitro release of PLGA-PEG_P1 and PLGA-PEG_P3 plotted as a function of percent peptide release versus days from PLGA-PEG nanoparticles. The graph represents one of the two experiments performed.

Figure 4

Immunoblotting (IB) analysis of the time-dependent effect of NP1 and NP3 on the phosphorylation of endogenous LPL. The equal amount of lysates made from osteoclasts treated with NP1 (lanes 1 and 3) and NP3 (lanes 3 and 4) for 4h (lanes 1 and 2) and 6h (lanes 3 and 4) was used for immunoprecipitation (IP) with an LPL antibody. IP with a nonimmune serum is shown in lane 5. Immunoprecipitates were subjected to IB with an antibody to p-Serine (top panel). Blot was stripped and reprobed with an antibody to LPL (bottom panel). Percent inhibition of phosphorylation of the representative experiment with NP1 is 78% at 4h and 53% at 6h as compared with corresponding NP3 control in lanes 2 and 4. These results represent one of the two experiments performed with similar results.

Figure 5

MTT assay and TRAP-staining of osteoclasts after treatment with TAT-fused sNT-LPL peptides (P1 and P3) and nanoparticles (NP1 and NP3). Osteoclasts were treated with indicated peptides or nanoparticles for 4h in the presence of TNF-α and bone particles. Next, cells were subjected to the calorimetric MTT assay as described in the Methods to determine the proliferation effects (a). TRAP staining was performed to determine the cell morphology or phenotype of osteoclasts (b). Osteoclasts untreated with peptides but treated with TNF-α and bone particles were used as controls (-). MTT assay confirmed that proliferation activity is not affected. Morphological characterization by TRAP-staining and attachment of osteoclasts to culture dishes suggest that cells are active and viable. Magnification-40X. These results represent one of the two experiments performed with similar results.

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).

Figure 7

Resorption assay using dentine matrix and immunoblotting analyses for resorption markers. Analysis of the effects of transduced TAT-LPL peptides (P1 and P3) and nanoparticles (NP1 and NP3) on resorption by osteoclasts using dentine slices. (a) Osteoclasts were cultured on dentine slices for 10–12h in the presence of TNF-α and indicated peptides. Osteoclasts untreated with any peptide but treated with TNF-α were used as controls (-). Pits were scanned in a Bio-Rad confocal microscopy. Scale bar- 25μm. Resorbed areas are seen as dark areas. (b) and (c) Statistic measurements of the pit area are provided as a graph: ∗∗∗p < 0.001 versus respective controls (P3 or NP3). The resorbed pit areas (8-10 pits/slice) were quantified, and data were compiled from three dentine slices per treatment (b). The data showed in the graph is the mean ± SD of one experiment performed. Pit area measurements are also given as scatterplots for the number of pits scanned (c). Experiments were repeated three times with three different osteoclast preparations. (d) Immunoblotting analyses: equal amount of lysate proteins (15μg) made from osteoclasts untreated or treated with peptides in the presence of TNF-α and bone particles for 12-14h was used for immunoblotting analyses with TRAP (top panel) and cathepsin K (Cath K; middle panel) antibodies. Immunoblotting with a GAPDH antibody was used as a loading control (bottom panel). Experiments were repeated twice.

Figure 8

Schematic representation of the effects of small MW L-plastin (LPL) peptides on osteoclast bone resorption. Biodegradable and biocompatible PLGA-PEG nanocarriers were used to deliver and release the small molecular weight L-plastin peptides (NP1 or NP3) of interest in a controlled and sustained fashion. Osteoclasts were incubated with these nanoparticles of interest in the presence of TNF-α. The released peptide P1 attenuated TNF-α induced phosphorylation of cellular LPL. Therefore, the formation of NSZs and sealing rings as well as resorption activity are significantly reduced (red arrows) in osteoclasts in vitro. No inhibition was found with the control peptide (P3). Sealing ring (SR) is indicated with an arrow in C.