Go green: Isobutyric acid is a high‐volume industrial chemical produced from non‐sustainable petroleum feedstocks and toxic materials. A synthetic metabolic pathway is constructed in E. coli to enable the biosynthesis of this compound from glucose, a renewable carbon source. The biosynthetic approach is an attractive option from both an economical and environmental point of view.

The liver is an ideal target for nucleic acid therapeutic applications (i.e., siRNA, gene therapy, and genome editing) due to its ability to secrete proteins into the blood. In this work, we present the first synthesis of a novel monomer derived from N-acetyl-d-galactosamine (GalNAc) and its polymerization as a facile route to create multivalent delivery vehicles with exceptional targeting efficiency to asialoglycoprotein receptors (ASGPRs) on liver hepatocytes. A series of cationic diblock GalNAc glycopolymers composed of a GalNAc-derived block of fixed length (n = 62) and cationic 2-aminoethylmethacrylamide (AEMA) blocks of varying lengths (n = 19, 33, and 80) have been synthesized and characterized. In addition, nontargeted control polymers consisting of either glucose or polyethylene glycol-derived neutral blocks with an AEMA cationic block were also created and examined. All polymeric vehicles were able to bind and encapsulate plasmids (pDNA) into polymer-pDNA complexes (polyplexes). The GalNAc-derived polyplexes were colloidally stable and maintained their size over a period of 4 h in reduced-serum cell culture media. The GalNAc-derived homopolymer effectively inhibited the uptake of Cy5-labeled asialofetuin (a natural ligand of ASGPRs) by cultured hepatocyte (HepG2) cells at lower concentrations (IC50 = 20 nM) than monomeric GalNAc (IC50 = 1 mM) and asialofetuin (IC50 = 1 μM), suggesting highly enhanced ASGPR binding due to multivalency. These polymers also showed cell type-specific gene expression in cultured cells, with higher protein expression in ASGPR-presenting HepG2 than HeLa cells, which lack the receptor. Biodistribution studies in mice show higher accumulation of pDNA and GalNAc-derived polymers in the liver compared with the glucose-derived nontargeted control. This study demonstrates the first facile synthesis of a multivalent GalNAc-derived block copolymer architecture that promotes enhanced delivery to liver and offers insights to improve targeted nanomedicines for a variety of applications.

The complexation of linear double stranded DNA and poly(styrenesulfonate) (PSS) with cationic poly(dimethylamino ethyl methacrylate)-block-poly(n-butyl methacrylate) micelles was compared in aqueous solutions at various pH values and ionic strengths. The complexation process was monitored by turbidimetric titration, as a function of the ratio (N/P) of amine groups in the micelle corona to the number of phosphates (or sulfonates) in the polyanion. The size, structure and stability of the resulting micelleplexes were studied by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM). In the short chain regime, where the contour lengths of the polyanions are shorter than or comparable to the micelle corona thickness, micelleplexes with DNA oligomers show very similar behavior to complexes with short PSS chains, in terms of titration curves and structural evolution of the complexes as a function of charge ratio. However, in the long chain regime, where the contour length of the polyanion far exceeds the micelle radius, micelleplexes of linear DNA show titration curves shifted toward lower N/P ratios, reduced stability at N/P < 1, and a higher percentage of small complexes at N/P > 1 compared to complexes with long chain PSS. Furthermore, at 1 M ionic strength, the cationic micelles could still complex with long chain PSS, but not with DNA of the same total charge. These differences are attributed to the flexibility difference between the polyanion chains, and possible mechanisms are proposed. This work highlights the importance of chain flexibility in complexation of dissimilar polyelectrolyte pairs, a factor that could therefore help guide the future design of micelleplexes for various applications.

A series of 3-guanidinopropyl methacrylamide (GPMA)-based polymeric gene delivery vehicles were developed via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers have been evaluated for their cellular internalization ability, transfection efficiency, and cytotoxicity. Two homopolymers: P(GPMA), P(GPMA), were synthesized to study the effect of guanidium polymer length on delivery efficiency and toxicity. In addition, an N-acetyl-d-galactosamine (GalNAc)-based hydrophilic block was incorporated to produce diblock polymers, which provides a neutral hydrophilic block that sterically protects plasmid-polymer complexes (polyplexes) from colloidal aggregation and aids polyplex targeting to hepatocytes via binding to asialoglycoprotein receptors (ASGPRs). Polyplexes formed with P(GPMA) (x = 20, 34) homopolymers were shown to be internalized via both energy-dependent and independent pathways, whereas polyplexes formed with block polymers were internalized through endocytosis. Notably, P(GPMA) polyplexes enter cells very efficiently but are also very toxic to human hepatocellular carcinoma (HepG2) cells and triggered cell apoptosis. In comparison, the presence of a carbohydrate block in the polymer structures reduced the cytotoxicity of the polyplex formulations and increased gene delivery efficiency with HepG2 cells. Transfection efficiency and toxicity studies were also carried out with HEK 293T (human embryonic kidney) cells for comparison. Results showed that polyplexes formed with the P(GPMA) homopolymers exhibit much higher transfection efficiency and lower toxicity with HEK 293T cells. The presence of the carbohydrate block did not further increase transfection efficiency in comparison to the homopolymers with HEK 293T cells, likely due to the lack of ASGPRs on the HEK 293T cell line. This study revealed that although guanidinium-based polymers have high membrane permeability, their application as plasmid delivery vehicles may be limited by their high cytotoxicity to certain cell types. Thus, the use of cell penetrating structures in polyplex formulations should be used with caution and carefully tailored toward individual cell/tissue types.

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