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Abstract: 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.

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Abstract: Short bioactive peptide sequences are of great interest in biomaterials development. We investigate the self-assembly of a lipopeptide containing both the highly cationic CSK4 toll-like receptor agonist hexapeptide sequence and RGDS integrin-binding motif, i.e., C16-CSK4RGDS, as well as the control containing a scrambled terminal sequence C16-CSK4GRDS. Both lipopeptides are found to form micelles, as revealed by small-angle X-ray scattering and cryogenic transmission electron microscopy, and modeled using atomistic molecular dynamics simulations. We carefully examined methods to probe the aggregation of the molecules, i.e. to obtain the critical micelle concentration (CMC). Fluorescent probe assays using 1-anilino-8-naphthalenesulfonate (ANS) reveal low CMC values, 1–2 μM, which contrast with consistent values more than 2 orders of magnitude larger obtained from surface tension and electrical conductivity as well as unexpected UV/vis absorption spectra discontinuities and fluoresccence probe assays using Nile red. The anomalous results obtained from an ANS fluorescence probe are ascribed to the effect of ANS binding to the cationic (lysine and arginine) residues in the lipopeptide, which leads to a conformational change, as shown by circular dichroism, even at low concentrations below the actual CMC. Despite the small change in the peptide sequence (swapping of G and R residues), there is surprisingly a significant difference in the aggregation propensity and association number, both of which are greater for C16-CSK4GRDS. Both lipopeptides are cytocompatible (with fibroblasts and myoblasts) at low concentration, although cytotoxicity is noted at higher concentration.

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Abstract: Lipopeptides can self-assemble into diverse nanostructures which can be programmed to incorporate peptide sequences to achieve a remarkable range of bioactivities. Here, the influence of peptide sequence and chirality on micelle structure and interactions is investigated in a series of lipopeptides bearing two lysine or D-lysine residues and tyrosine or tryptophan residues, attached to a hexadecyl lipid chain. All molecules self-assemble into micelles above a critical micelle concentration (CMC). Small-angle x-ray scattering (SAXS) is used to probe micelle shape and structure from the form factor and to probe inter-micellar interactions via analysis of structure factor. The CMC is obtained consistently from surface tension and electrical conductivity measurements. We introduce a method to obtain the zeta potential from the SAXS structure factor which is in good agreement with directly measured values. Atomistic molecular dynamics simulations provide insights into molecular packing and conformation within the lipopeptide micelles which constitute model self-assembling colloidal systems and biomaterials.

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Abstract: The conformation and self-assembly of two pairs of model lipidated tripeptides in aqueous solution are probed using a combination of spectroscopic methods along with cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). The palmitoylated lipopeptides comprise C16-YKK or C16-WKK (with two l-lysine residues) or their respective derivatives containing d-lysine (k), i.e., C16-Ykk and C16-Wkk. All four molecules self-assemble into spherical micelles which show structure factor effects in SAXS profiles due to intermicellar packing in aqueous solution. Consistent with micellar structures, the tripeptides in the coronas have a largely unordered conformation, as probed using spectroscopic methods. The molecules are found to have good cytocompatibility with fibroblasts at sufficiently low concentrations, although some loss of cell viability is noted at the highest concentrations examined (above the critical aggregation concentration of the lipopeptides, determined from fluorescence dye probe measurements). Preliminary tests also showed antimicrobial activity against both Gram-negative and Gram-positive bacteria.

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Abstract: Bradykinin (BK) is a peptide hormone that plays a crucial role in blood pressure control, regulates inflammation in the human body, and has recently been implicated in the pathophysiology of COVID-19. In this study, we report a strategy for fabricating highly ordered 1D nanostructures of BK using DNA fragments as a template for self-assembly. We have combined synchrotron small-angle X-ray scattering and high-resolution microscopy to provide insights into the nanoscale structure of BK-DNA complexes, unveiling the formation of ordered nanofibrils. Fluorescence assays hint that BK is more efficient at displacing minor-groove binders in comparison with base-intercalant dyes, thus, suggesting that interaction with DNA strands is mediated by electrostatic attraction between cationic groups at BK and the high negative electron density of minor-grooves. Our data also revealed an intriguing finding that BK-DNA complexes can induce a limited uptake of nucleotides by HEK-293t cells, which is a feature that has not been previously reported for BK. Moreover, we observed that the complexes retained the native bioactivity of BK, including the ability to modulate Ca2+ response into endothelial HUVEC cells. Overall, the findings presented here demonstrate a promising strategy for the fabrication of fibrillar structures of BK using DNA as a template, which keep bioactivity features of the native peptide and may have implications in the development of nanotherapeutics for hypertension and related disorders.