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Along with the celebration of the new year, we celebrate many successes at Bioconjugate Chemistry. This includes additions and renewal of our editors, who play a critical role in service to our research community of authors, reviewers, and readers. As highlighted in our January 2023 editorial, (1) we sincerely thank Prof. Vince Rotello, who stepped down after a decade of success as Editor-in-Chief. We also extend a sincere thank you to Prof. Bradley Smith, who also stepped down after 10 years of service as Associate Editor for the journal. With great enthusiasm, we introduce the 2023 editorial team at Bioconjugate Chemistry, which includes five new additions to the editorial staff: Prof. Gonçalo Bernardes from the University of Cambridge, Prof. Peng Zou, from the Peking University, Mr. Nathan Quinn and Dr. Christina MacLaughlin from ACS Publications, and Prof. Theresa Reineke from the University of Minnesota. We also introduce and celebrate the team of four editors that are continuing in dedicated service to our community and ACS Publications: Prof. Zhifei Dai from Peking University, Prof. Jan van Hest from Eindhoven University of Technology, Prof. Erin Lavik from the University of Maryland Baltimore Country, and Prof. Gang Zheng from the University of Toronto. We wish our entire scientific community a happy, healthy, and productive 2023 and look forward to working with you all to disseminate many exciting advances in Bioconjugate Chemistry.

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition–fragmentation chain transfer (RAFT) polymerization to yield pMAT43. The pMAT43 macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT-b-AEMA cationic-block copolymers, pMAT-b-AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT-b-AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT-b-AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo.

A new facile fabrication approach to generate polymeric nanostructures is described. Block copolymers containing immiscible segments can self-assemble to generate ordered nanostructures, such as cylinders of one block in a matrix of the other in the bulk, which can then be sectioned on the nanoscale using a microtome (nanoskiving). Dispersing these sections in a selective solvent for the matrix block results in nanocylinders. In one example, we utilized a poly(N,N-dimethylacrylamide)-block-poly(styrene) (PDMA-PS) copolymer containing 36% by volume of PS. This composition was selected as it self-assembles into cylinders of PS in a matrix of PDMA. Following a previously described procedure, the cylinders were aligned using a channel die. The aligned samples were subsequently sectioned using a microtome containing a diamond knife and dispersed in water, a selective solvent for the PDMA matrix, affording PS nanocylinders with a PDMA corona. This technique allows tuning of nanocylinders without the requirement of specialty fabrication equipment.

Polymer composition and morphology can affect the way polymers interact with biomolecules, cell membranes, and intracellular components. Herein, diblock, triblock, and statistical polymers that varied in charge center type (primary and/or tertiary amines) were synthesized to elucidate the role of polymer composition on plasmid DNA complexation, delivery, and cellular toxicity of the resultant polyplexes. The polymers were synthesized via RAFT polymerization and were composed of a carbohydrate moiety, 2-deoxy-2-methacrylamido glucopyranose (MAG), a primary amine group, N-(2-aminoethyl) methacrylamide (AEMA), and/or a tertiary amine moiety, N,N-(2-dimethylamino)ethyl methacrylamide (DMAEMA). The lengths of both the carbohydrate and cationic blocks were kept constant while the primary amine to tertiary amine ratio was varied within the polymers. The polymers were characterized via nuclear magnetic resonance (NMR) and size exclusion chromatography (SEC), and the polyplex formulations with pDNA were characterized in various media using dynamic light scattering (DLS). Polyplexes formed with the block copolymers were found to be more colloidally stable than statistical copolymers with similar composition, which rapidly aggregated to micrometer sized particles. Also, polymers composed of a higher primary amine content were more colloidally stable than polymers consisting of the tertiary amine charge centers. Plasmid DNA internalization, transgene expression, and toxicity were examined with each polymer. As the amount of tertiary amine in the triblock copolymers increased, both gene expression and toxicity were found to increase. Moreover, it was found that increasing the content of tertiary amines imparted higher membrane disruption/destabilization. While both block and statistical copolymers had high transfection efficiencies, some of the statistical systems exhibited both higher transfection and toxicity than the analogous block polymers, potentially due to the lack of a hydrophilic block to screen membrane interaction/disruption. Overall, the triblock terpolymers offer an attractive composition profile that exhibited interesting properties as pDNA delivery vehicles.

A series of diblock glycopolycations were created by polymerizing 2-deoxy-2-methacrylamido glucopyranose (MAG) with either a tertiary amine-containing monomer, N-[3-(N,N-dimethylamino) propyl] methacrylamide (DMAPMA), or a primary amine-containing unit, N-(2-aminoethyl) methacrylamide (AEMA). Seven structures were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization that varied in the block lengths of MAG, DMAPMA, and AEMA along with two homopolymer controls of DMAPMA and AEMA that lacked a MAG block. The polymers were all able to complex plasmid DNA into polyplex structures and to prevent colloidal aggregation of polyplexes in physiological salt conditions. In vitro transfection experiments were performed in both HeLa (human cervix adenocarcinoma) cells and HepG2 (human liver hepatocellular carcinoma) cells to examine the role of charge type, block length, and cell type on transfection efficiency and toxicity. The glycopolycation vehicles with primary amine blocks and PAEMA homopolymers revealed much higher transfection efficiency and lower toxicity when compared to analogs created with DMAPMA. Block length was also shown to influence cellular delivery and toxicity; as the block length of DMAPMA increased in the glycopolycation-based polyplexes, toxicity increased while transfection decreased. While the charge block played a major role in delivery, the MAG block length did not affect these cellular parameters. Lastly, cell type played a major role in efficiency. These glycopolymers revealed higher cellular uptake and transfection efficiency in HepG2 cells than in HeLa cells, while homopolycations (PAEMA and PDMAPMA) lacking the MAG blocks exhibited the opposite trend, signifying that the MAG block could aid in hepatocyte transfection.

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization to yield pMAT

Nucleic acid therapeutics are attracting renewed interest due to recent clinical advances and product approvals. Most leading programs use chemical conjugates, or viral vectors in the case of gene therapy, while several use no delivery system at all. Polymer systems, which have been at the periphery of this renaissance, often involve greater molecular complexity than competing approaches, which must be justified by their advantages. Advanced analytical methods, along with biological tools for characterizing biotransformation and intracellular trafficking, are increasingly being applied to nucleic acid delivery systems including those based on polymers. These frontiers of investigation create the opportunity for an era where highly defined polymer compositions are optimized based on mechanistic insights in a way that has not been previously possible, offering the prospect of greater differentiation from alternatives. This will require integrated collaboration between polymer scientists and those from other disciplines.

A series of nine poly(2-deoxy-2-methacrylamido glucopyranose)-b-poly(methacrylate amine) diblock copolycations has been synthesized as new colloidally stable polynucleotide vehicles. The cationic block was varied in length and in the degree of methyl group substitution (secondary, tertiary, quaternary) on the pendant amine in an effort to optimize the structure and activity for plasmid DNA (pDNA) delivery. Upon a thorough kinetic study of polymerization for each polymer, the glycopolymers were prepared with well-controlled Mn and Đ. The binding and colloidal stability of the polymer–pDNA nanocomplexes at different N/P ratios and in biological media have been investigated using gel electrophoresis and light scattering techniques. The toxicity and transfection efficiency of the polyplexes have been evaluated with Hep G2 (human liver hepatocellular carcinoma) cells; several polymers displayed excellent delivery and toxicity profiles justifying their further development for in vivo gene therapy.

Using a new class of nontoxic and degradable glycopolymer-based vehicles termed poly(glycoamidoamine)s, we demonstrate virus-like delivery efficacy of oligodeoxynucleotide (ODN) decoys to cardiomyoblasts (H9c2), primary cardiomyocytes, and the mouse heart. These glycopolymers bind and compact ODN decoys into nanoparticle complexes that are internalized by the cell membrane and mediate nuclear uptake of DNA in 90+% of cultured primary cardiomyocytes and 87% of the mouse myocardium. Experimental results reveal that decoys delivered via these glycopolymers block the activation of the transcription factor NF-κB, a major contributor to ischemia/reperfusion injury. Decoy complexes formed with glycopolymer T4 significantly blocked downstream gene expression of Cox-2 and limited myocardial infarction in vivo, phenocopying a transgenic mouse model. These promising delivery vehicles may facilitate high-throughput genetic approaches in animal models. Additionally, the low toxicity, biodegradation, and outstanding delivery efficacy suggest that these nanomedicines may be clinically applicable for gene regulatory therapy.

Rational design of RNA ligands continues to be a formidable challenge, but the potential powerful applications in biology and medicine catapults it to the forefront of chemical research. Indeed, small molecule and macromolecular intervention are attractive approaches, but selectivity and cell permeability can be a hurdle. An alternative strategy is to use molecules of intermediate molecular weight that possess large enough surface area to maximize interaction with the RNA structure but are small enough to be cell-permeable. Herein, we report the discovery of nontoxic and cell-permeable branched peptide (BP) ligands that bind to TAR RNA in the low micromolar range from on-bead high-throughput screening of 4,096 compounds. TAR is a short RNA motif in the 5'-UTR of HIV-1 that is responsible for efficient generation of full RNA transcripts. We demonstrate that BPs are selective for the native TAR RNA structure and that "branching" in peptides provides multivalent interaction, which increases binding affinity to RNA.