mRNA is a natural information-carrying molecule that cells read to make a specific protein. An mRNA vaccine contains information for making a key protein of a pathogen: for example, the spike protein of SARS-CoV-2, the virus that causes COVID-19. Inside the body, the mRNA is translated to a protein to teach the immune system about the pathogen in advance, preparing the body in case of an infection.
However, the mRNA molecule is too unstable to be used for pharmaceutical application without protection. The challenge is delivering the mRNA into the target cells in a form that is not immediately degraded in the body. In saRNAs (self-amplifying RNAs), a format of mRNAs that could be developed in potential new RNA vaccines, polymer-based systems have been identified to be particularly effective for the delivery.
One of these polymer systems is polyethylenimine (PEI), a clinically established gene delivery vehicle. It forms polyplex nanoparticles when mixed with RNA, where the polymer to RNA ratio (N/P) and the size of the nanoparticles formed are important for determining their biological performance. To develop the control over how the mRNA is delivered to the cells, a greater understanding of the assembly of these lipid nanoparticles is needed. As PEI can also be toxic at high concentrations, it's crucial to understand how the ratio of PEI and saRNA should be balanced.
In this study, published in Nature Nanotechnology, researchers from BioNTech used beamlines at ISIS to harness the unique properties of neutrons, working with beamline scientist Leide Cavalcanti. Using experiments on Zoom and SANS2D, the group studied the formation of polyplexes from PEI and saRNA at a range of N/P ratios. By combining these results with other characterisation techniques and testing the samples' biological activity, they aimed to investigate how the way that the nanoparticles are assembled effects their behaviour.
They are able to do this due to the different interactions of 'normal' water and deuterated 'heavy' water with neutrons. Using a combination of the two for different components of the nanoparticle system, Small Angle Neutron Scattering (SANS) is able to create contrast pictures of the structure, enabling the researchers to see what's going on inside. The team previously used Small Angle X-ray Scattering (SAXS) at the European Molecular Biology Laboratory (EMBL) beamline P12 of synchrotron DESY in Hamburg, and applied the neutron scattering experiments to build a complete picture of the system.
They found that, unexpectedly, the saRNA molecules could be stabilised in the form of very small nanoparticles in solution. They are much smaller than the lipid nanoparticles, comprise mostly only one saRNA molecule, and performed well in experiments against influenza.
These formulations are easy to manufacture and are low cost, which indicates they could be a good choice for developing into clinical products, especially if there is a need for vaccines that can be stored in conditions that do not require complex cooling systems.
“Combining experiments at X-ray and neutron scattering facilities can create new insights with practical impact for development of future pharmaceuticals," explains one of the corresponding authors on the study, Heinrich Haas.
The full paper can be found at DOI: 10.1038/s41565-025-01961-w
