Stereochemistry can alter small-molecule pharmacokinetics, safety and efficacy. However, it is unclear whether the stereochemistry of a single compound within a multicomponent colloid such as a lipid nanoparticle (LNP) can influence its activity in vivo. Here we report that LNPs containing stereopure 20α-hydroxycholesterol (20α) delivered mRNA to liver cells up to 3-fold more potently than LNPs containing a mixture of both 20α- and 20β-hydroxycholesterols (20mix). This effect was not driven by LNP physiochemical traits. Instead, in vivo single-cell RNA sequencing and imaging revealed that 20mix LNPs were sorted into phagocytic pathways more than 20α LNPs, resulting in key differences between LNP biodistribution and subsequent LNP functional delivery. These data are consistent with the fact that nanoparticle biodistribution is necessary, but not sufficient, for mRNA delivery, and that stereochemistry-dependent interactions between LNPs and target cells can improve mRNA delivery.
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All RNA sequencing data are available at GEO (GSE181333). All other data are represented in the main figures or supplementary figures.
All code used to analyse the data is available at https://github.com/Jack-Feldman/barcode_count.
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The authors thank S. Durham, R. Hughley, D. Gulick, N. Djeddar at Georgia Tech, and D. Alexis at the Emory Winship Cancer Institute. The authors thank K. Tiegren. This work was funded by the National Institutes of Health (R01GM132985, awarded to J.E.D., and UG3-TR002855, awarded to J.E.D. and P.J.S.).
J.E.D. is an advisor to GV. All other authors declare no competing interests.
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Hatit, M.Z.C., Dobrowolski, C.N., Lokugamage, M.P. et al. Nanoparticle stereochemistry-dependent endocytic processing improves in vivo mRNA delivery. Nat. Chem. 15, 508–515 (2023). https://doi.org/10.1038/s41557-023-01138-9