Get the skinny on LNPs with Stunner

Importance of the topic

The characterization of lipid nanoparticles (LNPs) is vital for the rapid development of mRNA-based therapies and vaccines. Accurate measurement of particle size distribution and encapsulated RNA content ensures formulation quality, stability assessment, and regulatory compliance.

Objectives and Overview of the Study/Article

This application note introduces the Stunner platform, which integrates high-throughput dynamic light scattering (DLS) with short-pathlength UV/Vis spectroscopy. The study demonstrates the platform’s capability to deliver simultaneous data on particle size, size distribution, total RNA concentration, and turbidity of RNA-LNP samples, comparing its performance with established dye-based assays.

Methodology and Instrumentation

• Platform: Stunner by Unchained Labs, enabling DLS and UV/Vis measurements on 2 µL samples in 96-well SBS-format plates.
• Sample preparation: Fluc-mRNA-LNPs and empty LNP stocks from Precision NanoSystems; LNPs assembled with GenVoy-ILM lipid mix using NanoAssemblr® Ignite System.
• Measurement parameters: RNA concentration determined via deconvoluted absorbance at 260 nm with a 40× factor; DLS calculation using viscosity of 1.002 and refractive index of 1.334 at 20 °C.
• Regulatory compliance: Optional 21 CFR Part 11 tools and UV/Vis performance verification against US and European Pharmacopeia standards.

Main Results and Discussion

• Reagent-free RNA quantification: Spectral unmixing separates turbidity and RNA absorbance, achieving strong agreement with RiboGreen® assay (R² > 0.99; slope ≈ 1) down to 1.2 µg/mL.
• High-throughput DLS: Analysis of 96 samples in under one hour produced an average hydrodynamic diameter of 79 nm (CV 1%) and a PDI of 0.14 (SD 0.02), indicating moderate monodispersity.
• Turbidity-based concentration: Linear correlation observed between turbidity (A260) and known particle counts for empty and full LNPs, supporting potential non-destructive concentration estimation.
• Data management: Graphical overview, Excel export, and PDF reporting streamline workflow and QA/QC.

Benefits and Practical Applications of the Method

• Combines particle sizing and total RNA quantification in a single, rapid assay without dyes or detergents.
• Minimizes sample volume (2 µL) and eliminates dilution errors, conserving reagents and samples.
• Enables high-throughput screening of LNP formulations and stability studies.
• Supports both research and regulated manufacturing environments with automation and compliance features.

Future Trends and Applications

• Development of calibration-free turbidity standards for robust particle concentration assays.
• Extension of high-throughput multimodal analysis to diverse nanoparticle and biologic systems.
• Integration into continuous manufacturing and real-time release testing for next-generation vaccines and gene therapies.
• Application of AI-driven analytics to further accelerate formulation optimization.

Conclusion

The Stunner platform offers a unique, integrated solution for comprehensive characterization of RNA-LNPs. By uniting high-throughput DLS with UV/Vis spectral deconvolution, it delivers rapid, reagent-free measurements of size, RNA content, and turbidity—streamlining development, quality control, and regulatory workflows in mRNA therapeutics.

References

• RiboGreen assay protocol, C. Walsh et al. Precision NanoSystems User Guide PNI-SOP-S9-001-EXT, 2016.
• C. Roces et al. Manufacturing considerations for the development of lipid nanoparticles using microfluidics. Pharmaceutics, 2020;12(11):1–19.
• L. Ribeiro et al. Use of nanoparticle concentration as a tool to understand the structural properties of colloids. Scientific Reports, 2018;8(1):1–8.