Skip the mess with dye-free LNP payload quant on Stunner

Importance of the Topic

Lipid nanoparticles (LNPs) play a central role in mRNA therapeutics and vaccine delivery, where precise quantification of encapsulated RNA and particle characteristics underpins product quality, batch consistency, and regulatory compliance. Traditional fluorescent dye assays for RNA content and encapsulation efficiency (EE%) are laborious, time-consuming, and expensive, prompting the need for streamlined, dye-free alternatives.

Objectives and Study Overview

This application note evaluates the Unchained Labs Stunner platform as a combined sizing and dye-free RNA quantification tool, comparing its performance with standard RiboGreen®-based assays. Three LNP formulations (X, Y, Z) were prepared in citrate and acetate buffers, loaded with PolyA or luciferase mRNA, and assessed for particle size, polydispersity, count, total/free RNA, EE%, and process yield.

Methodology and Instrumentation

• Conventional workflow: RiboGreen assays require separate calibration curves for free and total RNA, surfactant incubation, multiple plate setups, and over 266 pipetting steps for 12 samples.
• Stunner workflow: Uses 96-well microfluidic plates with 2 µL samples; integrates rotating-angle dynamic light scattering (RADLS) at seven angles for size, PDI, and count; and UV/Vis spectroscopy with an Unmix algorithm to resolve RNA, lipid, and turbidity signals. A single RiboGreen calibration set measures free RNA.
• Sample preparation: Automated microfluidic mixing (Sunscreen) in 20 mM acetate or citrate buffers; dialysis against PBS; monitoring of RNA concentration from aqueous input through post-dialysis.

Main Results and Discussion

• Particle size and PDI: Acetate buffer produced marginally smaller, monodisperse LNPs (Z-average < 100 nm; PDI < 0.2) compared to citrate, especially for formulation Z.
• Particle count: Consistent counts (~9 × 10¹¹ particles/mL), with reduced counts for LNP-Z in citrate due to aggregation.
• RNA quantification: Total RNA and EE% from Stunner closely matched RiboGreen results (difference < 1%); Stunner triplicates showed CV < 1%, indicating high precision.
• Process yield: Real-time tracking revealed ~98% RNA retention for LNP-Z versus ~78% for LNP-Y after dialysis, guiding formulation improvements.

Benefits and Practical Applications

• Time and cost efficiency: Stunner sizing (35 min for 12 samples) and hybrid quantification (15 min) versus traditional protocols (>120 min).
• Reduced complexity: Fewer pipetting steps and calibration curves; elimination of surfactant interference for total RNA.
• Comprehensive data: Simultaneous measurement of size, PDI, particle count, total/free RNA, and EE% facilitates high-throughput screening, process development, and quality control.

Future Trends and Applications

With the expanding landscape of LNP-based therapeutics, integrated analytical platforms will be essential. Anticipated developments include:

• Custom RNA analytes for diverse payload types and sizes.
• Deeper automation for in-line process analytics and real-time monitoring.
• Enhanced deconvolution algorithms for complex biotherapeutic formulations.
• Scaling workflows to meet GMP-level QC demands in industrial settings.

Conclusion

The Stunner platform delivers a powerful, dye-free solution for LNP characterization by combining advanced RADLS sizing with UV/Vis quantification. It significantly reduces assay time, cost, and complexity while enhancing data quality and throughput, making it an invaluable tool for both academic research and industrial LNP development.