Dynamics 8

RNA was packaged into LNPs using a NanoAssemblr microfluidic system (Precision Nanosystems, Vancouver, Canada) according to manufacturer instructions. Briefly, LNP formulations were formed by injecting 12.5 mM of the lipid solution and 0.173 μg/μl of RNA in formulation buffer at a flow rate of 12 ml/min. After mixing, LNP suspension was immediately diluted in PBS (Corning, Manassas, USA). Then, the formulation was reconcentrated by centrifugation at 2000 g in Amicon filters (30,000 MWCO, Amicon Ultra-15 Centrifugal Filter Unit, Millipore Sigma, Burlington, USA). Finally, the LNP suspension was filtered using a 0.22 micron syringe filter and kept at 4°C until use. Free and total RNA concentration were determined by Ribogreen assay (Quant-iT RiboGreen RNA, Invitrogen, Carlsbad, USA). For obtaining total RNA concentration, LNPs were lysed for 30 min at 37°C in Triton X-100 1%. Encapsulation was calculated as (total RNA-Free RNA)/(total RNA x 100). Particle sizes were measured by Dynamic Light Scattering in a DynaPro NanoStar instrument (Wyatt Technology, Santa Barbara, USA) and analyzed with Dynamics 8.0 software (Wyatt Technology, Santa Barbara, USA). Samples were diluted in PBS (Corning, Manassas, USA) until full laser power could be used to record the signal. For each sample, 3 measurements were conducted, each consisting of 10 recordings of 10 s.