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

Lipid nanoparticles (LNPs) have emerged as the most promising vectors to deliver nucleic acids to mammalian cells. Advanced delivery strategies involving multiple nucleic acid species require a reliable determination of RNA payload and LNP size. Here, we use total internal reflection fluorescence (TIRF) microscopy and fluorescence correlation spectroscopy (FCS) to determine mRNA payload and siRNA/mRNA codelivered payload. Particularly, FCS proves to be a reliable method for payload determination, independently of fluorophore properties that can be altered during encapsulation. By varying the PEG-lipid fraction relative to the reference ONPATTRO formulation, we find that mRNA payload of enhanced green fluorescent protein (eGFP) coding mRNA increases from ∼10 with LNP size according to a power law with an exponent 1.3. Cryogenic electron microscopy (Cryo-EM) reveals that in this case blebs occur more frequently in small LNPs. For siRNA/mRNA coencapsulation the siRNA fraction within LNPs exceeds that in the initial RNA mixture, suggesting that coencapsulation is competitive and constrained by nucleic acid diffusion. Together, our findings highlight the importance of particle size and encapsulation kinetics in controlling the loading and composition of RNA payloads in LNPs.