Seeing the Building Blocks: Direct Imaging of Payload Distribution of DNA and RNA in Lipid Nanoparticles

Lipid nanoparticles (LNPs) have gained significant traction in the therapeutic world due to their ability to encapsulate and deliver a variety of substances to specific target cells or tissues. LNPs are used to deliver both DNA and RNA for therapeutic formulations including vaccines, such as recent mRNA COVID-19 vaccines, gene delivery and editing, and drug delivery for neurodegenerative, cardiovascular, and other rare diseases. 

LNPs are ideal for delivering DNA and RNA due to their biocompatibility, as they are often composed of materials that are biocompatible, meaning they are well-tolerated by the body and have minimal toxic effects. This is crucial for safe and effective gene and drug delivery. Further, LNPs are efficient carriers for delivering DNA into cells, as they can fuse with the cell membrane, allowing the DNA payload to enter the cells' cytoplasm, where it can be utilized for gene expression. LNPs are able to shield delicate DNA and RNA from degradation by enzymes or nucleases, increasing its stability and allowing it to reach its target cells intact.

When selecting whether to use DNA or RNA in LNPs, researchers and developers need to consider several factors. Both DNA- and RNA-based technologies can be used as vaccine or therapeutic treatments. DNA is often used for gene replacement and gene editing and has long-term effects, but encapsulation and delivery is challenging due to its large size and structural complexity. RNA (with mRNA, siRNA, CRISPR-Cas9 components, cRNA as examples used for protein expression, gene silencing, gene editing, etc) on the other hand is preferred for rapid protein expression and is less likely to trigger an immune response, but has a transient effect.

Cryo transmission electron microscopy (cryo-TEM), can be used to image LNPs and visualize the payload distribution of DNA or mRNA encapsulated in LNPs. Other indirect methods including SEC-MALS-UV-dRI and nano Flow Cytometry can be used to perform size-dependent RNA content analysis of LNPs. However, these techniques do not take into account that certain particles have fewer nucleic acid molecules encapsulated or none at all compared to others. Currently, the only technique to study per-particle payload, without the need for labeling, is cryo-TEM.

It is vital to understand if DNA or RNA payload is distributed evenly throughout your lipid nanoparticles and to assess the percent of full, and empty LNPs in order to corroborate other analytical techniques and assist in calculating dosage of final formulations. 

As the only GMP compliant cryo-TEM lab in North America, NIS is uniquely positioned to help analyze your final formulation. Contact us to schedule a meeting with our expert scientists to learn more.

A Snapshot of DNA in Lipid Nanoparticles

Cryo-TEM studies can image LNPs with DNA in order to show if they are evenly encapsulated. In the cryo-TEM image below, an evenly striated pattern reveals that DNA has been evenly distributed throughout the unilamellar lipid nanoparticles.

Direct Visualization of RNA in Lipid Nanoparticles

mRNA encapsulation can be viewed directly with cryo-TEM imaging. In the below images, cryo-TEM imaging reveals LNPs loaded with mRNA (Figure A, loaded particles = pink arrows, nonloaded particles = green arrows) and an empty formulation of nonloaded particles (Figure B). Further, cryo-TEM can identify lamellarity and other morphological features including whether individual particles are multivesicular, multicompartmental, unilamellar, and whether it is round and evenly shaped, or irregularly shaped as shown in Figure C.

To learn more about how cryo-TEM imaging can help you, contact us to schedule a meeting.