Lipid Nanoparticles for RNA Delivery

RNA-based therapeutics have been under development for over 30 years and the first medication (ONPATTROTM) was approved in 2018 for treatment of patients suffering from transthyretin-mediated amyloidosis. However, the COVID-19 pandemic has pushed them into the limelight with the rapid development of mRNA-based vaccines against the SARS-Cov2 virus by Moderna and BioNTech/Pfizer. These vaccines have proven to be remarkably effective and safe. A key part of the technology are the lipid nanoparticles (LNPs) used to deliver RNA. Naked RNA cannot simply be injected as it is immunogenic, easily susceptible to enzymatic degradation, and is not taken up by cells. To overcome these problems, RNA is packaged up in LNPs that protect it from degradation while circulating, allow it to enter cells, then release the contents into the cytoplasm so the RNA can be used by ribosomes to synthesize the desired protein(s).

Decades of research have gone into developing and formulating the lipids that make up effective LNPs. Essentially, 4 types of lipids are required to formulate LNPs (approximate proportions in brackets):

  • Ionizable Lipid. This is the key component of the LNP (35-50%) and has two main roles: bind the RNA and allow release of the RNA in the cell. The pKa of the lipid is an important factor as the lipid needs to be positively charged at low pH in the LNP to bind RNA but uncharged at neutral pH so the LNP does not cause toxicity. Hundreds of lipids were synthesized and screened by many research groups to identify those with the desired properties and effects. Some successful candidates include ALC-0315, cKK-E12, SM-102, and Dlin-MC3-DMA.
  • PEGylated lipid. A small amount of a PEG derivatized lipid (0.5-3%) is incorporated to increase the circulatory half-life in the body. PEG-lipids have also been used for years in liposomal drug delivery systems creating so-called “stealth” liposomes. In addition, the percentage of PEG-lipid influences the size of the LNP. Examples include ALC-0159, DSPE-mPEG, and DMG-mPEG.
  • Cholesterol. Cholesterol is a structural “helper” lipid that makes up a significant part of the LNP (40-50%) and improves efficacy possibly by promoting membrane fusion and promoting endosomal escape.
  • Neutral phospholipid. Synthetic phospholipids such as DSPC, DPPC, and DOPE (~10%) are also commonly used as structural “helper” lipids in LNP formulation to promote cell binding.

To manufacture RNA-containing LNPs, the lipids in ethanol and the RNA in low pH buffer are rapidly mixed in a microfluidic mixer. The ionizable lipid is protonated and begins to bind and encapsulate the RNA. The pH is gradually increased to 7.4 and the ethanol is removed, completing the formation of the LNPs (Figure 1). The efficiency of the RNA encapsulation is quite high using microfluidic mixing compared with traditional extrusion used for liposomes. The characteristics of the LNP formulation such as particle size, surface charge, tissue targeting, etc. can be tailored by the type and ratio of lipids in the LNP.

Lipid Nanoparticle for RNA - Echelon Biosciences

Figure 1: An example of the components of a lipid nanoparticle (LNP). An outer lipid coat composed of ionizable lipids, PEGylated lipids, cholesterol, and helper phospholipids surrounds a core of ionizable lipids encapsulating the RNA cargo.

After the LNPs are injected, the particles are taken up by cells in endosomes. The pH decreases during endosomal maturation protonating the ionizable lipids and causing the endosomes to burst, releasing the LNPs into the cytosol. The higher pH of the cytosol triggers the LNPs to dissociate, releasing the RNA for ribosomal translation and protein expression to produce the desired effect.

The RNA-based vaccines currently being administered to prevent COVID-19 are the culmination of two decades of research and their incorporation into LNPs is a key factor in their success. In addition, there are countless applications for other vaccines and therapeutics beyond the current pandemic.  With increasing numbers of lipids for LNP generation now available to scientists, how will you be able to use them for your research?


1. Buschman MD, Carrasco, MJ, et al. (2021) “Nanomaterial Delivery Systems for mRNA Vaccines” Vaccines 9:65

2. Tenchov, R, Bird, R, et al. “Lipid Nanoparticles – From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement” ACS Nano DOI:10.1021/acsnano.1c04996 

3. Guevara ML, Persano F and Persano S (2020) “Advances in Lipid Nanoparticles for mRNA-Based Cancer Immunotherapy.” Front. Chem. 8:589959. doi: 10.3389/fchem.2020.589959 

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