Efficient viral or nonviral delivery of nucleic acids is the key step of genetic nanomedicine. Both viral and synthetic vectors have been successfully employed for genetic delivery with recent examples being DNA, adenoviral, and mRNA-based Covid-19 vaccines. Viral vectors can be target specific and very efficient but can also mediate severe immune response, cell toxicity, and mutations. Four-component lipid nanoparticles (LNPs) containing ionizable lipids, phospholipids, cholesterol for mechanical properties, and PEG-conjugated lipid for stability represent the current leading nonviral vectors for mRNA. However, the segregation of the neutral ionizable lipid as droplets in the core of the LNP, the "PEG dilemma", and the stability at only very low temperatures limit their efficiency. Here, we report the development of a onecomponent multifunctional ionizable amphiphilic Janus dendrimer (IAJD) delivery system for mRNA that exhibits high activity at a low concentration of ionizable amines organized in a sequence-defined arrangement. Six libraries containing 54 sequence-defined IAJDs were synthesized by an accelerated modular-orthogonal methodology and coassembled with mRNA into dendrimersome nanoparticles (DNPs) by a simple injection method rather than by the complex microfluidic technology often used for LNPs. Forty four (81%) showed activity in vitro and 31 (57%) in vivo. Some, exhibiting organ specificity, are stable at 5 °C and demonstrated higher transfection efficiency than positive control experiments in vitro and in vivo. Aside from practical applications, this proof of concept will help elucidate the mechanisms of packaging and release of mRNA from DNPs as a function of ionizable amine concentration, their sequence, and constitutional isomerism of IAJDs.

Viral and synthetic vectors for delivery of nucleic acids impacted genetic nanomedicine by aiding the rapid development of the extraordinarily efficient Covid-19 vaccines. Access to targeted delivery of nucleic acids is expected to expand the field of nanomedicine beyond most expectations. Both viral and synthetic vectors have advantages and disadvantages. The major advantage of the synthetic vectors is their unlimited synthetic capability. The four-component lipid nanoparticles (LNPs) are the leading nonviral vector for mRNA used by Pfizer and Moderna in Covid-19 vaccines. Their synthetic capacity inspired us to develop a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) delivery system for mRNA. The first experiments on IAJDs provided, through a rational-library design combined with orthogonal-modular accelerated synthesis and sequence control in their hydrophilic part, some of the most active synthetic vectors for the delivery of mRNA to lung. The second experiments employed a similar strategy, generating, by a less complex hydrophilic structure, a library of IAJDs targeting spleen, liver, and lung. Here, we report preliminary studies designing the hydrophobic region of IAJDs by using dissimilar alkyl lengths and demonstrate the unexpectedly important role of the primary structure of the hydrophobic part of IAJDs by increasing up to 90.2-fold the activity of targeted delivery of mRNA to spleen, lymph nodes, liver, and lung. The principles of the design strategy reported here and in previous publications indicate that IAJDs could have a profound impact on the future of genetic nanomedicine.

Targeted and efficient delivery of nucleic acids with viral and synthetic vectors is the key step of genetic nanomedicine. The four-component lipid nanoparticle synthetic delivery systems consisting of ionizable lipids, phospholipids, cholesterol, and a PEG-conjugated lipid, assembled by microfluidic or T-tube technology, have been extraordinarily successful for delivery of mRNA to provide Covid-19 vaccines. Recently, we reported a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) synthetic delivery system for mRNA relying on amphiphilic Janus dendrimers and glycodendrimers developed in our laboratory. Amphiphilic Janus dendrimers consist of functional hydrophilic dendrons conjugated to hydrophobic dendrons. Co-assembly of IAJDs with mRNA into dendrimersome nanoparticles (DNPs) occurs by simple injection in acetate buffer, rather than by microfluidic devices, and provides a very efficient system for delivery of mRNA to lung. Here we report the replacement of most of the hydrophilic fragment of the dendron from IAJDs, maintaining only its ionizable amine, while changing its interconnecting group to the hydrophobic dendron from amide to ester. The resulting IAJDs demonstrated that protonated ionizable amines play dual roles of hydrophilic fragment and binding ligand for mRNA, changing delivery from lung to spleen and/or liver. Replacing the interconnecting ester with the amide switched the delivery back to lung. Delivery predominantly to liver is favored by pairs of odd and even alkyl groups in the hydrophobic dendron. This simple structural change transformed the targeted delivery of mRNA mediated with IAJDs, from lung to liver and spleen, and expands the utility of DNPs from therapeutics to vaccines.

The mixed-ligand system consisting of tris(2-aminoethyl)amine (TREN) and tris(2-dimethylaminoethyl)amine (Me 6 -TREN) during the Cu(0) wire-catalyzed single electron transfer-living radical polymerization (SET-LRP) of methyl acrylate (MA) in "programmed" biphasic mixtures of the dipolar aprotic solvents NMP, DMF, and DMAc with H 2 O is reported. Kinetic and chain end analysis studies by NMR and MALDI-TOF before and after thio-bromo "click" reaction demonstrated that Me 6 -TREN complements and makes the less expensive TREN a very efficient ligand in the absence of externally added Cu(II)Br 2 . Statistical analysis of the kinetic data together with control experiments demonstrated that this mixed-ligand effect enhanced the apparent rate constant of propagation, monomer conversion, and molecular weight control. The most efficient effect was observed at a 1/1 molar ratio between these two ligands, suggesting that in addition to a fast exchange between the two ligands, a new single dynamic ligand generated by hydrogen bonding may be responsible for the mixed ligand observed.

A mixed-ligand effect was observed for mixtures of tris(2-dimethylaminoethyl)amine (Me 6 -TREN) with tris(2aminoethyl)amine (TREN) ligands during Cu(0) wire-catalyzed, single-electron transfer-living radical polymerization (SET-LRP) of methyl acrylate (MA) initiated with bis(2-bromopropionyl)ethane (BPE) in DMSO. The external order of reaction of SET-LRP both in the presence of Me 6 -TREN, TREN and of the mixedligand Me 6 -TREN/TREN, in DMSO, demonstrated a catalytic activity for DMSO similar to that reported in the presence of Cu(0) powder. The catalytic activity of DMSO, with close to 100% chain-end functionality, facilitates the much less expensive TREN to act as a very efficient ligand that is competitive with Me 6 -TREN and with the mixed-ligand and revitalizes TREN into an excellent ligand. The highest activity of the mixed-ligand at 1/1 ratio between ligands suggests that in addition to a fast exchange between these two ligands, a new single dynamic ligand stabilized by hydrogen-bonding, may generate these results.