Several new classes of pyridinium cationic lipids were synthesized and tested as gene delivery agents. They were obtained through a procedure that generates simultaneously the heterocyclic ring and the positively charged nitrogen atom, using lipophilic pyrylium salts as key intermediates that react with primary amines, yielding pyridinium salts. The choice of the appropriately substituted primary amine, diamine or polyamine, allows the design of the shape of the final lipids, gemini surfactants, or lipophilic polycations. We report also a comprehensive structure-activity relationship study that identified the most efficient structural variables at the levels of the hydrophobic anchor, linker, and counterion for these classes of pyridinium cationic lipids. This study was also aimed at finding the best liposomal formulation for the new transfection agents.

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LL-23 is a natural peptide corresponding to the N-terminal 23 amino acid residues of human host defense cathelicidin LL-37. LL-23 demonstrated, compared to LL-37, a conserved ability to induce the chemokine MCP-1 in human peripheral blood mononuclear cells, a lack of ability to suppress induction of the pro-inflammatory cytokine TNF-α in response to bacterial lipopolysaccharides (LPS), and reduced antimicrobial activity. Heteronuclear multidimensional NMR characterization of LL-23 revealed similar secondary structures and backbone dynamics in three membrane-mimetic micelles: SDS, dodecylphosphocholine (DPC), and dioctanoylphosphatidylglycerol. The NMR structure of LL-23 determined in perdeuterated DPC contained a unique serine that segregated the hydrophobic surface of the amphipathic helix into two domains. To gain further understanding, Ser9 of LL-23 was changed to either Ala or Val based on homologous primate cathelicidins. These changes made the hydrophobic surface of LL-23 continuous and enhanced antibacterial activity. While identical helical structures did not explain the altered activities, a reduced hydrogen-deuterium exchange from LL-23, LL-23A9, to LL-23V9 suggested a deeper penetration of LL-23V9 into the interior of the micelles, which correlated with enhanced activities. Moreover, these LL-23 variants had discrete immunomodulatory activities. Both restored the TNF-α dampening activity to the level of LL-37. Furthermore, LL-23A9, like LL-23, maintained superior protective MCP-1 production, while LL-23V9 was strongly immunosuppressive, preventing baseline MCP-1 induction and substantially reducing LPS-stimulated MCP-1 production. Thus, these LL-23 variants, designed based on a structural hot spot, are promising immune modulators that are easier to synthesize and less toxic to mammalian cells than the parent peptide LL-37.

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