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

Abstract

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.