siRNA/mRNA payload of lipid nanoparticles quantified using fluorescence microscopy and correlation spectroscopy

Lipid nanoparticles (LNPs) serve as the leading vectors for delivering nucleic acids to mammalian cells. To accurately measure the RNA content and size of LNPs, advanced techniques like total internal reflection fluorescence (TIRF) microscopy and fluorescence correlation spectroscopy (FCS) are employed. FCS proves to be particularly reliable in determining the payload of mRNA and siRNA/mRNA coencapsulated in LNPs, regardless of fluorophore properties that may change during encapsulation.

By adjusting the proportion of PEG-lipid relative to a control formulation called ONPATTRO, researchers observed that the mRNA payload of an enhanced green fluorescent protein (eGFP) coding mRNA increased from around 10 per LNP as the size of the LNPs grew, following a power law relationship with an exponent of 1.3. Cryogenic electron microscopy (Cryo-EM) analysis revealed that small LNPs tend to have more "blebs" compared to larger ones.

In the case of coencapsulating both siRNA and mRNA within LNPs, the siRNA fraction within the LNPs was consistently higher than that found in the original RNA mixture. This indicates that the coencapsulation process is competitive and limited by the diffusion of nucleic acids. Overall, these findings emphasize the critical roles of particle size and encapsulation kinetics in determining the loading and composition of RNA payloads in LNPs.