Neutron scattering and selective deuteration reveal the structure and composition of mRNA-containing LNPs and the effects induced by the serum protein ApoE

Decades of research into messenger RNA (mRNA) and lipid nanoparticles (LNPs) as delivery vehicles laid the foundation for the rapid development of mRNA-LNP-based SARS-CoV-2 vaccines by Moderna and Pfizer/BioNTech. Authorised in 2020, they were the first mRNA vaccines to reach the market for any indication and have now been used worldwide. Further investigation, however, is required in order to optimise the disruptive potential of this new and innovative technology. The structure and composition of mRNA-containing LNPs, in addition to the effects induced by the serum protein apolipoprotein E (ApoE), were recently revealed by combining the powerful techniques of neutron scattering and selective deuteration through academic-industrial collaboration between Malmö University and AstraZeneca, funded by the Knowledge Foundation in Sweden.

The COVID-19 pandemic highlighted the major advantages of RNA-based technology, in particular the ability to target rare or difficult-to-treat diseases, quick and cost-effective manufacturing and their potential for personalised medicine. The technology, however, is very new. “There is significant room for improvement with mRNA-based vaccines,” explains  Federica Sebastiani, Tenure Track Assistant Professor at the University of Copenhagen. “One of the bottlenecks, for example, is that a very small proportion of the mRNA is released into the cytosol to ultimately affect protein expression. If that efficiency could be improved then the dose could be lowered, reducing both costs and side effects.”

A precise structural and compositional characterisation of mRNA-LNPs is crucial in order to continue expanding our knowledge about how they function and comprehend their distribution and cellular uptake after administration. Previous work carried out using cryo-electron microscopy (cryo-EM) was unable to clearly distinguish different components due to their near equivalent electron density. Small-angle neutron scattering (SANS), if used alone, would also suffer from such a problem due to the similar scattering density and thus contrast of components. “The strength of neutrons for this study is the strong contrast between hydrogen and its isotope deuterium,” explains Sebastiani. “Selective deuteration involves the substitution of one component with its deuterated form, thus enabling various parts of the LNP structure to be masked or highlighted.” The combination of SANS with full exploitation of selective deuteration provides the means to determine the overall structure of mRNA-LNPs, in addition to the distribution of the different components within the LNP.

For this study, mRNA-containing LNPs were formulated using a cationic ionizable lipid (CIL, MC3), phospholipids (DSPC), cholesterol, a PEG-lipid (DMPE-PEG) and mRNA. “Cholesterol and DSPC mainly stabilise the nanoparticle. The PEG-lipid also contributes to this stabilisation, in addition to controlling the size of the LNP and ensuring that aggregation does not occur prior to administration,” explains Sebastiani. “Once intravenous administration has occurred, the PEG-lipid is shed and the CIL, which encapsulates the RNA, helps the LNP to bind and fuse with the endosomal membrane, facilitating mRNA release.” Four distinct LNP samples were prepared with the same formulation but with different components substituted with their deuterated versions. Deuterated cholesterol and CIL compounds were provided by the deuteration laboratories at AstraZeneca, the Australian Nuclear Science and Technology Organisation (ANSTO) and the Institut Laue-Langevin (ILL).

Small-angle neutron scattering (SANS) experiments were carried out using the D22 instrument at the ILL and the KWS-2 instrument at FRM-II (Germany). The data confirmed the core−shell structure previously suggested for LNPs. “The suggested structure was derived from knowledge of the components, their solubility and how they mix,” explains Sebastiani. “In comparison, the data we acquired constitutes the first real experimental proof.” The exact composition and distribution of all four lipid components across the LNP shell and core was also determined; this information was previously completely unknown. “Though some variation in size was found among different LNP samples, the composition across shell and core remained constant,” explains Sebastiani. “We quantified that cholesterol is approximately two to four times more concentrated in the shell than in the core, whereas MC3 is almost twice as concentrated in the core than in the shell. The shell was also found to contain CIL and DMPE-PEG.”

ApolipoproteinE (ApoE) is a serum protein that binds to LNPs and is known to be critical for cellular uptake and protein production in the liver. “While it is well known that serum proteins bind to LNPs once administered, little work has been done to study whether this binding actually affects the LNP,” explains Sebastiani. “A novel aspect of our work was the acquisition of complementary SANS and SAXS data of mRNA-LNPs after 3 hours incubation with ApoE in order to investigate the potential structural and compositional effects induced by binding.” The results demonstrated that ApoE binding causes a rearrangement of both the LNP surface and core structures, in particular, cholesterol is transferred from the LNP core to the surface, increasing the LNP surface cholesterol concentration. At a certain critical concentration, the LNP core packing fails and mRNA is released.

The researchers hope to continue to build upon these findings, published in ACS Nano in 2021, by moving from a characterisation carried out in static conditions and at physiological pH to parameters that are more realistic and biologically-relevant post-administration. “It would be interesting, for example, to study mRNA-containing LNPs at different pH conditions as it is known that the cellular uptake of particles is associated with a drop in pH and that this influences mRNA release. I’d also like to investigate LNP behaviour under flow conditions as studies have shown that for LNPs administered in blood, the protein corona composition is different in static or dynamic conditions,” explains Sebastiani.