mRNA vaccines delivered by lipid nanoparticles (LNPs) have enabled clinical success in combating the COVID-19 pandemic, demonstrating LNPs as a promising nanocarrier for mRNA delivery. Despite this advance, use of PEGylated lipid (PEG-lipid) in LNPs has raised concerns about PEG-associated immunogenicity upon repeated dosing. Recent studies reported hydrophilic polymer lipids as replacement of PEG-lipid, which demonstrated the potential to reduce PEG-associated immune recognition. Most of these PEG-lipid alternatives are based on polymers with non-degradable polymer backbone. In this study, we synthesized a series of oligo(ethylene glycol)-functionalized polycarbonate lipids (OPL-1 to OPL-8) based on a polycarbonate backbone with reported biodegradability and biocompatibility to replace PEG-lipid such as ALC-0159 used in Comirnaty mRNA vaccine for attenuating PEG immunogenicity. Unlike ALC-0159 with 50 EG units existing in a linear form, oligo(ethylene glycol) (OEG) in OPLs is in a brush-like conformation. OEG length played a key role in formulating LNPs with desirable physicochemical properties. OPL-7 and OPL-8 with EG9 produced LNPs with nanosize, narrow size distribution, near-neutral surface charge and high encapsulation efficiency, comparable to ALC-0159 LNPs. OPL LNPs mediated significantly higher mRNA transfection efficiency in HEK293T and two model antigen-presenting cells (APCs) than ALC-0159 LNPs, which was associated with enhanced cellular uptake as evidenced by flow cytometry analysis. Inhibitor studies suggested that clathrin-mediated endocytosis was a major pathway for cellular uptake of LNPs, with caveolae-dependent endocytosis also contributing. A confocal microscopic study indicated comparable endosomal escape of OPL and ALC-0159 LNPs. In vivo and ex vivo bioimaging at 6 h post-administration demonstrated that OPL-7 and OPL-8 LNPs exhibited significantly reduced hepatic mRNA transfection and relatively higher lymph node mRNA transfection, resulting in significantly greater lymph node-to-liver mRNA transfection ratios. In a mouse vaccination study, OPL-7 and OPL-8 LNPs loaded with mRNA encoded for the spike glycoprotein of SARS-CoV-2 Delta variant elicited robust humoral immune responses without causing significant toxicity. Repeated dosing of OPL-7 or OPL-8 LNPs induced significantly lower anti-PEG IgM response than ALC-0159 LNPs. Moreover, OPL-7 or OPL-8 avoided recognition by pre-existing anti-PEG antibodies. Collectively, these findings identify OPL-7 and OPL-8 as promising PEG-lipid alternatives for mRNA delivery that maintain delivery performance while attenuating anti-PEG antibody responses.
