In shape and structure, lipid nanoparticles turn out to be more like jelly beans than spheres, as previously thought. Credit: Bella Ciervo. CC BY-SA.

Breakthrough in characterising lipid nanoparticles provides blueprint for engineering more effective RNA therapies.

Lipid nanoparticles (LNPs) are the delivery vehicles of modern medicine, carrying cancer drugs, gene therapies and vaccines into cells.

Until recently, many scientists assumed that all LNPs followed more or less the same blueprint, like a fleet of trucks built from the same design. 

Now, researchers from the University of Pennsylvania, Brookhaven National Laboratory and Waters Corporation have characterised the shape and structure of LNPs in unprecedented detail, revealing that the particles come in a surprising variety of configurations.

That variety isn’t just cosmetic: as the researchers found, a particle’s internal shape and structure correlates with how well it delivers therapeutic cargo to a particular destination.

Penn Engineering’s Michael J Mitchell, Associate Professor in Bioengineering and a co-senior author of the paper, said: “Treating LNPs like one model of car has worked, as evidenced by the millions of people these particles have helped, but LNPs are not one-size-fits-all for every RNA therapy.

“Just as pickups, delivery vans and freight trucks best suit different journeys, we can now begin to match LNP designs to particular therapies and tissues, making these particles even more effective.”

Co-senior author Kushol Gupta, Research Assistant Professor in Biochemistry and Biophysics in Penn’s Perelman School of Medicine, added: “These results deliver a more fundamental understanding of how the composition and shape of these therapeutic particles relate to their biology.

“These particles have already proven themselves in the clinic, and these insights will make them even more powerful by helping us tailor delivery to specific diseases more quickly.”

Illuminating the ‘black box’

In recent years, the Mitchell Lab, among others, has found that different LNP formulations have varying biological effects. 

Adding phenol groups, for instance, reduces inflammation, while branched ionisable lipids improve delivery.

First author Marshall Padilla, a bioengineering postdoctoral fellow, said: “It’s almost like recipe development.

“We’ve known that different ingredients and techniques change the outcomes.”

But understanding why certain chemical tweaks lead to particular biological effects has proved challenging.

Padilla added: “These particles are something of a ‘black box’; we’ve had to develop new formulations mostly by trial and error.” 

The varied shapes and structures of lipid nanoparticles could lead to smarter medicines. Credit: Bella Ciervo. CC BY-SA.

Bringing LNPs into focus

To visualise the particles, the researchers employed multiple techniques. Past studies, by contrast, typically relied on a single method, like freezing the particles in place. 

Because of the particles’ size — it would take thousands of LNPs to encircle a human hair — prior work also frequently tagged the particles with fluorescent materials and averaged measurements, at the risk of altering the particles’ shape and obscuring variations. 

Gupta said: “We needed to combine multiple, fundamentally dissimilar techniques that left the particles intact in solution.

“That way, we could be confident that agreement between the methods showed us what the particles really looked like.”

Three techniques, one study

The researchers examined four ‘gold-standard’ LNP formulations, including those used in the COVID-19 vaccines and Onpattro, an FDA-approved therapy for a rare genetic disease.

One visualisation technique, sedimentation velocity analytical ultracentrifugation (SV-AUC), involved spinning the LNPs at high speeds to separate them by density. 

Another, field-flow fractionation coupled to multi-angle light scattering (FFF-MALS), gently separated the LNPs by size and measured how the nucleic acid was distributed across the different particles. 

A third, size-exclusion chromatography in-line with synchrotron small-angle x-ray scattering (SEC-SAXS), allowed the researchers to study the internal structure of LNPs by hitting them with powerful beams of x-rays at the National Synchrotron Light Source II (NSLS-II), a US Department of Energy (DOE) Office of Science user facility at DOE’s Brookhaven National Lab.

Gupta, summarising the results, said: “We used to think LNPs looked like marbles, but they’re actually more like jellybeans, irregular and varied, even within the same formulation.” 

The X-ray source. Equipment in the National Synchrotron Light Source II. Credit: Timothy Kuhn/ Brookhaven National Laboratory. CC BY-SA.

The power of collaboration 

The results would have been unattainable without bringing together academia, industry and a national laboratory. 

Martin Kurnik, Wyatt Technology Principal Scientist at Waters Corporation, who led the FFF-MALS experiments, said: “We’ve been developing methods to measure both lipid nanoparticle size and their drug content without breaking the particles apart.”

James Byrnes, a beamline scientist at NSLS-II, who conducted the SEC-SAXS experiments, added:  “The capabilities at Brookhaven National Lab enabled a unique experiment that combined x-rays with ultraviolet light to quantify the particles’ geometric characteristics.

“This paves the way to characterising particle formulations at scale and highlights the exciting potential for deeper collaborations between synchrotron facilities and LNP developers.” 

Padilla said: “This entire project speaks to the power of different institutions pooling their resources and expertise.

“We were only able to visualise the particles in such detail because each partner saw them from a different angle.” 

Future directions

The results open the door to a new era of rational LNP design, moving beyond today’s trial-and-error approach. 

Rather than assuming a single ‘best’ formulation, the study shows that particle size, shape, internal structure and preparation method must be matched to the therapeutic context.

While some of the tools used in the experiments — like a particle accelerator — are difficult to access, many of the steps can be reproduced with more common equipment.

As additional labs generate structural and functional data, the field could even assemble the data sets needed to train AI for LNP design.

Ultimately, the findings point toward a future in which nanoparticles can be engineered with the same precision as drugs themselves.

The study is published in Nature Biotechnology.