Self-Catalyzed Transesterification Transforms Ionizable Amphiphilic Janus Dendrimers into Multicomponent Vectors and Generates Complex Design Strategies for mRNA Delivery

Abstract Genetic nanomedicine delivers nucleic acids by viral and synthetic vectors to produce drugs directly in the body’s own cells. Each approach presents relative advantages, but synthetic vectors are distinguished by their effectively unlimited synthetic capabilities. The leading synthetic vectors are the four-component lipid nanoparticles (LNPs) used in the Pfizer and Moderna Covid-19 vaccines, which deliver mRNA to the liver. Inspired by this concept, our laboratories developed a one-component, multifunctional, sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) that delivers mRNA predominantly to the more desirable spleen, as well as to other organs. Benzoyl, benzyl, and aliphatic biodegradable esters connect the hydrophobic and hydrophilic domains of IAJDs. IAJDs have already been used in preclinical studies of therapeutics and vaccines, and several are commercially available. Here we report that, unexpectedly, the least stable benzyl-ester-based IAJDs, which contain the 1-(2-hydroxyethyl)piperazine ionizable amine (IA), undergo an extraordinarily slow, self-catalyzed transesterification at room temperature that transforms the one-component system into a multicomponent one. This reaction progressively extends the hydrophilic ionizable-amine domain from monomer to dimer, trimer, tetramer, and pentamer, generating a mixture of the original IAJD, its oligomers, and the released benzyl alcohol. Remarkably, at up to 50% transesterification, the in vivo activity of this multicomponent system remains similar to that of the parent one-component IAJD. Beyond providing a simple in situ method to convert one-component IAJDs into an unprecedented multicomponent IAJDs system, these results establish a new design strategy to construct the hydrophilic domain of one-component IAJDs through a complex, “vertical”, sequence-defined arrangement of IAs.