(LNPs) containing exhibit a disordered inverse hexagonal internal structure with a characteristic distance of approximately 6 nm.
LNPs modified with mRNA show significant structural differences compared to those without mRNA.
The size and surface composition of LNPs significantly influence the production of proteins inside human adipocytes and hepatocytes.
For 130 nm LNPs, protein expression levels can vary by as much as 50-fold based on surface characteristics.
The localization of DSPC mainly at the surface of mRNA-containing LNPs may affect their interaction with cell membranes.
Simplified
The development of safe and efficacious gene vectors has limited greatly the potential for therapeutic treatments based on messenger RNA (). (LNPs) formed by an ionizable cationic lipid (here DLin-MC3-DMA), helper lipids (distearoylphosphatidylcholine, DSPC, and cholesterol), and a poly(ethylene glycol) (PEG) lipid have been identified as very promising delivery vectors of short interfering RNA (siRNA) in different clinical phases; however, delivery of high-molecular weight RNA has been proven much more demanding. Herein we elucidate the structure of hEPO modified mRNA-containing LNPs of different sizes and show how structural differences affect of human adipocytes and hepatocytes, two clinically relevant cell types. Employing small-angle scattering, we demonstrate that LNPs have a disordered inverse hexagonal internal structure with a characteristic distance around 6 nm in presence of mRNA, whereas LNPs containing no mRNA do not display this structure. Furthermore, using contrast variation small-angle neutron scattering, we show that one of the lipid components, DSPC, is localized mainly at the surface of mRNA-containing LNPs. By varying LNP size and surface composition we demonstrate that both size and structure have significant influence on intracellular protein production. As an example, in both human adipocytes and hepatocytes, protein expression levels for 130 nm LNPs can differ as much as 50-fold depending on their surface characteristics, likely due to a difference in the ability of LNP fusion with the early endosome membrane. We consider these discoveries to be fundamental and opening up new possibilities for rational design of synthetic nanoscopic vehicles for mRNA delivery.
Key numbers
10×
Increase in hEPO production
Comparison of hEPO production in hepatocytes vs. adipocytes.
50×
Increase in protein production
Protein expression in adipocytes for optimized .
45 to 135 nm
LNP diameter range
Size range of – used in experiments.
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