The Constitutional Isomerism of One-Component Ionizable Amphiphilic Janus Dendrimers Orchestrates the Total and Targeted Activities of mRNA Delivery.

Constitutional isomerism has been previously demonstrated by one of our laboratories to represent a powerful design strategy for the elaboration of complex functional self-organizations. Here we report the design, synthesis, and characterization of 14 positional, skeletal, and functional constitutional isomeric one-component, multifunctional, sequence-defined, amphiphilic ionizable Janus dendrimers (IAJDs). Their coassembly by simple injection with luciferase mRNA (Luc-mRNA) to form dendrimersome nanoparticles (DNPs) was studied. Subsequently, the resulting DNPs were employed to investigate, with screening experiments, the delivery of Luc-mRNA in vivo. Constitutional isomerism was shown to produce changes of up to two orders of magnitude of the total-body luciferase activity and targeted luciferase activity to the spleen and liver, of up to three orders of magnitude difference in targeted luciferase activity to the lungs and up to six orders of magnitude to lymph nodes. These results indicate that constitutional isomerism may represent not only a simple but also an important synthetic strategy that most probably may impact the activity of all components of synthetic vectors used in RNA-based nanomedicine, including in mRNA vaccines and therapeutics.

Screening Libraries to Discover Molecular Design Principles for the Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers Derived from Plant Phenolic Acids.

Viral and synthetic vectors to deliver nucleic acids were key to the rapid development of extraordinarily efficient COVID-19 vaccines. The four-component lipid nanoparticles (LNPs), containing phospholipids, PEG-conjugated lipids, cholesterol, and ionizable lipids, co-assembled with mRNA via a microfluidic technology, are the leading nonviral delivery vector used by BioNTech/Pfizer and Moderna to access COVID-19 mRNA vaccines. LNPs exhibit a statistical distribution of their four components when delivering mRNA. Here, we report a methodology that involves screening libraries to discover the molecular design principles required to realize organ-targeted mRNA delivery and mediate activity with a one-component ionizable multifunctional amphiphilic Janus dendrimer (IAJD) derived from plant phenolic acids. IAJDs co-assemble with mRNA into monodisperse dendrimersome nanoparticles (DNPs) with predictable dimensions, via the simple injection of their ethanol solution in a buffer. The precise location of the functional groups in one-component IAJDs demonstrated that the targeted organs, including the liver, spleen, lymph nodes, and lung, are selected based on the hydrophilic region, while activity is associated with the hydrophobic domain of IAJDs. These principles, and a mechanistic hypothesis to explain activity, simplify the synthesis of IAJDs, the assembly of DNPs, handling, and storage of vaccines, and reduce price, despite employing renewable plant starting materials. Using simple molecular design principles will lead to increased accessibility to a large diversity of mRNA-based vaccines and nanotherapeutics.