Nanoparticle Characterization Samples

Lipid Nanoparticle (LNP) Characterization

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Lipid Nanoparticles (LNPs) have become a leading non-viral delivery system for therapeutics and vaccines.  LNP size, size distribution, lamellarity, cargo encapsulation, and stability are critical factors which influence their in vivo effectiveness. Cryo-TEM can be used to assess all of these factors, as well as provide confidence in sample uniformity and purity, and evaluate the influence of storage conditions such as pH, temperature, and time on lipid nanoparticle morphology.

Cryo-TEM can be used to reveal sample information at multiple levels - from the LNP population as a whole, down to minute structural details, such as the leaflets of lipid bilayers. In addition, cryo-TEM can visualize bleb formation in LNPs and the lamellarity of your lipid nanoparticle, showing if your LNP is unilamellar or multilamellar.

To read an in-depth study showing how cryo-TEM per-particle analysis of LNPs can reveal subtle structural changes including bleb formations, that other analytical methods can not, download the whitepaper here.

Microscopic image of lipid nanoparticle characterization by cryo-tem | nanoimaging services
Visualize mRNA-loaded (left) and unloaded (right) lipid nanoparticle samples with cryo-TEM imaging.

Visualize the unexpected with cryo-EM.

Microscopic image of lipid nanoparticle characterization by vryo-tem | nano imaging services

While typically spherical and ~20-100 nm in diameter, lipid nanoparticles (LNPs) could unexpectedly be produced in a variety of shapes and sizes, as seen in the image. Along with particle size distribution, the fraction of particles which have multiple bilayers, multiple compartments (pink arrows), or contain other vesicles (green arrow) can be determined.

When a detailed 3-dimensional understanding of particle morphology and contents is required, cryo-electron tomography is used to image a small subset of particles.

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Frequently Asked Questions

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How many particles do you count, or images do you analyze per sample or grid?

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We collect ~60 images from different sections of each grid, usually including different ice thicknesses, and analyze the agreed upon number of particles (between 250 to over 1,000).

What additional information about LNPs can cryo-TEM detect that DLS cannot?

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DLS can provide titer, particle size, and polydispersity values for a sample. Cryo-TEM imaging can provide the same and more: average size, size distribution, shape, cargo encapsulation, morphology and lamellarity, aggregation, and more in one data set. Cryo-TEM provides direct, per-particle images of lipid nanoparticles in their near-native state. This allows us to visualize particle morphology and provide a quantitative analysis of morphological features such as the percentage of particles with bleb features, whether particles are multi- or unilamellar, and whether your formulation contains multicompartmental or multivesicular LNPs. To learn more about what can be seen with cryo-TEM, you can download our whitepaper here.

How well does cryo-TEM data correlate with DLS data for particle size distribution analysis of LNPs?

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Generally the average size as measured in cryo-TEM correlates with DLS data closely. However, we often see that DLS values are higher than those seen in cryo-TEM. DLS measures hydrodynamic diameter and large particles dominate the average value in DLS, as they scatter more. To see an in-depth comparison of cryo-TEM and DLS, you can read the paper “Dynamic light scattering and transmission electron microscopy in drug delivery: a roadmap for correct characterization of nanoparticles and interpretation of results” by Filippov et al.

What additional information about LNPs can cryo-TEM detect that the RiboGreen assay cannot?

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The RiboGreen assay can provide encapsulation efficiency, but will not tell you how many particles are empty versus contain nucleic acid. In addition, a Ribogreen assay will not tell you where the nucleic acid is located in an LNP. Cryo-TEM can give you an idea of nucleic acid distribution, and in addition can provide average size, size distribution, shape, morphology and lamellarity, aggregation, and more for LNPs in one data set.

How do you currently measure payload distribution for lipid nanoparticles?

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Currently, we are determining payload distribution manually but we are actively developing an automated, AI based program that will automatically provide these ratios. The presence of nucleotides in LNPs manifests as discernible puncta and/or striations. We visually classify the particles in your samples with cargo as either ‘loaded’ or ‘non-loaded’ based on the internal appearance of each particle. If you have non-loaded LNP’s we can use as a reference, please let your project manager know and we can set this up for you.

Can you tell the difference between RNA or DNA encapsulation in my LNPs?

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At this time, we cannot differentiate between RNA and DNA. However, DNA is generally easier to detect in cryo-TEM images than RNA as DNA is larger, and as a result we often see that LNPs that encapsulate DNA are generally also larger in size than LNPs that encapsulate RNA. Cryo-TEM imaging is able to reveal the existence and distribution of nucleic acid molecules throughout the oily (or lipid) phase as well as the aqueous phase in individual LNP particles. To learn more about how cryo-TEM can enable direct visualization of LNP payloads, you can read more here.

Can we measure RNA outside/inside/on the individual lipid nanoparticles?

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We can detect the presence of nucleotides inside the particles (e.g. payload distribution analysis), but we cannot determine the copy number or amount. It is possible to detect RNA outside particles; how well we can detect RNA outside particles likely depends on its length, amount, and type of RNA. We do not know the minimum detection limit (we have been able to easily detect a mRNA that was a few hundred bp long).

Is it possible to distribute between mRNA-loaded and aqueous blebs with cryo-TEM?

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Using cryo-TEM we can easily distinguish between aqueous blebs and lipidic compartments. Aqueous blebs have an internal density that is very much the same as the surrounding buffer in the images, whereas lipidic compartments (oily phase) is much more dense. If these aqueous blebs and lipidic compartments are evenly not dense/dense they are usually devoid of nucleic acid (or contain too little to detect by cryo-TEM). If these compartments show very dense puncta/striations then this is usually representative of nucleic acid being present.

Is the ability to detect and report an accurate percentage of blebbed areas more difficult as the particles become smaller?

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Not necessarily. However, reporting an accurate percentage of blebbed areas will be more challenging when blebs are smaller (independent of overall particle size). However, smaller blebs may in turn also affect overall functionality less than larger blebs. It is important to mention that blebs on top/bottom of LNPs (in viewing direction) cannot be seen in 2D images. Therefore, determining bleb percentages is most useful when comparing to a baseline batch or other batches.

How do you measure the PEG layer in lipid nanoparticle samples?

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We cannot directly visualize a PEG layer with cryo-TEM imaging, but oftentimes we can give an estimated PEG layer thickness in the summary based on the distance between regularly packed particles. The distance is measured between regularly packed particles (if present) across several images and a range is provided.

Are you able to see the PEG layer on LNPs with cryo-TEM? If so, how can you tell the difference between the PEG layer and lipid bilayer?

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It is typically not possible to directly visualize the PEG layer in cryo-TEM images. However, the packing of the particles and the distance between the particles can be used to infer its presence and potential thickness. However, if LNPs have a high surface charge this may be a bit more complicated. The lipid bilayer is clearly visible in cryo-TEM images, however, we cannot distinguish PEGylated lipid from other lipids in the bilayer.

Can cryo-TEM determine the number of antibodies conjugated on LNP surfaces?

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Generally no. LNPs are heterogeneous in size and often in morphology, so averaging techniques do not work here. However, if antibody binding/conjugation is high enough, densities around the LNP could possibly be detected. Another method to visualize bound antibodies is cryo electron tomography. Using this technique we are able to look at a 3-dimensional reconstruction of LNP(s). Gold conjugated secondary antibodies can be used as an alternative method to evaluate the presence of antibodies on the LNP surface.

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