Abstract/Summary

Toll-like receptor (TLR) agonists are of interest in immunotherapy and cancer vaccines. The most common agonists of TLR2 are based on Pam2Cys or Pam3Cys. In the former, two palmitoyl (Pam) fatty acids are linked to a glycerylcysteine motif by ester linkages. Pam3Cys is analogous but contains an extra Pam group on the α-amine. Here, we compare the self-assembly in aqueous solution of the parent Pam2CysOH and Pam3Cys amino acid conjugates to that of Pam2CysSK4 and Pam3CysSK4 which are potent TLR2 agonists bearing the CysSK4 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. Pam2CysOH and Pam3CysOH 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 Pam2CysSK4 and Pam3CysSK4, which are spherical micelles or predominantly wormlike micelles, respectively [Hamley, I. W.; et al. Toll-like Receptor Agonist Lipopeptides Self-Assemble into Distinct Nanostructures. Chem. Comm. 2014, 50, 15948-15951]. We also examine the effect of introduction in the bulky N-terminal Fmoc [fluorenylmethoxycarbonyl] group on the self-assembly of Fmoc-Pam2CysOH. This compound also forms vesicles (above a critical aggregation concentration, determined from dye probe fluorescence experiments) in aqueous solution, larger than those for Pam2CysOH and with a population of perforated/compound vesicles. The carboxyl-coated (and amino-coated for Pam2CysOH) vesicles demonstrated here represent a promising system for future development toward bionanotechnology applications such as immune therapies. Conjugates Pam2CysOH, Pam2CysSK4, and Pam3CysSK4 show good cytocompatibility at low concentrations, and in fact, the cell compatibility extends over a wider concentration range for Pam2CysOH. 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 Pam2CySOH shows significant activity, although not to the extent of Pam2CysSK4 or Pam3CysSK4. Thus, Pam2CysOH in particular is of interest as a vesicle-forming TLR2/6 agonist and stimulator of immune response.