The advent of the vaccines for SARS-COV-2 / COVID19 has generated an explosion in interest in creating new medicines based on their core technology: lipid nanoparticles (LNPs). LNPs package nucleic acids in a lipid coat that protects it from degradation. This allows the cargo to enter cells where it can then be released into the cytoplasm for synthesis of putatively therapeutic proteins.
While schematically simple, care must be taken in the design of both the cargo and the lipid formulation of the LNP in order to ensure sufficient delivery of potentially therapeutic mRNA, siRNA, or in some cases plasmid DNA.
LNP Components
The building blocks of LNPs are remarkably simple. A typical LNP will include an ionizable or cationic lipid, cholesterol, at least one phospholipid, and a polyethylene glycol (PEG) modified lipid. These lipid components are used to shield or encapsulate a nucleic acid cargo that encodes a therapeutic protein.
The type of cargo ranges from mRNA or siRNA to plasmid DNA and the choice of lipids in the formulation is influenced by the cargo type. The primary concern here is the choice of the ionizable lipid.
Ionizable Lipids
Ionizable lipids are the key component to building a fully functional lipid nanoparticle. These specialized lipids obtain a positive charge in acidic pH, which allows them to interact with and encapsulate nucleic acids which contain negative charges via their phosphate groups. Screening and selection of ionizable lipids is based on the type of nucleic acid cargo, the target tissue, and therapeutic application. Plasmid DNA (pDNA) and mRNA or siRNA may preferentially interact with structurally different classes of ionizable lipids. The relative molar percentage of the ionizable lipid or the addition of another charged lipid to the formulation can influence the surface charge of the LNP, which can in turn affect the biodistribution of the LNP.
| Name | Catalog # | Name | Catalog # |
|---|---|---|---|
| SM-102 | N-1102 | DLIN-MC3-DMA | N-1282 |
| ALC-0315 | N-1020 | LP-01 | N-1007 |
| Lipid 5 | N-1005 | cKK-E12 | N-1012 |
| 4A3-SC8 | N-1438 | C12-200 | N-1220 |
| 306Oi10 | N-1010 | YSK05 | N-1050 |
| 9A1P9 | N-1919 | Lipid 319 | N-1319 |
| Lipid 10 | N-1110 | AA3-Dlin | N-1003 |
| YHS-12 | N-1009 | PPZ-A10 | N-1410 |
| TT3 | N-1113 | ATX100 | N-1100 |
The need for new ionizable lipids is growing as the types of cells targeted for therapy and varieties of RNA continue to expand. Echelon’s chemists have years of expertise in lipid synthesis and are actively adding new ionizable lipids to our product catalog. Synthesis of novel ionizable or cationic lipid structures is also available through our chemistry group. Our LNP group can then pre-screen any new ionizable lipids with benchmark reporter mRNAs for encapsulation and transfection efficiency.
LNP Formulation
Once lipids have been selected and cargo prepared, LNP formulation can move forward. The current standard for this step is the use of automated microfluidic mixers that ensure reproducibility. These mixers allow for precise, individual control at which the organic phase lipids are combined with the aqueous phase cargo. This also allows for a great deal of flexibility in terms of the ratios at which the two components are mixed, which can be critical when dealing with unique RNAs.
At this point, it is critical to screen any potential lipid mixes with the desired cargo. Different ionizable lipids may not interact with a given cargo at similar efficacies. Therefore screening properties such as the N:P ratio (nitrogen content of the lipids:phosphate content of the nucleic acid) and the molar percentage of the ionizable lipid should be performed at minimum.
The type and amount of the helper lipid should also be evaluated before scaling up any formulation.
Analysis
Following formulation, the LNPs are subjected to an array of analytical methods to determine various physiochemical properties. These include encapsulation efficiency, surface charge (zeta potential), polydispersity, diameter, and molecular weight. Collectively, these measurements can be used as quality control indicators for batch to batch reproducibility as well as give insights into how specific compositions will affect biodistribution.
Additional assays to assess RNA integrity via chromatography and LNP morphology through cryo-EM may also be performed. While these additional analytical methods may not be immediately necessary in early stage R&D, they provide important data points that are not available through basic spectrophotometry methods and can reveal potential issues to address ahead of clinical development.
Ready to start your formulation project? Contact us about our LNP Services.