This study systematically combined eight types of headgroups with two tail chain variants to design and synthesize sixteen ionizable lipids featuring symmetric dual-tail structures, which were subsequently formulated into corresponding lipid nanoparticles (LNPs). The aim was to investigate their structure-performance relationships and develop efficient mRNA delivery vectors. Comprehensive characterization revealed that LNPs with alkane tails generally exhibited smaller particle sizes (mostly within 100-150 nm), uniform distribution (PDI < 0.2), and encapsulation efficiencies exceeding 80%. The apparent pKa of these LNPs was co-modulated by the protonation capability of the headgroup and the particle size. In terms of cytotoxicity, at concentrations up to 200 μM, alkane-tailed LNPs showed no significant toxicity toward HepG2 cells, whereas some olefin-tailed LNPs displayed toxicity at higher concentrations. In A549 cells, all tested LNPs inhibited cell proliferation, and the positive control DLin-MC3-DMA (MC3) exhibited reduced toxicity at elevated concentrations. In Huh-7 cells, MC3 promoted proliferation, while compounds 4 and 5 demonstrated significant toxicity at 400 μM. During the evaluation of mRNA delivery performance, small-sized LNPs - particularly compounds 4 and 5 - showed excellent cellular uptake in HepG2 cells. The transfection efficiency of compound 5 reached 67.80%, slightly exceeding that of the positive control MC3 (53.16%). Further investigation of intracellular trafficking indicated that after internalization of LNP-mRNA complexes, compound 5 led to partial premature release and degradation of mRNA during the endosomal acidification stage, whereas MC3 mainly released mRNA into the cytoplasm following proton sponge-triggered endosomal escape. This difference resulted in distinct kinetics of EGFP expression. The study systematically elucidates the combined effects of tail structure, headgroup basicity, particle size, and other key factors on the delivery efficiency and safety of LNPs, providing a rational basis and experimental support for the design of highly efficient and low-toxicity mRNA lipid nanoparticles.
