Soybean oil-derived lipids for efficient mRNA delivery

Introduction: Lipids have been increasingly utilized as a delivery platform for mRNA. Varying lipid compositions, surface modifications, charges, and pKa values can impact mRNA expression and allow organ-specific targeting for effective disease treatment. Current lipid synthesis methods involve Michael addition, epoxide ring-opening, reductive amination, and thiol-ene reactions. Notably, a common soybean oil derivative, rich in epoxy groups, can readily undergo ring-opening reactions with amino-containing compounds. Since soybean oil is FDA-approved and has excellent biocompatibility, it may give rise to a new class of synthetic lipid materials with the potential for mRNA delivery applications.

Methods: In our synthesis, epoxidized soybean oil and amino-containing compounds with varying carbon chain lengths, structures, and numbers of N elements were used as the sole reactants. An array of lipids (a total of 30 formulations) was synthesized via epoxide ring-opening reactions and subsequently purified through heating and rotary evaporation, all without the use of solvents. We then encapsulated Fluc mRNA in lipid nanoparticles (LNP) and characterized them using DLS and TEM. We assessed in vitro LNP delivery of Fluc mRNA and EGFP mRNA in HCT116 cells via confocal microscopy and bioluminescence imaging. Next, we investigated in vivo delivery of Fluc mRNA LNP in both ICR and C57 mice and in vivo gene editing potential of Cre mRNA LNP in Ai9 mice via bioluminescence imaging using the IVIS Spectrum system. The major organs were collected for subsequent biosafety evaluation via H&E staining and blood chemistry analysis.

Results: Most of the LNPs had sizes greater than 200 nm in diameter with varied surface zeta potential. We found that most lipids derived from soybean oil showed high mRNA loading capacity (>30%), thereby enhancing mRNA expression in vitro (>75%), all the while demonstrating excellent biosafety. In our in vivo studies of Fluc mRNA delivery, we observed that certain lipid formulations could enhance mRNA expression in the spleen, while others could achieve high expression in the lungs or liver. This highlights the potential utility of these lipids for precise and tunable organ targeting in disease-specific treatments. In addition, soybean oil-derived lipids displayed exceptional performance in delivering Cre mRNA for gene editing. We observed strong fluorescence signal in the livers of all lipid groups and obvious red fluorescence signal (indicative of tdTomato expression) in the lungs of one lipid formulation and in the spleen of another lipid formulation.

Conclusions: Our findings demonstrate the significant potential of soybean oil-derived lipids for organ-targeted delivery of functional mRNA. Encouraged by these results, we anticipate that soybean oil-derived lipid materials will assume a more prominent role in the realm of mRNA delivery for a diverse range of bioapplications. The synthesis of novel lipids utilizing FDA-approved natural substances remains a promising domain yet to be fully explored.

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