How a century-old technology could revolutionise LNP characterisation

The global market for lipid nanoparticles (LNPs) hit $786 million in 2024 and is projected to grow 14% a year to 2030, driven by their increasing popularity as drug delivery vehicles.[1] The success of two LNP-based mRNA vaccines to protect against COVID-19 fuelled increased development of the technology, which is now in demand for use in drugs to treat cancer, cardiovascular disease, and other chronic conditions.[2]

LNPs offer several advantages over other drug-delivery methods. For example, LNPs facilitate the delivery of therapeutic drug payloads to tissues and organs of the body, as well as to tumours. Moreover, innovations in LNP formulations have improved not only their targeting ability, but also their drug-loading efficiency and stability.[3]

Developing LNPs does present challenges, not the least of which is characterising them early in the development process to assess their quality, efficacy, and safety. LNPs are inherently heterogeneous, making it difficult to determine the homogeneity and size of LNP formulations - characteristics that are thought to influence treatment potency and safety. Traditional characterisation techniques such as dynamic light scattering (DLS) are limited in their ability to fully characterise LNPs.

Analytical ultracentrifugation (AUC) could be the ideal solution. This technology, which was invented nearly 100 years ago, uses centrifugal forces to separate samples based on sedimentation and diffusion coefficients, resulting in high-resolution characterisation of the samples. The utility of AUC in LNP characterisation is growing, thanks to ongoing efforts to improve the technology.

A deeper understanding of particle size

The most widely used technique for LNP characterisation is DLS, which measures size and size distribution based on Brownian motion. One limitation of this technology is that it can only measure average size range, so if there are different populations with similar sizes they usually only appear as one population, therefore not revealing the true heterogeneity of a given sample. Another commonly used technology, transmission electron microscopy (TEM), is useful for studying the size and shape of LNPs, but that requires removing them from solution and putting them on slides - a tedious process.

One of the biggest advantages of AUC is the ability to derive more precise data on LNP size and heterogeneity than technologies allow. Measuring sedimentation and diffusion parameters reveals vital information, such as size distributions, cargo loading, and molar mass. It also provides high-resolution data on the hydrodynamic radius of the particles. This deep understanding of the lipid nanoparticles improves the characterisation of such factors as the amount of mRNA that’s loaded into them, the distribution of full versus empty LNPs, the number of mRNA copies per LNP, and other critical attributes.

“Generally, the characterisation of these lipid-containing structures is challenging due to the heterogeneous composition and the potential interaction of lipids with matrices,” said Klaus Richter, group leader of UC at Coriolis Pharma Research in Munich, during a webinar earlier this year. This complexity “makes it important that we use methods that are not disruptive,” he said.

AUC workflows offer several other advantages that help improve the accuracy of characterisation. For example, AUC can be run over a larger concentration range than what’s possible with other technologies. That’s important, because diluting samples can alter the behaviour of LNP formulations, masking characteristics that could be detrimental in the final formulation. And AUC doesn’t require the use of a column to separate the particles, which is a downside of another widely used technology for LNP characterisation: size exclusion chromatography. This method requires running the nanoparticles over a column, which can shear the LNPs, negatively influencing data or, at worst, destroying the LNPs altogether.

Researchers at Coriolis tested the utility of AUC versus DLS in characterising LNPs exposed to various stress conditions, including freezing and thawing. They tested LNP formulation samples using buffers with different densities. By quantifying factors such as the flotation speed of the particles, AUC allowed them to “characterise LNPs in more detail, focusing on the density of the particles,” Richter said.

CRISPR generating new interest in LNPs

Another rapidly advancing application of lipid nanoparticles (LNPs) is in the delivery of CRISPR-based gene editing therapies. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enables precise, targeted modifications to DNA within living organisms. As these therapies continue to evolve, LNPs have emerged as a critical component in enabling their clinical application. LNPs serve as the primary delivery system, or vector, for transporting CRISPR components - such as Cas nucleases and guide RNAs - into target cells. This targeted delivery ensures that the gene-editing machinery reaches the appropriate intracellular compartments efficiently and safely, minimising off-target effects. By encapsulating and protecting these molecular tools, LNPs facilitate precise gene editing and support the development of personalised genetic treatments.

Looking ahead

We’re at a pivotal moment in the evolution of lipid nanoparticle (LNP) research, with CRISPR serving as just one compelling example of the transformative potential these technologies hold. While the field is still in its early stages, the momentum is undeniable. Realising the full promise of LNPs will require strong cross-disciplinary collaboration, particularly in simplifying complex workflows and enhancing efficiency. Innovations in automation and advanced analytical techniques - such as analytical ultracentrifugation - will be key to accelerating development, improving reproducibility, and ultimately bringing novel therapies to market faster. The path ahead is full of opportunity, and the work we do now will lay the foundation for the next generation of breakthroughs.

About the author

Amy Henrickson, PhD, is global market development manager at Beckman Coulter Life Sciences. She earned her PhD in Biological Sciences in 2023 with a focus on Biophysics in the lab of Dr Borries Demeler. Henrickson's doctoral research focused on advancing and refining analytical ultracentrifugation (AUC) methods for the characterisation of AAVs and LNPs. Her outstanding academic achievements have been recognised through numerous scholarships and grants, including the prestigious NSERC CGS-D. This involved the application of multiwavelength, density matching, and analytical buoyant density equilibrium (ABDE, also known as DGE-AUC) methods to the AUC. Her contributions include applying these innovative strategies to systems beyond gene therapy drugs, with a focus on characterising complex biopolymer interactions, including protein-nucleic acid and RNA-RNA interactions.

References

[1] Grand View Research, “Lipid Nanoparticle Market Size & Trends,” https://www.grandviewresearch.com/industry-analysis/lipid-nanoparticle-market-report.

[2] BioSpace, “Lipid Nanoparticles Market Anticipated to Surge to US$ 2,387.98 Million by 2032,” https://www.biospace.com/lipid-nanoparticles-market-anticipated-to-surge-to-us-2-387-98-million-by-2032.

[3] Grand View Research, “Lipid Nanoparticle Market Size & Trends,” https://www.grandviewresearch.com/industry-analysis/lipid-nanoparticle-market-report.