Construction of LP-LNP with novel lipopeptides as adjuvants and its enhancing effects on mRNA vaccines

Objective To construct lipid nanoparticles (lipopeptide-lipid nanoparticle, LP-LNP) with novel lipopeptides as adjuvants, and initially explore their synergistic effect on mRNA vaccines. Methods Two novel lipopeptides, SS-10 and SQ18, were designed and synthesized. Microfluidic technology was used to encapsulate lipopeptides in different proportions, as well as mRNAs encoding enhanced green fluorescent protein (eGFP), firefly luciferase (F-luc), and ovalbumin (OVA) into lipid nanoparticles to construct an mRNA delivery system with novel lipopeptides as adjuvants (LP-LNP). The particle size and polydispersity coefficient of LP-LNP were measured using dynamic light scattering. The activation effect on Toll-like receptors 2 (TLR2) was detected using HEK-BlueTM mTLR2 reporter cells to screen the optimal lipopeptide ratio. The preferred LP-LNP-eGFP-mRNA was transfected into HEK293T cells, and the expression of eGFP was observed under a fluorescence microscope. In vivo imaging was used to investigate the expression level of LP-LNP-F-luc-mRNA in mice. Flow cytometry was used to evaluate the ability of LP-LNP-OVA-mRNA to induce the maturation of dendritic cells (DCs) in draining lymph nodes and cross-presentation of antigens after immunization. Results Lipopeptides SQ18 and SS-10 were incorporated into LNP at 0.50% and 0.75% molar ratios, respectively, to obtain LP-LNP with uniform particle size, high encapsulation efficiency, and good in vitro safety. The ability of this formulation to activate TLR2 was significantly stronger than the positive control Pam2CSK4 (P < 0.01). The preferred LP-LNP obtained effective in vitro transfection, and LP-LNP prepared with SQ18 at 0.50% molar ratio had significantly better in vivo transfection efficiency than traditional LNP (P < 0.01), and significantly promoted the maturation of DCs in draining lymph nodes and cross-presentation of antigens (P < 0.05). Conclusion LP-LNP with novel lipopeptides as adjuvants can enhance the delivery capacity of mRNA and further improve the immune effect of mRNA vaccines.