Investigation of the internal structure of lipid nanoparticles for gene delivery

Lipid nanoparticles (LNPs) are very small self-assembled particles of approximately 100 nanometres (0.1 micrometres) in diameter, which are able to enclose and protect biologically active molecules such as nucleic acids (RNA or DNA). They are able to deliver their payload intact to the inside of cells, which has led to their use for COVID-19 mRNA vaccines, with spectacular success. However, how it all works is still something of a 'black-art', due to a lack of detailed understanding at a molecular level of the factors which control each stage of this very complex process. It is now well known that four types of lipid are needed for the LNP, with the most critical of these being an ionizable lipid (CIL), which changes its charge state upon being acidified inside compartments (endosomes) within cells, destabilising the endosome wall, and allowing the mRNA to escape into the cell interior, where it can carry out its work.
Although the Astrazeneca team have extensive knowledge of LNPs, and have synthesized and screened hundreds of lipids, the relationship between LNP lipid composition, LNP structure, and its biological activity is not well understood. This information is needed to help rationale the design of the optimal lipids to enable more efficient and safer intracellular delivery of mRNAs and other nucleic acids for vaccines and other advanced treatments such as gene editing.
In this project we aim to take advantage of the state of the art facilities at Rutherford Appleton Laboratories to generate high quality structural data from LNPs formulated from a set of promising lipid candidates developed by Astrazeneca. The techniques we will use are Small Angle X-ray Scattering(SAXS) and Small Angle Neutron Scattering (SANS), and cryo- Transmission Electron Microscopy (Cryo-TEM). Synchrotron SAXS at Diamond Light Source is a very powerful way of probing the self-assembled structures, identifying and characterising liquid-crystalline or other types of ordering within the LNPs. SANS at the neutron scattering facility ISIS has the unique ability to determine where the different lipids are located within the LNPs. This approach takes advantage of a unique feature of neutron scattering, whereby molecules can be 'highlighted' by substituting deuterium for hydrogen in the chemical structure of the synthesized molecule. Cryo-TEM is able to give additional structural detail, particularly with regard to the release of mRNA inside cells. The experimental work will be complemented by computer simulation studies of the interaction of mRNA with the lipids.