SignificancemRNA treatments represent an exciting approach to cure diseases that cannot be tackled with current therapeutics. However, the delivery of mRNA to target cells remains a challenge, but among the existing alternatives, lipid nanoparticles (LNPs) offer a promising answer to this. Here we determine the structure of LNPs encapsulating mRNA, consisting of a lipid mixture already evaluated in clinical trials. We show that the lipids are not homogeneously distributed across the LNP, and one of the lipids is localized mainly at its surface. The structural information enabled us to design LNPs that successfully modify intracellular protein production in two clinically relevant cell types. Our findings and approach provide a framework for understanding and optimizing vehicles for mRNA delivery.
Emerging
therapeutic treatments based on the production of proteins
by delivering mRNA have become increasingly important in recent times.
While lipid nanoparticles (LNPs) are approved vehicles for small interfering
RNA delivery, there are still challenges to use this formulation for
mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine
(DSPC), cholesterol, and a PEG-lipid. The structural characterization
of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding
of the way in which they function, but this information alone is not
enough to predict their fate upon entering the bloodstream. The biodistribution
and cellular uptake of LNPs are affected by their surface composition
as well as by the extracellular proteins present at the site of LNP
administration, e.g., apolipoproteinE (ApoE). ApoE,
being responsible for fat transport in the body, plays a key role
in the LNP’s plasma circulation time. In this work, we use
small-angle neutron scattering, together with selective lipid, cholesterol,
and solvent deuteration, to elucidate the structure of the LNP and
the distribution of the lipid components in the absence and the presence
of ApoE. While DSPC and cholesterol are found to be enriched at the
surface of the LNPs in buffer, binding of ApoE induces a redistribution
of the lipids at the shell and the core, which also impacts the LNP
internal structure, causing release of mRNA. The rearrangement of
LNP components upon ApoE incubation is discussed in terms of potential
relevance to LNP endosomal escape.
We present how dramatically the nonequilibrium nature of an oppositely charged polyelectrolyte/surfactant mixture can affect the interfacial properties. We show for the first time that the cliff edge peak in the surface tension of the poly(diallyldimethylammonium chloride)/sodium dodecyl sulfate system is produced as a direct result of depletion of surface-active material from the bulk solution due to a slow precipitation process in the phase separation region. Simple illustrations are given of how to control the production of the peak, to eliminate the feature for equivalent aged solutions through the use of different sample handling methods, and even to change its characteristics at short surface ages. The potential to tune nonequilibrium, steady-state interfacial properties for such strongly associating systems is clearly demonstrated. We propose that our findings in general may be applicable to a broad range of mixtures containing surfactants and oppositely charged macromolecules such as polymers, proteins, and DNA.
We show for the oppositely charged system poly(diallyldimethylammonium chloride)/sodium dodecyl sulfate that the cliff edge peak in its surface tension isotherm results from the comprehensive precipitation of bulk complexes into sediment, leaving a supernatant that is virtually transparent and a depleted adsorption layer at the air/water interface. The aggregation and settling processes take about 3 days to reach completion and occur at bulk compositions around charge neutrality of the complexes which lack long-term colloidal stability. We demonstrate excellent quantitative agreement between the measured surface tension values and a peak calculated from the surface excess of surfactant in the precipitation region measured by neutron reflectometry, using the approximation that there is no polymer left in the liquid phase. The nonequilibrium nature of the system is emphasized by the production of very different interfacial properties from equivalent aged samples that are handled differently. We go on to outline our perspective on the "true equilibrium" state of this intriguing system and conclude with a comment on its practical relevance given that the interfacial properties can be so readily influenced by the handling of kinetically trapped bulk aggregates.
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