The Bleb Blog: A Deep Dive with Cryo-TEM

Questions About Blebs?

While round, unilamellar particles are often thought of as typical lipid nanoparticles (LNPs), particles with multiple compartments are also commonly seen. These particles have one or more aqueous compartments referred to as blebs - a funny name for an important LNP feature.   

There are many open questions about LNP blebs: 

• How are they formed?
• Why do some particles have more or larger blebs? 
• What impact do they have on potency?
• What about empty blebs that don’t have any cargo?

Although much remains to be learned about blebs, what we do know has been learned using cryogenic transmission electron microscopy (cryo-TEM). Electron microscopy is the only technique currently available that can visualize blebs or determine the fraction of blebbed particles within a sample. TEM images can also show us the number and approximate size of blebs as well as whether blebs are empty or loaded.

What can we observe about LNP morphology and organization with cryo-TEM?

Blebs, Bilayers, & Beyond

The cryo-TEM image below (Figure 1) shows several unilamellar LNPs (teal), and LNPs with one bleb (green) or multiple blebs (pink).

There is a clear difference in morphology between the round unilamellar particles, and the blebbed particles with protruding compartments.

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Within the blebbed particles, we can also see variation in interior electron density below (Figure 2). Like the unilamellar particles, blebbed particles have one compartment that’s dense and representative of the lipid phase (pink arrows).

In addition, they also have aqueous compartments that are either not dense (teal arrows) or have a grainy or mottled texture (green arrows). Particles with multiple blebs can have a mixture of empty and loaded blebs.

This texture is the key to determining whether a bleb is loaded with nucleic acid or empty. Using an electron-dense stain that binds RNA in "Encapsulation State of Messenger RNA Inside Lipid Nanoparticles", Brader et. al. showed that the mottled texture they observed in blebs corresponded to RNA.

Finally, if we look closely at the boundaries of the blebs, we can see lipid bilayers (green arrow). These bilayers are thought to be rich in the lipids that form liposomal leaflet structures like DSPC and cholesterol.

Cryo-TEM can also reveal answers about:

• Automated Particle Size Distribution Analysis
• Particle Homogeneity
• PEG Layer & Bilayer Thickness
• Payload Distribution & Encapsulation Efficiency
• Formulation Impurities
• Sample Stability

What Does the Literature Say About Blebs?

Several studies have shown that the presence and proportion of blebbed particles is affected by conditions such as buffer and storage parameters. Are blebs good or bad for potency? There is no single answer.

In "Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles" Henderson et. al. imaged LNPs after dialysis into different buffers and found unilamellar particles regardless of whether TRIS, phosphate, or HEPES buffered saline was used. After a freeze thaw cycle, all three buffer conditions showed an increase in blebbed particles, but differences were seen in the extent of blebbing and whether the blebs contained RNA. All conditions showed reduced potency after freeze thaw.

On the other hand, in "Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency", Cheng et. al. showed that increased blebbing in freshly made LNPs was correlated with greater in vivo potency. LNPs formulated with pH 4 sodium citrate showed the majority of particles were blebbed. In contrast, only ~5% of LNPs were blebbed when formulated with sodium acetate of equivalent ionic strength. These and other experiments led the authors to propose a model for bleb formation that involves fusion of small vesicles with mRNA-LNPs at low pH, followed by phase separation and a shift of mRNA to the aqueous bleb upon neutralization.

The two studies of course have many distinctions that can potentially explain the different impact of blebs on potency. Two important ones are the method of bleb induction and the extent of RNA encapsulation seen in the blebs. The study finding a positive effect of blebs, looked at fresh LNPs and saw blebs which contained RNA, while the study that found decreased potency, induced blebs by freezing, and saw blebs which were often empty.

Ready to learn more about studies related to LNP blebs? Read Jens Simonsen’s insightful discussion of blebs in “A perspective on bleb and empty LNP structures”.

Unlock Your LNP Formulations with Cryo-TEM

Blebs may have a funny name, but their impact on lipid nanoparticle structure and function is no joke. Cryo-TEM continues to provide unparalleled insights into LNP morphology. As we continue to uncover how blebs form, their potential role in potency, and how formulation conditions influence their presence, one thing is clear - Cryo-TEM remains the gold standard for visualizing and characterizing the unexpected in LNPs.

Want to learn more about how cryo-TEM can help optimize your LNP formulations? Check out our whitepapers or reach out to set up a conversation!