In-vitro evaluation of cationic Lipopeptides as adjuvant candidate for DNA plasmid vaccine

Lipopeptides with different fatty acids (palmitic, palmitoleic, stearic, oleic, and linoleic acids) conjugated to CHSPKKKRKV were synthesised by a solid-phase peptide method using the Fmoc strategy (Fmoc-SPPS) on 2-CTC (2-chlorotritylchloride) resin. The lipopeptides were purified by RP-HPLC and characterised by ToF-ESI-MS and 1D-NMR. The capability of the lipopeptide to interact with the plasmid was evaluated by DNA agarose gel electrophoresis. The particle size of the lipopeptide/DNA complexes was determined by dynamic light scattering assay and TEM analysis. The biological activities including cytotoxicity, nitrite oxide (NO) release, and IL-6 and TNF-α production were evaluated in RAW 264.7 cells. ToF-ESI-MS revealed [M + 2H]²⁺ and [M + 3H]³⁺ ion peaks which were validated by ¹H NMR and ¹³C NMR, confirming the lipopeptide molecular structure. All lipopeptides condensed and protected the DNA plasmid from enzymatic degradation at the lipopeptide/DNA mass ratio of 2:1. In addition, the size of the cationic lipopeptide/DNA complexes ranged from ∼250 to 700 nm. The lipopeptides showed moderate cytotoxicity with IC₅₀ values ranging from 120 to 190 ppm, induced NO release (275-1060 ppm) and IL-6 (40-497 pg) and TNF-α (150-270 pg) production with the highest level achieved by C_(18,0)-CHSPKKKRKV. In conclusion, CHSPKKKRKV-based lipopeptides with different fatty acids are potential adjuvant candidates but further evaluation in animal models is required.