• • A library of 124 ionizable lipids synthesized via Ugi-4CR yielded multiple NLA candidates; in vitro screening based on TNF-α production from splenocytes identified leading formulations that induced robust cytokine secretion, enabling rapid down-selection without costly in vivo testing.
• • The optimized NLA formulation, when co-administered with RSV pre-F antigen, elicited significantly enhanced central and effector memory T cell responses (p < 0.01) and provided protection against viral challenge comparable to an AS01e-like liposome adjuvant, demonstrating potential to replace costly AS01 in RSV vaccines.
• • Safety evaluations including hematology, blood biochemistry, and histopathology confirmed good biocompatibility, with no observed systemic toxicity, addressing a critical barrier for adjuvant translation.
• • NLRP3 gene knockout cells showed that the inflammatory properties of NLAs rely mainly on the NLRP3 inflammasome pathway, providing a mechanistic basis for rational design and mitigating off-target inflammation risks.