Breaking Boundaries: Exploring the Advantages of Flexible Lipid Nanoparticle Formulations

Using dramatic graphics to show Earth in flames or shattering like glass seeks to amplify scientific and mainstream understanding of human impact on the planet.

Lipid nanoparticles (LNPs) based on ionizable lipids have demonstrated advantages, including high nucleic acid encapsulation and transfection efficiency, low toxicity, and immunogenicity.


Lipid nanoparticles have the potential to revolutionize mRNA delivery. However, mRNA-LNPs are still relatively new and face several challenges that have to be overcome to gain clinical application, such as lipid nanoparticle stability and mRNA innate immune activation.

Most mRNA-LNP vaccines must be stored under low temperatures due to their instability. Understanding the root cause of this instability is essential to optimize mRNA-LNP formulations and ease temperature requirements for storage. The latest structural data suggest that mRNA-LNPs comprise a core surrounded by ionizable cationic lipids and water, with neutral helper lipids positioned in an outer encapsulating wall. This means that mRNA is exposed to the milieu inside the LNP core, and a better understanding of how this affects mRNA stability could reduce the requirement for low temperatures.

Additionally, mRNA not encapsulated within the LNPs degrades quickly and cannot be taken up by cells for translation. It is, therefore, essential to identify which of the two components of mRNA-LNPs is the bottleneck for stability, as this may lead to targeted optimization of excipients or formulation milieu in mRNA-LNP vaccines.


While it can improve mRNA stability by optimizing the 5′ cap, poly-A tail, and untranslated regions (UTRs) of mRNA to avoid unwanted innate immune activation, it can also be essential to optimize formulation excipients to enable lyophilization. This may allow for stable storage of mRNA encapsulated within LNPs.

In addition, flexible lipid nanoparticle is a promising drug delivery system for mRNA due to their lipid bilayer composition and ability to protect mRNA from degradation in harsh conditions. However, it is critical to understand the physicochemical properties of mRNA and the lipid composition of the resulting lipid-nanoparticle complexes to improve stability.

For example, a high GC content in the mRNA coding sequence and codon optimization by selecting ‘frequent’ codons increase mRNA translation in vivo and its stability in the presence of LNPs. In addition, an excellent buffering system and an osmolyte are essential for maintaining the pH stability of mRNA-LNP formulations. This will be particularly important for cold chain applications. A sound buffering system should withstand pH drops of up to 3.5 units in the mRNA-LNP solution upon cooling below 0 deg C.


mRNA in its naked state degrades very quickly in aqueous solution. Thus, it requires specialized storage and freezing techniques to keep it stable for longer.

When encapsulated in LNPs, however, the mRNA is protected from degradation. It is also able to bind to a variety of cell surface receptors and initiate translation.

It is important to note, though, that in some assays, the mRNA needs to be released from the LNP to be measured. This process may impact the integrity of the mRNA. Therefore, the mRNA should be measured in its encapsulated form to avoid false positives.

Physical degradation of mRNA-LNPs is usually due to aggregation, fusion, or leakage of the mRNA payload. These properties can be controlled by modifying the lipid composition and shape of the NP. For example, ellipsoids and discoid shapes can localize to blood vessels more effectively than spheres because they have higher aspect ratios. An explicitly designed to research and develop lipid nanoparticle formulations, allowing scientists to optimize their process conditions before scaling up to a GMP-compliant system.


The platform is flexible enough to allow researchers to screen various formulations. The instrument was used here to prepare mRNA-encapsulating liposomes using a rationally selected lipid mix (926027). The microfluidic system generates uniform particles with a controlled and precise size distribution, allowing scientists to identify formulations with an optimal zeta potential for complexation with negatively charged mRNA.

The lipid-mRNA complex is then subjected to a simple, high-throughput fluorescence assay (standard curve measured by measuring the luminescence of mRNA-dye vs. a known mRNA concentration) to determine the mRNA’s encapsulation efficiency. The dilution protocol can be changed to produce mRNA-encapsulated lipid nanoparticles at the required attention. This is especially useful for proof of concept testing, as pure nucleotides are expensive.

Once the mRNA-LNP formulation has been optimized, production can be scaled up. The system was designed with lipid-mRNA process development and optimization in mind – the liquid flow path of the instrument can be configured to include or exclude dilution following lipid nanoparticle formation, allowing you to produce lipid nanoparticles containing your mRNA of choice in quantities that are precisely what is needed for pre-clinical studies.


In general, mRNA is highly unstable in the body. It needs to be encapsulated for long-term delivery to the cell. In contrast to protein therapeutics that target receptors on cells, mRNA must enter the cell to activate its function. This makes mRNA-LNPs even more challenging to formulate.

The stability of mRNA-LNPs can be influenced by their core composition, lipid bilayer, and the mRNA itself. The mRNA must be protected long enough to reach the cell and initiate its function. It also must be stable against innate immune activation.

One prevalent idea is to optimize the 5′ cap and poly-A tail of mRNA, which controls stability and translation so RNase can cleave it. Another approach is to increase translation efficiency by optimizing mRNA’s untranslated regions (UTRs).

Another challenge is determining if the problem with the stability of mRNA-LNPs is the mRNA itself or the lipid bilayer. It is possible to use gel electrophoresis to assess mRNA quality. Depending on the degradation or strand breaks, the mRNA bands will either broaden or disappear completely, and new bands appear.