High-Throughput Synthesis of Antimicrobial Copolymers and Rapid Evaluation of Their Bioactivity

The growing trend in antimicrobial resistance is a potential threat to our society. Due to this, the development of new antimicrobial compounds is urgently required. High-throughput compositional analysis, combined with recent advances in polymerization protocols, allows for rapid production of potentially antimicrobial compounds with minimal expertise. This can provide the impetus for correlating activity with composition and functionality. In this study, we have used high-throughput photoinduced electron transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization to test the combinations of seven different monomers using 120 different formulations against three distinct bacterial species: Gram-negative Pseudomonas aeruginosa, Gram-positive Staphylococcus aureus, and Mycobacterium smegmatis. Through variations in composition, we have demonstrated the potential of high-throughput PET-RAFT for highly reproducible products, as well as simultaneous testing of multiple variables. Results indicate that primary amines work best against Gram-negative P. aeruginosa, while quaternary ammonium provides activity versus M. smegmatis. Copolymers of these provide avenues for further optimization, especially in the case of quaternary ammonium functionalities

Visible Light Mediated Controlled Radical Polymerization in the Absence of Exogenous Radical Sources or Catalysts

The application of external stimuli such as light to induce controlled radical polymerization reactions has important implications in the field of materials science. In this study, the photoactivation of trithiocarbonates (TTCs) (i.e., conventional RAFT agents) by visible light (∼460 nm) is investigated, and the ability of TTCs to control radical polymerization under visible light in the complete absence of exogenous photoinitiators or catalysts is demonstrated for the first time. By selectively exciting the spin-forbidden n → π* electronic transition, polyacrylates and polyacrylamides of low dispersity and high end group fidelity were obtained. In addition, this approach allows for the efficient synthesis of well-defined linear, (multi)­block, and network (co)­polymers. This study demonstrates the versatility of our strategy to generate polymers with controllable properties by visible light, which may be highly useful for applications such as surface patterning

Controlled Formation of Star Polymer Nanoparticles via Visible Light Photopolymerization

A recently developed visible light mediated photocontrolled radical polymerization technique using trithiocarbonates (i.e., conventional RAFT agents) as the sole control agent in the absence of additional photoinitiators or catalysts is utilized for the synthesis of core cross-linked star (CCS) polymer nanoparticles. The attractive features of this photopolymerization system, including high end-group fidelity at (near) complete monomer conversion, are exploited to facilitate a high-yielding, one-pot pathway toward well-defined star polymer products. Moreover, reinitiation of the photoactive trithiocarbonate moieties from within the star core is demonstrated to form (pseudo)­miktoarm stars via an “in–out” approach, showing extremely high initiation efficiency (95%)

Modulating Antimicrobial Activity and Mammalian Cell Biocompatibility with Glucosamine-Functionalized Star Polymers

The development of novel reagents and antibiotics for combating multidrug resistance bacteria has received significant attention in recent years. In this study, new antimicrobial star polymers (14–26 nm in diameter) that consist of mixtures of polylysine and glycopolymer arms were developed and were shown to possess antimicrobial efficacy toward Gram positive bacteria including methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) and vancomycin-resistant <i>Enterococcus</i> (VRE) (with MIC values as low as 16 μg mL^–1) while being non-hemolytic (HC₅₀ > 10 000 μg mL^–1) and exhibit excellent mammalian cell biocompatibility. Structure function analysis indicated that the antimicrobial activity and mammalian cell biocompatibility of the star nanoparticles could be optimized by modifying the molar ratio of polylysine to glycopolymers arms. The technology described herein thus represents an innovative approach that could be used to fight deadly infectious diseases

Highly Efficient and Versatile Formation of Biocompatible Star Polymers in Pure Water and Their Stimuli-Responsive Self-Assembly

This study demonstrates the rapid and efficient formation of functional core cross-linked star polymers via copper-mediated reversible-deactivation radical polymerization (RDRP) in pure water using fully soluble monomers and cross-linkers. This high throughput “arm-first” methodology allows the generation of complex nanoarchitectures with tailored core, shell, or periphery- functionalities and is potentially well-suited for biomedical applications given that the macromolecular synthesis is performed entirely in water. To exemplify this approach, different homo- and miktoarm star polymers composed of either poly­(<i>N</i>-isopropylacrylamide) (PNIPAM), poly­(2-hydroxyethyl acrylate) (PHEA), and poly­(ethylene glycol) (PEG) as the polymeric arms are formed. The star products are generated in high yield (88–96%) in one-pot and require short reaction times (1–3 h) and minimal purification steps (dialysis and lyophilization). In addition, the thermal responsivity of PNIPAM-based miktoarm star polymers leading to reversible supramolecular self-assembly is confirmed by DLS and 2D-NOESY NMR analysis. Furthermore, cytotoxicity studies using human embryonic kidney (HEK239T) cells as the model mammalian cells revealed that the star polymers are nontoxic even up to high polymer concentrations (2 mg mL^–1). The simplistic product formation and isolation, combined with the use of water as the polymerization medium, mean that this procedure is highly attractive as a low-cost pathway toward functional, biocompatible organic nanoparticles for commercial applications

Structure Governs the Deformability of Polymer Particles in a Microfluidic Blood Capillary Model

Particle stiffness is a design parameter that affects bionano interactions, including biodistribution kinetics and cellular processing. Herein, we develop soft polysaccharide (hyaluronic acid, HA) replica particles and capsules with tunable stiffness and sizes similar to human red blood cells (RBCs) via atom transfer radical polymerization-mediated continuous assembly of polymers (CAP_ATRP) and investigate their stiffness and deformability using colloidal-probe atomic force microscopy (CP-AFM) and a microfluidic blood capillary model, respectively. We demonstrate that HA replica particles and capsules with comparable nanoscale stiffness exhibit significantly different behaviors in a microfluidic blood capillary model. HA capsules behaved as RBCs, while HA replica particles had difficulty passing through the capillaries. These results (i) demonstrate how flow-based deformability measurements can be used to complement nanoscale stiffness measurements and (ii) provide important insight into the role of particle structure on the flow-based deformability of soft replica particles and capsules in a physiologically relevant microfluidic model

Rational Design of Single-Chain Polymeric Nanoparticles That Kill Planktonic and Biofilm Bacteria

Infections caused by multidrug-resistant bacteria are on the rise and, therefore, new antimicrobial agents are required to prevent the onset of a postantibiotic era. In this study, we develop new antimicrobial compounds in the form of single-chain polymeric nanoparticles (SCPNs) that exhibit excellent antimicrobial activity against Gram-negative bacteria (e.g., Pseudomonas aeruginosa) at micromolar concentrations (e.g., 1.4 μM) and remarkably kill ≥99.99% of both planktonic cells and biofilm within an hour. Linear random copolymers, which comprise oligoethylene glycol (OEG), hydrophobic, and amine groups, undergo self-folding in aqueous systems due to intramolecular hydrophobic interactions to yield these SCPNs. By systematically varying the hydrophobicity of the polymer, we can tune the extent of cell membrane wall disruption, which in turn governs the antimicrobial activity and rate of resistance acquisition in bacteria. We also show that the incorporation of OEG groups into the polymer design is essential in preventing complexation with proteins in biological medium, thereby maintaining the antimicrobial efficacy of the compound even in in vivo mimicking conditions. In comparison to the last-resort antibiotic colistin, our lead agents have a higher therapeutic index (by ca. 2–3 times) and hence better biocompatibility. We believe that the SCPNs developed here have potential for clinical applications and the information pertaining to their structure–activity relationship will be valuable toward the general design of synthetic antimicrobial (macro)­molecules

Stereoregular High-Density Bottlebrush Polymer and Its Organic Nanocrystal Stereocomplex through Triple-Helix Formation

We report the synthesis of a well-defined molecular bottlebrush polymer with stereoregular side chains (i.e., syndiotactic PMMA). The simultaneous control over the molecular weight, side-chain tacticity, and architecture allows the macromolecule to stereocomplex with the complementary linear stereoregular polymers (i.e., isotactic PMMAs) in controlled manners. By modulating the feed ratio of the complexing materials and chain length of the linear assembling component, a variety of crystalline materials with different sizes and morphologies, including discrete spherical nanoparticle, multiple-particle assembly, and cross-linked network structure, can be produced. Among these, uniformed sized, stable nanocrystals that exhibit temperature-induced solution assembly and disassembly properties can be derived from a combined process of PMMA triple-helix stereocomplex formation and polymer architecture-directed intramolecular crystallization. This work has established a new, facile synthetic protocol toward stimuli-responsive organic nanocrystals, which is applicable to the fabrication of a wide variety of functional crystal nanomaterials with practical applications

Nitric Oxide-Loaded Antimicrobial Polymer for the Synergistic Eradication of Bacterial Biofilm

Bacterial biofilms are often difficult to treat and represent the main cause of chronic and recurrent infections. In this study, we report the synthesis of a novel antimicrobial/antibiofilm polymer that consists of biocompatible oligoethylene glycol, hydrophobic ethylhexyl, cationic primary amine, and nitric oxide (NO)-releasing functional groups. The NO-loaded polymer has dual-action capability as it can release NO which triggers the dispersion of biofilm, whereas the polymer can induce bacteria cell death via membrane wall disruption. By functionalizing the polymers with NO, we observed a synergistic effect in biofilm dispersal, planktonic and biofilm killing activities against Pseudomonas aeruginosa. The NO-loaded polymer results in 80% reduction in biofilm biomass and kills >99.999% of planktonic and biofilm P. aeruginosa cells within 1 h of treatment at a polymer concentration of 64 μg mL^–1. To achieve this synergistic effect, it is imperative that the NO donors and antimicrobial polymer exist as a single chemical entity, instead of a cocktail physical mixture of two individual components. The excellent antimicrobial/antibiofilm activity of this dual-action polymer suggests the advantages of combination therapy in combating bacterial biofilms

Photocontrolled Cargo Release from Dual Cross-Linked Polymer Particles

Burst release of a payload from polymeric particles upon photoirradiation was engineered by altering the cross-linking density. This was achieved via a dual cross-linking concept whereby noncovalent cross-linking was provided by cyclodextrin host–guest interactions, and irreversible covalent cross-linking was mediated by continuous assembly of polymers (CAP). The dual cross-linked particles (DCPs) were efficiently infiltrated (∼80–93%) by the biomacromolecule dextran (molecular weight up to 500 kDa) to provide high loadings (70–75%). Upon short exposure (5 s) to UV light, the noncovalent cross-links were disrupted resulting in increased permeability and burst release of the cargo (50 mol % within 1 s) as visualized by time-lapse fluorescence microscopy. As sunlight contains UV light at low intensities, the particles can potentially be incorporated into systems used in agriculture, environmental control, and food packaging, whereby sunlight could control the release of nutrients and antimicrobial agents