mRNA-loaded lipid nanoparticles (mRNA-LNPs) hold great potential for disease treatment and prevention. LNPs are normally made from four lipids including ionizable lipid, helper lipid, cholesterol, and PEGylated lipid (PEG-lipid). Although PEG-lipid has the lowest content, it plays a crucial role in the effective delivery of mRNA-LNPs. However, previous studies have yet to elucidate the key factors of PEG-lipid that influence the properties of LNPs. This study reported how PEG-lipid content, lipid tail length, and chemical linkage between PEG and lipid affected in vitro and in vivo properties of mRNA-LNPs. Forty-eight LNP formulations were prepared and characterized. The results revealed that a PEG-lipid molar content exceeding 3.0 % significantly reduced the encapsulation efficiency of mRNA in LNPs via manual mixing. An increased PEG-lipid content also significantly decreased mRNA translation efficiency. Although the chemical linkage had minimal impact, the lipid tail length of PEG-lipid significantly affected the properties of mRNA-LNPs, irrespective of whether the LNPs were prepared using manual or microfluidic mixing. mRNA-LNPs made from ALC-0159 with C14 lipid tails, which is used in Pfizer/BioNTech COVID-19 mRNA vaccines, or C16-Ceramide-PEG preferably accumulated in the liver, while mRNA-LNPs prepared from C8-Ceramide-PEG were largely found in the lymph nodes. In a mouse SARS-CoV-2 Delta variant spike protein-encoded mRNA vaccine model, mRNA-LNPs made from either C8-Ceramide-PEG or C16-Ceramide-PEG yielded comparable vaccination efficacy to mRNA-LNPs made from ALC-0159, while mRNA-LNPs formulated with DSPE-PEG with C18 lipid tails mediated lower vaccination efficacy. C16-Ceramide-PEG LNPs and DSPE-PEG LNPs induced higher anti-PEG antibody response than C8-Ceramide-PEG and ALC-0159 LNPs. All the LNPs tested did not cause significant toxicity in mice. These results offer valuable insights into the use of PEG-lipid in LNP formulations and suggest that C8-Ceramide-PEG holds potential for use in the formulation of mRNA vaccine-loaded LNPs.
