Introduction
Distribution of nucleic acid cargo among lipid nanoparticles (LNPs) has important implications for the dosage, efficacy, and safety of a vaccine or therapeutic. Uneven payload distribution may lead to higher than necessary dosage of components such as PEG-lipids, while also making it difficult to predict the amount of RNA or DNA that will reach the cytosol of target cells.
Optimization of an LNP formulation requires reliable methods that can assess key characteristics. While LNP size, zeta potential, and encapsulation efficiency can be measured with established techniques with known strengths and weaknesses, options for determining LNP payload distribution are fewer and less tested.
We sought to develop a cryo transmission electron microscopy (cryo-TEM) image analysis method that would provide an estimate of the percentage of loaded and empty LNPs. First, we imaged LNPs prepared with or without mRNA in order to visualize and identify cargo encapsulation. Particles in the mRNA-LNP formulation were then visually classified as loaded or non-loaded based on their internal appearance.
While this provides an estimate of loading percentage, visual classification by an analyst is a subjective process. To automate classification, we developed an analysis method that uses a metric of particle texture and a quantitative loading threshold to estimate the percentage of loaded LNPs. Next, we asked whether payload distribution varied with size or particle type (i.e. unilamellar vs blebbed particles). To test whether the textural analysis method provided internally consistent estimates, we used a second LNP formulation and mixed LNPs prepared with and without mRNA at known ratios.
Finally, we collaborated with Spectradyne to compare cryo-TEM results with payload distribution measurements made using an ARCTM particle analyzer. Before diving into these experiments, we’ll first take a look at previous studies which measured LNP loading percentage, existing commercially available methods, and cryo-TEM studies which examined RNA encapsulation.
Commercially Available Methods for Loading Percentage Measurement
Three methods which use single particle fluorescence to measure the percentage of loaded particles are commercially available. Two instruments, Spectradyne’s ARC particle analyzer and NanoFCM’s Flow NanoAnalyzer, use a flow-based system to measure LNPs as they individually pass a fluorescence detector; while ONI’s Nanoanalyzer uses super resolution microscopy to precisely locate individual LNPs on a slide. All three methods use a fluorescent nucleic acid stain to detect cargo.
- Spectradyne’s ARC particle analyzer measures fluorescence when a particle is detected with resistive pulse sensing, which also provides size and concentration information.
- NanoFCM’s NanoAnalyzer uses light scattering to estimate particle size and concentration. Scattering events and fluorescence peaks are correlated to detect empty and loaded LNPs as well as the presence of free nucleic acid.
- ONI’s Nanoanalyzer uses dSTORM imaging of fluorescently labelled LNPs to pinpoint the locations of individual particles. Although a dSTORM compatible nucleic acid stain is not available, the less precise fluorescence signal of the cargo can be computationally matched with the dSTORM resolved LNPs.
Cryo-TEM Studies Examining LNP Cargo
Cryo Transmission Electron Microscopy (cryo-TEM) is an orthogonal method which can be used to simultaneously evaluate several LNP characteristics. It has been used extensively to characterize LNP size, morphology, and lamellarity. Cryo-TEM has also been used to investigate the presence of RNA cargo in LNPs.
Internal details of LNPs, such as a dense mottled or grainy appearance, are often evident in cryo-TEM images. Association between a dense mottled interior appearance and nucleic acid loading was supported with cryo-TEM imaging of LNPs labeled with thionine, a cationic dye that binds RNA (Brader, 2021).
The presence of RNA has also been inferred from the spacing of repeated structures seen in LNPs specifically designed to form lamellar or hexagonal superstructures (Pattipeiluhu, 2024). Cryo-TEM images of LNPs formulated with and without siRNA were analyzed using fast Fourier transforms to determine lattice spacing. One siRNA-LNP formulation showed two populations with different lattice spacing of structures - one population had spacing similar to that of the formulation without RNA, while the other population showed greater lattice spacing. Because the difference in spacing between populations matched the calculated width of a siRNA molecule, the two particle populations were interpreted as empty and siRNA-filled particles. While this type of analysis is useful for LNP formulations that have regularly repeating structures, it is not relevant to many LNP formulations which have particles with an amorphous lipid phase.
To test whether cryo-TEM could feasibly be used to estimate loading percentage, we analyzed images of LNPs using both a visual semi-quantitative and fully quantitative approach. In collaboration with Spectradyne, the same samples were measured with an ARC particle analyzer to understand how cryo-TEM image analysis compares with a fluorescence-based technique.
Read the Whitepaper
To see the experiments, including quantitative estimation of loading percentage, cargo loading by particle size and lamellarity, download the whitepaper.
Conclusions
Current and emerging methods for measuring payload distribution and loading percentage in LNP formulations require further testing. While investigation of these methods is ongoing, single particle fluorescence instruments, such as Spectradyne’s ARC particle analyzer, represent the only type of methodology available to measure the percentage of particles which contain cargo. Using textural analysis of cryo-TEM images as a quantitative readout for loading, may represent another method to estimate the percentage of loaded particles.
Because there is no gold standard technique for loading percentage and no standard LNP samples with defined loading percentages to test new techniques, the use of multiple complimentary orthogonal techniques is useful in understanding payload distribution. Further testing will help to determine how different methods and instruments compare and the best use cases for each. Cryo-TEM loading analysis is likely to be suitable for identifying loading differences between batches of the same formulation and can provide added value when used alongside other cryo-TEM analysis methods which measure particle size, lamellarity, and morphology.
Collaboration with Spectradyne
This study was done in collaboration with Spectradyne. To learn more about Spectradyne, visit www.nanoparticleanalyzer.com or email info@spectradynellc.com.
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