Lipid Nanoparticles - Delivering Nucleic Acids

Lipid nanoparticles (LNPs) have gained so much attention due to the launch of vaccines and therapeutics in the past few years. Comprised typically of four lipid components, not all LNPs are encapsulated with nucleic acids cargo, though the ratios of encapsulated/unencapsulated LNPs remain high.

In a recent publication, Munter et al. (J. Colloid Inter. Sci., 2024, 674, 139-144) examined and characterized the nucleic acid encapsulation within the nanoparticle assemblies. Using two different fluorescent probes, one having the affinity for lipids (e.g. DiIC18 (5)-DS). The other for nucleic acid (e.g. Cy3-RNA), the authors were able to determine the nucleic acid distribution between individual LNPs.

LNPs with Nucleic Acids

For example, in LNPs that are 40 nm in size prepared by microfluidic technology and containing SM-102 (Moderna vaccine) or ALC-0315 (Pfizer/BioNTech vaccine) ionizable lipids, only 15.6% were found to be empty. It was also true for Onpattro^® containing DLin-MC3-DMA, where only 10% were empty, further supporting that a significantly large proportions of LNPs are loaded with nucleic acid cargo, whether 996 nucleotides mRNA or 21 nucleotide siRNA.

In contrast with larger nucleotides cargo, e.g. 2700 nucleotides plasmid DNA in LNPs containing C12-200 ionizable lipid, an average of 26% LNPs were empty. With mRNA and siRNA using the same C12-200 ionizable lipid, only 7% and 2% of LNPs were empty, respectively.

The shear mixing rate also had an impact on empty vs loaded LNPs. For example, LNPs containing ALC-0315 ionizable lipid and mRNA, and prepared by low shear mixing (vortex) vs T-mixing (microfluidic NanoAssemblr), the ratio of empty and encapsulated LNPs was 3:1. This further demonstrates that the spontaneous self-assembly of LNPs yields higher loading efficiency.

Figure 1: NanoAssemblr Microfluidic Systems (Image courtesy of Cytiva).

Ascendia’s LNP Expertise and cGMP Capabilities

Taken collectively, this study sheds light on encapsulation efficiency of the nucleic acid cargo in lipids, which is dependent on nucleotide molecular weight, type and composition of cationic lipids, and also on the method for preparation. Ascendia^® Pharma’s expertise and cGMP capabilities, along with maintaining the library of novel ionizable lipids, can help formulate the nucleic acids and plasmids in LNPs by employing our state-of-the-art NanoAssemblr^® microfluid systems from Cytiva. Our team uses Ignite, Blaze and GMP equipment (Figure 1) to manufacture preclinical and clinical batches for Phase 1 trials and later phases.

Ascendia recently published an article in Drug Development & Delivery to shed light on LNPs/mRNA delivery. To learn more contact us today.