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Abstract The introduction of more effective and selective mRNA delivery systems is required for the advancement of many emerging biomedical technologies including the development of prophylactic and therapeutic vaccines, immunotherapies for cancer and strategies for genome editing. While polymers and oligomers have served as promising mRNA delivery systems, their efficacy in hard-to-transfect cells such as primary T lymphocytes is often limited as is their cell and organ tropism. To address these problems, considerable attention has been placed on structural screening of various lipid and cation components of mRNA delivery systems. Here, we disclose a class of charge-altering releasable transporters (CARTs) that differ from previous CARTs based on their beta-amido carbonate backbone (bAC) and side chain spacing. These bAC-CARTs exhibit enhanced mRNA transfection in primary T lymphocytes in vitro and enhanced protein expression in vivo with highly selective spleen tropism, supporting their broader therapeutic use as effective polyanionic delivery systems. 

Organocatalyzed ring-opening polymerization (ROP) is a versatile technique for synthesizing biodegradable polymers, including polyesters and polycarbonates. We introduce o-phenylene bisurea (OPBU) (di)anions as a novel class of organocatalysts that are fast, easily tunable, mildly basic, and exceptionally selective. These catalysts surpass previous generations, such as thiourea, urea, and TBD, in selectivity (kp/ktr) by 8 to 120 times. OPBU catalysts facilitate the ROP of various monomers, achieving high conversions (>95%) in seconds to minutes, producing polymers with precise molecular weights and very low dispersities (Đ ≈ 1.01). This performance nearly matches the ideal distribution expected from living polymerization (Poisson distribution). Density functional theory (DFT) calculations reveal that the catalysts stabilize the oxyanion transition state via a hydrogen bond pocket similar to the "oxyanion hole" in enzymatic catalysis. Both experimental and theoretical analyses highlight the critical role of the semi-rigid o-phenylene linker in creating a hydrogen bond pocket that is tight yet flexible enough to accommodate the oxyanion transition state effectively. These new insights have provided a new class of organic catalysts whose accessibility, moderate basicity, excellent solubility, and unparalleled selectivity and tunability open up new opportunities for controlled polymer synthesis. 

Organocatalyzed ring-opening polymerization is a powerful tool for the synthesis of a variety of functional readily degradable polyesters and polycarbonates. We report the use of (thio)ureas in combination with cyclopropenimine bases as unique catalyst for the polymerization of cyclic esters and carbonates with a large span of reactivities. Methodologies of exceptionally effective and selective cocatalyst combinations were devised to produce polyesters and polycarbonates with narrow dispersity (Đ = 1.01 – 1.10). Correlations of the pKa of the various ureas and cyclopropenimine bases revealed the critical importance of matching the pKa of the two cocatalysts to achieve the most efficient polymerization conditions. It was found that promoting strong H-bonding interactions with a noncompetitive organic solvent, such as CH2Cl2, enabled greatly accelerated polymerization rates. The stereoselective polymerization of rac-lactide afforded stereoblock poly(lactides) that crystallize as stereocomplexes, as confirmed by wide-angle x-ray scattering. 

A library of structurally related heterocycles containing N-H motifs are explored as ring-opening polymerization (ROP) pre-catalysts. Upon deprotonation of these heterocycles with appropriate bases, corresponding salts are formed, which catalyze the ROP of various lactones and cyclic carbonates, affording polymers with dispersity values ranging from 1.01 to 1.12. These catalysts exhibit a wide range of catalytic activities, spanning over seven orders of magnitude (>107), with their relative rates generally correlating to the pKa of the N-H group in the neutral heterocycle. Despite apparent structural and electronic similarities, these heterocycle catalysts display markedly different kinetic behaviors regarding the identity of different cations. Kinetic and NMR studies have revealed two distinct sets of mechanisms: small alkali metal cations such as Li+ and Na+ reduce the activity of imidazol(in)e derived catalysts due to their tendency to associate with the alkoxide chain-end, thus inhibiting its propagation; conversely, these cations form robust cation-π assemblies with indolocarbazole anions, simultaneously binding and activating monomer carbonyls towards the nucleophilic attack, resulting in a significant rate enhancement. This distinctive activation motif of the indolocarbazole sets it apart from other catalysts by utilizing cations as a potent handle for modulating polymerization reactivity. Coupled with its high availability, good solubility, high activity, moderate basicity, and high selectivity, the indolocarbazole heterocycle emerges as one of the most versatile organocatalysts for ring-opening polymerization. 

Targeted delivery of nucleic acid therapeutics to the lungs could transform treatment options for pulmonary disease. We have previously developed oligomeric charge-altering releasable transporters (CARTs) for in vivo mRNA transfection and demonstrated their efficacy for use in mRNA-based cancer vaccination and local immunomodulatory therapies against murine tumors. While our previously reported glycine-based CART-mRNA complexes (G-CARTs/mRNA) show selective protein expression in the spleen (mouse, >99%), here, we report a new lysine-derived CART-mRNA complex (K-CART/mRNA) that, without additives or targeting ligands, shows selective protein expression in the lungs (mouse, >90%) following systemic IV administration. We further show that by delivering siRNA using the K-CART, we can significantly decrease expression of a lung-localized reporter protein. Blood chemistry and organ pathology studies demonstrate that K-CARTs are safe and well-tolerated. We report on the new step economical, organocatalytic synthesis (two steps) of functionalized polyesters and oligo-carbonate-co-α- aminoester K-CARTs from simple amino acid and lipid-based monomers. The ability to direct protein expression selectively in the spleen or lungs by simple, modular changes to the CART structure opens fundamentally new opportunities in research and gene therapy.