Why SMLM for lipid nanoparticles?

The idea of delivering drugs inside a “capsule” made of protective lipids has been around for a few decades: much like their naturally occurring counterparts, extracellular vesicles, lipid nanoparticles (LNPs) can naturally associate into vesicles, encapsulating bioactive cargo, and once encounter a natural cell membrane, they can fuse with it, releasing their content into the target cell. The idea was particularly appealing when the “drug” in question involved nucleic acid content, particularly RNA, which is notoriously sensitive to degradation. Even though the idea of LNPs sounds easy enough, many efforts were invested in order to get the correct lipid composition that will be safe, stable and effective.

LNPs became the frontrunner of the biomedical world in 2020 when the world witnessed the unprecedented speed of vaccine development and production. It is not an overstatement that LNP-based COVID-19 vaccines saved millions of lives worldwide, and therefore it is not surprising that the scientists that pioneered their development were awarded the Nobel Prize in 2023. This remarkable success inspired many others to harness LNPs for multiple applications, from seasonal influenza vaccines to gene-therapy of severe genetic disorders. 

While there is no dispute that LNP-based therapies are safe and effective, their development, production and distribution still face some challenges, especially around their long-term stability. LNPs are often quite sensitive and require strict storage conditions in order to remain intact. Scientists invest many efforts in optimizing production processes and storage conditions, however, many of the current analytical methods for LNP quality control have some substantial limitations. Namely, while methods like dynamic light scattering (DLS) or electron microscopy (EM) are very good for sizing of LNP, they are not compatible with analyzing the cargo of the LNP, nor the loading of targeting ligands on their surface. Meanwhile, mass-spectrometry is excellent for quantification of surface ligands and cargo, but it does not provide information on the LNP structural integrity, which is an important predictor of its effectiveness. 

SMLM offers a solution for these concerns. Using a dSTORM-compatible surface marker, it is possible to accurately size LNPs with resolution of 20 nm, well within the range of a single LNP. Visualizing single LNPs provides information on their structural integrity, as well as additional valuable information, such as, that very large LNPs are likely aggregates, which are less likely to be taken up by cells, while very small entities are probably debris, rather than cargo-containing vesicles. Equally, assuming that specific antibodies are available, SMLM can identify the ligand on the LNP surface and inform the scientists on the success of their ligand loading, a process that is essential for targeting LNPs to specific cells. Combining these two together provides valuable insights on the size and integrity of the LNP, as well as how effective  the ligand loading is.

To this end, there is no RNA-cargo dye that is compatible with dSTORM, namely, a dye that can penetrate LNPs, is specific to RNA and is also blinking for a long time. Alternatives to that include other fluorescent RNA dyes that are only compatible with standard diffraction-limited microscopy. Nonetheless, it is possible to computationally match the larger, blurry spots of the RNA staining with the accurate localizations detected for the surface marker and the ligand. This imaging strategy is very powerful, thanks to its ability to collect comprehensive data on many many individual LNPs and get quantitative information on the numbers, the size distribution (polydispersity) and the loading efficiency of both cargo and ligand. This, of course, can be monitored over time and under different storage conditions, for example.

We are confident that over the next few years we will see more and more LNP-based therapeutic approaches. With better analytical tools, their production processes can be optimized, to make them more stable and more effective over time. Should a new pandemic threaten us, new vaccines may be available even faster, protecting many. Alongside that, conditions that are still considered incurable may well be treatable, thanks to the ability to deliver nucleic acids to specific target cells.   

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