Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicles for RNA and have enabled the development of RNA-based drugs such as the mRNA COVID-19 vaccines. Functional delivery of mRNA by an LNP greatly depends on the inclusion of an ionizable lipid, and small changes to these lipid structures can significantly improve delivery. However, the structure–function relationships between ionizable lipids and mRNA delivery are poorly understood, especially for LNPs administered intramuscularly. Here, we show that the iterative design of a novel series of ionizable lipids generates key structure–activity relationships and enables the optimization of chemically distinct lipids with efficacy that is on-par with the current state of the art. We find that the combination of ionizable lipids comprising an ethanolamine core and LNPs with an apparent pKa between 6.6 and 6.9 maximizes intramuscular mRNA delivery. Furthermore, we report a nonlinear relationship between the lipid-to-mRNA mass ratio and protein expression, suggesting that a critical mass ratio exists for LNPs and may depend on ionizable lipid structure. Our findings add to the mechanistic understanding of ionizable lipids and demonstrate that hydrogen bonding, ionization behavior, and lipid-to-mRNA mass ratio are key design parameters affecting intramuscular mRNA delivery. We validate these insights by applying them to the rational design of new ionizable lipids. Overall, our iterative design strategy efficiently generates potent ionizable lipids. This hypothesis-driven method reveals structure–activity relationships that lay the foundation for the optimization of ionizable lipids in future LNP-RNA drugs. We foresee that this design strategy can be extended to other optimization parameters beyond intramuscular expression.

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Memories are often categorized into types, reflecting their behavioural, anatomical and molecular diversity: these classifications both aid understanding of the differences among varieties of memory and help delineate the unifying cross-species principles underlying them. In the nematode wormCaenorhabditis elegans, we find that an associative memory of the pairing of the normally attractive odorant benzaldehyde and starvation depends on de novo transcription and translation, is independent of CREB, and is produced by massed training: a pattern which does not correspond to any of the well-characterized molecular categories of invertebrate memory. Further, as has been shown for many memories in vertebrates, but not previously in nematodes, we find that formation of this memory continues after removal of the stimuli initially causing it, and that it is labile to disruption through protein synthesis inhibition following training, but that inhibition of proteasomal activity does not extend the duration of the memory. Previous findings have implicated insulin pathway signalling as a key component of this benzaldehyde/starvation memory, however we find that the transcriptional activity required for the memory is likely to be independent of the transcription factors that function at the terminus of this pathway. These findings better characterize this model associative memory in relation to other invertebrate memory types and identify ways in which it both shares their traits and differs from them.

Lipid nanoparticles (LNPs) and ribonucleic acid (RNA) technology are highly versatile tools that can be deployed for diagnostic, prophylactic, and therapeutic applications. In this report, supramolecular chemistry concepts are incorporated into the rational design of a new ionizable lipid, C3‐K2‐E14, for systemic administration. This lipid incorporates a cone‐shaped structure intended to facilitate cell bilayer disruption, and three tertiary amines to improve RNA binding. Additionally, hydroxyl and amide motifs are incorporated to further enhance RNA binding and improve LNP stability. Optimization of messenger RNA (mRNA) and small interfering RNA (siRNA) formulation conditions and lipid ratios produce LNPs with favorable diameter (<150 nm), polydispersity index (<0.15), and RNA encapsulation efficiency (>90%), all of which are preserved after 2 months at 4 or 37 °C storage in ready‐to‐use liquid form. The lipid and formulated LNPs are well‐tolerated in animals and show no deleterious material‐induced effects. Furthermore, 1 week after intravenous LNP administration, fluorescent signal from tagged RNA payloads are not detected. To demonstrate the long‐term treatment potential for chronic diseases, repeated dosing of C3‐K2‐E14 LNPs containing siRNA that silences the colony stimulating factor‐1 (CSF‐1) gene can modulate leukocyte populations in vivo, further highlighting utility.