Self-Assembly of Toll-Like Receptor (TLR2/6) Agonist Lipidated Amino Acid or Peptide Conjugates: Distinct Morphologies and Bioactivities.

Toll-like receptor (TLR) agonists are of interest in immunotherapy and cancer vaccines. The most common agonists of TLR2 are based on PamCys or PamCys. In the former, two palmitoyl (Pam) fatty acids are linked to a glycerylcysteine motif by ester linkages. PamCys is analogous but contains an extra Pam group on the α-amine. Here, we compare the self-assembly in aqueous solution of the parent PamCysOH and PamCys amino acid conjugates to that of PamCysSK and PamCysSK which are potent TLR2 agonists bearing the CysSK peptide sequence. All four conjugates exhibit a critical aggregation concentration above which self-assembled structures are formed. We find through a combination of small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and confocal fluorescence microscopy remarkable differences in self-assembled nanostructures. PamCysOH and PamCysOH both form unilamellar vesicles, although these are larger for the latter compound, an effect ascribed to enhanced membrane rigidity. This is in contrast to previously reported morphologies for PamCysSK and PamCysSK4, which are spherical micelles or predominantly wormlike micelles, respectively [Hamley, I. W.; et al. . Chem. Comm. 2014, 50, 15948-15951]. We also examine the effect of introduction in the bulky -terminal Fmoc [fluorenylmethoxycarbonyl] group on the self-assembly of Fmoc-PamCysOH. This compound also forms vesicles (above a critical aggregation concentration, determined from dye probe fluorescence experiments) in aqueous solution, larger than those for PamCysOH and with a population of perforated/compound vesicles. The carboxyl-coated (and amino-coated for PamCysOH) vesicles demonstrated here represent a promising system for future development toward bionanotechnology applications such as immune therapies. Conjugates PamCysOH, PamCysSK, and PamCysSK show good cytocompatibility at low concentrations, and in fact, the cell compatibility extends over a wider concentration range for PamCysOH. The TLR2/6 agonist activity was assessed using an assay that probes secreted alkaline phosphatase (SEAP) in NF-κB-SEAP reporter HEK293 cells expressing human TLR2 and TLR6, and PamCySOH shows significant activity, although not to the extent of PamCysSK4 or PamCysSK. Thus, PamCysOH in particular is of interest as a vesicle-forming TLR2/6 agonist and stimulator of immune response.