Lipid nanoparticle-mediated mRNA delivery system into preimplantation embryos†

Lipid nanoparticles (LNPs) have emerged as a nonviral mRNA delivery vehicle for both basic and clinical applications. In the present study, we showed that the LNPs passed through the zona pellucida, the extracellular matrix surrounding the egg, and efficiently transduced preimplantation embryos. We first tested different types of surface polarity in LNPs carrying Enhanced Green Fluorescent Protein (EGFP) (LNP-EGFP) by changing cholesterol and found that the LNPs composed with neutral or anionic cholesterol are less toxic and more suitable for mRNA delivery into zygotes. Next, we transferred Cre mRNA using neutral LNP (LNP-Cre) and saw the floxed allele recombination in reporter mTmG transgenic embryos. After treatment with LNP-Cre at a 10 ng/μL concentration for 20 h at the zygote stage, about 76% mTmG embryos expressed the reporter EGFP at the blastocyst stage, and about 82% of embryos expressed EGFP in both the placenta and fetus at E12.5. Finally, we treated mTmG blastocyst stage embryos with LNP-Cre at a 50 ng/μL concentration for 20 h and found that about 86% of them expressed the reporter EGFP. Intriguingly, the EGFP fluorescence was only observed in trophectoderm cells, but not in the inner cell mass. Subsequently, we observed placenta-specific reporter EGFP expression in about 65% of the later-stage embryos (E12.5). Collectively, our study shows that LNPs provide a novel mRNA delivery system into preimplantation embryos that can be used for studying gene functions in pre-, peri-, and post-implantation development.