Lipid Nanoparticle Encapsulation Efficiency
The problem
When it comes to nanoparticle characterization, fluorescence encapsulation efficiency (EE%) assays that measure RNA payload inside vs. outside of a nanoparticle are a total grind. The classic fluorescent dye-based assay has tons of hands-on steps, reads only a few samples at a time, is difficult to optimize, struggles with reproducibility, and creates a big headache with toxic detergents.
Other methods typically require two preps to be measured for each LNP sample to determine encapsulation efficiency (EE%). Measure the amount of free RNA in the intact prep. Since fluorescent dyes can’t get inside the nanoparticle, this initial reading uses fluorescent dyes to measure RNA free in solution. Add detergents to break apart your nanoparticles and repeat the RNA concentration measurement. This will be your total RNA concentration.
A 96-well plate typically handles up to 12 samples in duplicate for this assay. The rest of the wells are taken up by blanks and multiple calibration curves for each prep - with and without the surfactant. Each plate requires over 266 individual pipetting events and takes an experienced technician over 1 hour to complete – and that’s only for the EE% part of LNP characterization.
The right tools for the job
Stunner Add Fluorescence (AF) brings together dye-based fluorescence and intelligent UV/Vis to deliver EE% and combines that with sizing and particle concentration data from rotating angle dynamic light scattering (RADLS) for up to 44 samples per plate.
The proof
Easier than ever encapsulation efficiency (EE%)
Total RNA is read using UV/Vis from 2 µL of LNP sample without any dilutions, reagents, detergents, dyes, or standards. Free RNA is read in a neighboring well and only needs a simple master mix dilution step before loading.
Full LNP characterization in record time
Stunner AF’s unique combination of UV/Vis, fluorescence and RADLS make it a no-brainer for nanoparticle characterization. Whether you’re looking for the perfect formulation or checking quality from multiple manufacturing runs, Stunner AF has the throughput to keep up. Run up to 44 samples on a single plate or run multiple plates to increase throughput even further. Stunner AF characterizes a whole plate of LNPs in about an hour with a fraction of the work of traditional methods while also adding sizing and aggregation data in the same assay.
Stunner
Stunner AF is the only system that pulls together UV/Vis concentration with rotating angle dynamic light scattering (RADLS) and dye-based fluorescence on the same 2 μL sample. Nail down your lipid nanoparticle quality by knocking size, encapsulation efficiency (EE%), particle concentration and aggregate detection off your list in one hit. Without skipping a beat, you’ll know if your nanoparticle is good to go.
Want more info?
Want to learn more about how Stunner helps you with encapsulation efficiency?