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Seeing the Forest AND the Trees: LNP Analyses with Cryo-TEM 

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Dr. Eric Bushong

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Seeing the Forest AND the Trees: LNP Analyses with Cryo-TEM 

Lipid Nanoparticles (LNPs) Deliver Novel Medicines

Scientific render of bilayer lipid nanoparticle

One of the biggest challenges for developing any new therapeutic agent is devising an effective delivery mechanism. A vitally important innovation in this area in the past few decades has been the advent of lipid nanoparticles (LNPs). There are several different types of lipid nanoparticles, including niosomes, liposomes, solid lipid nanoparticles, and nanostructured lipid carriers, each offering advantages for delivering different classes of drugs (Thi et al., 2021).

Liposomes consist of one or more lipid bilayers enclosing an aqueous compartment and can be used to deliver both hydrophilic and hydrophobic drugs. The development of ionizable lipids and novel formulation methods began to allow payloads of genetic material to be efficiently and safely delivered by solid lipid nanoparticles. These lipid nanoparticles can carry various forms of RNA or DNA to cells, allowing for regulation of protein expression. This technology is now being widely employed in the development of gene therapies and vaccines, including recently approved drugs such as patisiran and COVID-19 vaccines.

Visualizing LNPs: Then and Now

Cryotransmission electron microscopy (cryo-TEM) has long played a pivotal role in the characterization of lipid nanoparticles. In the earliest studies on the development of liposomes, negative staining TEM transmission electron microscopy (TEM) was typically used to show their basic shape and size (Thi et al., 2021). Unfortunately, negative staining leads to significant disruption of the morphology of lipid-based particles and does not allow for visualization of the internal structure and contents of particles. 

By the 1990s, cryo-TEM was being used to avoid these issues (Lasic et al., 1992; Templeton et al., 1997). By instantly vitrifying samples, particles are preserved in their near-native hydrated state. Since image contrast in cryo-TEM is based on the inherent density of the particles, instead of heavy metal staining, it is possible to see detailed features of the composition and structure of particles. Cryo-TEM has been critical for developing an understanding of how lipid nanoparticles form and how they encapsulate drugs (Kulkarni et al, 2018).

A Modern Toolkit for Analytical Characterization of LNPs

The proper characterization of nanoparticles requires an assessment of numerous properties, including size, morphology, encapsulation efficiency, aggregation, uniformity, and presence of contamination. There are a number of techniques that can provide important data on these features, including cryo-TEM, dynamic light scattering (DLS), zeta potential measurement, small angle x-ray scattering, small angle neutron scattering, and nuclear magnetic resonance. A combination of these approaches is necessary to completely characterize any sample.

Cryo-TEM distinguishes itself from other techniques by allowing for the examination of populations composed of individual particles, as opposed to bulk measurements on samples. By examining individual particles, it is possible to assess heterogeneity within samples more accurately. For example, it has been shown that using DLS to perform bulk measurement of the size of solid lipid nanoparticles can lead to significantly skewed results, with the smallest particles within the sample underrepresented (Crawford et al., 2011). 

Additionally, cryo-TEM excels compared to most other techniques in the range of questions that can be addressed in a single study. With a cryo-TEM study, it is possible to simultaneously assess particle size, particle morphology, drug encapsulation, particle uniformity, aggregation, and contamination. This can all be done with as little as 3 microliters of sample. Unlike any other characterization method, cryo-TEM leverages the power of focusing on individual particles (the trees) to help scientists better understand the different populations in any given sample (the forest).

Cryo-TEM is Within Your Reach

The investment in infrastructure and instrumentation to perform cryo-TEM is considerable, and the expertise needed to gain insights from the technique takes years of training. These factors have left people incorrectly believing the power of cryo-TEM is out of reach. NanoImaging Services has mastered all aspects of cryo-TEM and collaborates with researchers to quickly and affordably solve characterization challenges that cannot be addressed with other methods, helping clients gain a competitive edge in vaccine and therapeutics development.

Contact us now to schedule your cryo-EM consultation or learn about our Nanoparticle Characterization services.

Citations
  • Crawford, R.; Dogdas, B.; Keough, E.; Haas, R.M.; Wepukhulu, W.; Krotzer, S.; Burke, P.A.; Sepp-Lorenzino, L.; Bagchi, A.; Howell, B.J. Analysis of Lipid Nanoparticles by Cryo-EM for Characterizing siRNA Delivery Vehicles. Int J Pharm 2011, 403, 237. https://doi.org/10.1016/j.ijpharm.2010.10.025
  • Kulkarni, J.A.; Darjuan, M.M.; Mercer, J.E.; Chen, S.; van der Meel, R.; Thewalt, J.L.; Tam, Y.Y.C.; Culllis, P.R. On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA. ACS Nano 2018, 21, 4787. https://doi.org/10.1021/acsnano.8b01516
  • Lasic, D.D.; Frederik, P.M.; Stuart, M.C.A.; Barnholz, Y.; McIntosh, T.J. Gelation of Liposome Interior. A Novel Method for Drug Encapsulation. FEBS Lett 1992, 312, 255. https://doi.org/10.1016/0014-5793(92)80947-f
  • Templeton, N.S.; Lasic, D.D.; Frederik, P.M.; Strey H.H.; Roberts, D.D.; Pavlakis, G.N. Improved DNA:Liposome Complexes for Increased Systemic Delivery and Gene Expression. Nat Biotech 1997, 15, 647. https://doi.org/10.1038/nbt0797-647
  • Thi, T.T.H.; Suys, E.J.A.; Lee, J.S.; Nguyen, D.H.; Park, K.D.; Truong, N.P. Lipid-Based Nanoparticles in the Clinic and Clinical Trials: From Cancer Nanomedicine to COVID-19 Vaccines. Vaccines 2021, 9, 359. https://doi.org/10.3390/vaccines9040359

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