Characterisation of lipid nanoparticles under different osmotic stress using SANS and contrast variation

Treatments based on protein production and delivering messenger RNA (mRNA) represent a promising approach for therapeutic applications. One of the major challenges is to protect mRNA from enzymatic degradation and deliver it into the target cells. Lipid nanoparticles (LNPs) formed by a cationic ionisable lipid (CIL), DSPC, cholesterol and a pegylated lipid were approved for delivery of small interference RNA. In vitro and in vivo studies draw different conclusions when LNPs are involved and this may be due to the different environments where LNPs are immersed, not only in terms of molecular composition of the media but also due to responsiveness of LNPs to the exerted stress, which derive, for instance, from injection in the body. There is little known on the structural effects of these stress upon intravenous (IV) administration. We propose to use SANS to probe LNP structure under osmotic stress. Partially-deuterated polymer will be used to exert increasing stress on the LNPs but will be contrast matched by a proper mixture of water and heavy water. We plan to measure LNPs with and without the payload in two different mixtures of water and heavy water to selectively match either the shell or the content of the LNPs. We will use two partially-deuterated PEGs that will be matched by the same solvents to exert increasing osmotic stress. The measured LNP form factors will be fit using a core-shell model implemented in SASView and combining the results with computer simulations.