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Cell Adhesion Motif-Functionalized Lipopeptides: Nanostructure and Selective Myoblast Cytocompatibility
The conformation and self-assembly of four lipopeptides,
peptide
amphiphiles comprising peptides conjugated to lipid chains, in aqueous
solution have been examined. The peptide sequence in all four lipopeptides
contains the integrin cell adhesion RGDS motif, and the cytocompatibility
of the lipopeptides is also analyzed. Lipopeptides have either tetradecyl
(C14, myristyl) or hexadecyl (C16, palmitoyl)
lipid chains and peptide sequence WGGRGDS or GGGRGDS, that is, with
either a tryptophan-containing WGG or triglycine GGG tripeptide spacer
between the bioactive peptide motif and the alkyl chain. All four
lipopeptides self-assemble above a critical aggregation concentration
(CAC), determined through several comparative methods using circular
dichroism (CD) and fluorescence. Spectroscopic methods [CD and Fourier
transform infrared (FTIR) spectroscopy] show the presence of β-sheet
structures, consistent with the extended nanotape, helical ribbon,
and nanotube structures observed by cryogenic transmission electron
microscopy (cryo-TEM). The high-quality cryo-TEM images clearly show
the coexistence of helically twisted ribbon and nanotube structures
for C14-WGGRGDS, which highlight the mechanism of nanotube
formation by the closure of the ribbons. Small-angle X-ray scattering
shows that the nanotapes comprise highly interdigitated peptide bilayers,
which are also present in the walls of the nanotubes. Hydrogel formation
was observed at sufficiently high concentrations or could be induced
by a heat/cool protocol at lower concentrations. Birefringence due
to nematic phase formation was observed for several of the lipopeptides,
along with spontaneous flow alignment of the lyotropic liquid crystal
structure in capillaries. Cell viability assays were performed using
both L929 fibroblasts and C2C12 myoblasts to examine the potential
uses of the lipopeptides in tissue engineering, with a specific focus
on application to cultured (lab-grown) meat, based on myoblast cytocompatibility.
Indeed, significantly higher cytocompatibility of myoblasts was observed
for all four lipopeptides compared to that for fibroblasts, in particular
at a lipopeptide concentration below the CAC. Cytocompatibility could
also be improved using hydrogels as cell supports for fibroblasts
or myoblasts. Our work highlights that precision control of peptide
sequences using bulky aromatic residues within “linker sequences”
along with alkyl chain selection can be used to tune the self-assembled
nanostructure. In addition, the RGDS-based lipopeptides show promise
as materials for tissue engineering, especially those of muscle precursor
cells