Optimization of peptide amphiphile-lipid raft interaction by changing peptide amphiphile lipophilicity

Abstract

Various peptide amphiphile (PA) molecules have been developed to promote bone regeneration. Previously we discovered that a peptide amphiphile with a palmitic acid tail (C₁₆) attenuates the signaling threshold of leucine-rich amelogenin peptide (LRAP)-mediated Wnt activation by increasing membrane lipid raft mobility. In the current study, we found that treatment of murine ST2 cells with an inhibitor (Nystatin) or Caveolin-1-specific siRNA abolishes the effect of C₁₆ PA, indicating that Caveolin-mediated endocytosis is required. To determine whether hydrophobicity of the PA tail plays a role in its signaling effect, we modified the length of the tail (C₁₂, C₁₆ and C₂₂) or composition (cholesterol). While shortening the tail (C₁₂) decreased the signaling effect, lengthening the tail (C₂₂) had no prominent effect. On the other hand, the cholesterol PA displayed a similar function as the C₁₆ PA at the same concentration of 0.001% w/v. Interestingly, a higher concentration of C₁₆ PA (0.005%) is cytotoxic while cholesterol PA at the higher concentration (0.005%) is well-tolerated by cells. Use of the cholesterol PA at 0.005% enabled a further reduction of the signaling threshold of LRAP to 0.20 nM, compared to 0.25 nM at 0.001%. Caveolin-mediated endocytosis is also required for cholesterol PA, as evidenced by Caveolin-1 siRNA knockdown experiments. We further demonstrated that the noted effects of cholesterol PA are also observed in human bone marrow mesenchymal stem cells (BMMSCs). Taken together, these results indicate that the cholesterol PA modulates lipid raft/caveolar dynamics, thereby increasing receptor sensitivity for activation of canonical Wnt signaling. STATEMENT OF SIGNIFICANCE: Cell signaling involves not only the binding of growth factors (or other cytokines) and cognate receptors, but also their clustering on the cell membrane. However, little or no work has been directed thus far toward investigating how biomaterials can serve to enhance growth factor or peptide signaling by increasing diffusion of cell surface receptors within membrane lipid rafts. Therefore, a better understanding of the cellular and molecular mechanism(s) operating at the material-cell membrane interface during cell signaling has the potential to change the paradigm in designing future biomaterials and regenerative medicine therapeutics. In this study, we designed a peptide amphiphile (PA) with a cholesterol tail to enhance canonical Wnt signaling by modulating lipid raft/caveolar dynamics.

Keywords: Caveolin-mediated endocytosis; Lipid raft; Peptide amphiphile; Supramolecular nanofiber; Wnt signaling; leucine-rich amelogenin peptide (LRAP).