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Water-soluble and biocompatible polymers are of interest in biomedicine as the search for alternatives to PEG-based materials becomes more important. In this work, the synthesis of a new sulfoxide-containing monomer, 2-(methylsulfinyl)ethyl acrylamide (MSEAM), is reported. Well-defined polymers were prepared by photoinduced initiators for continuous activator regeneration atom transfer radical polymerization (PICAR ATRP). The polymerizations were performed in water under biologically relevant conditions in a small volume without degassing the reaction mixture. DNA-PMSEAM and protein-PMSEAM hybrids were also synthesized. The lower critical solution temperature (LCST) of PMSEAM was estimated to be approximately 170 °C by extrapolating the LCST for a series of copolymers with variable content of

A predictable and reproducible number of sulfuric acid sites have been achieved for cation exchange resins by employing a mild SuFEx-based reagent system to effect the hydrolysis of fluorosulfonated polymer beads. The resultant poly(hydrogen sulfate) beads effectively demonstrate their utility as cation exchange resins in ion-capture experiments. Furthermore, their polyfluorosulfonated precursors have also proven to be suitable substrates for traditional SuFEx-type click reactions in both small molecule and protein immobilization applications.

An industrially important enzyme, Candida antarctica lipase B (CalB), was modified with a range of functional polymers including hydrophilic, hydrophobic, anionic, and cationic character using a "grafting to" approach. We determined the impact of polymer chain length on CalB activity by synthesizing biohybrids of CalB with each polymer at three different chain lengths, using reversible addition-fragmentation chain transfer (RAFT) polymerization. The activity of CalB in both aqueous and aqueous-organic media mixtures was significantly enhanced for acrylamide (Am) and N,N-dimethyl acrylamide (DMAm) conjugates, with activity remaining approximately constant in 25 and 50% ethanol solvent systems. Interestingly, the activity of N,N-dimethylaminopropyl-acrylamide (DMAPA) conjugates increased gradually with increasing organic solvent content in the system. Contrary to other literature reports, our study showed significantly diminished activity for hydrophobic polymer-protein conjugates. Functional thermal stability assays also displayed a considerable enhancement of retained activity of Am, DMAm, and DMAPA conjugates compared to the native CalB enzyme. Thus, this study provides an insight into possible advances in lipase production, which can lead to new improved lipase bioconjugates with increased activity and stability.

Protein polymer hybrids play an increasingly important role in therapeutics and biocatalysis. Synthesis of these biohybrids can be achieved by either the grafting-from or grafting-to strategy and are generally thought to be interchangeable. Therefore, when choosing between these two strategies the decision is usually based on polymer accessibility and purification preference. However, in this study, we demonstrated that the choice of the polymer ligation strategy played a significant role in the stability and bioactivity of the final hybrid. Our goal was to prepare a thermally stable DNase I polymer hybrid by utilizing either synthetic strategies. We found that the grafting-from strategy using reversible addition-fragmentation chain transfer polymerization (RAFT) or atom transfer radical polymerization (ATRP) yielded DNase I biohybrids with no activity. Control reactions were used to demonstrate inherent protein deactivation caused by the grafting-from conditions for either ATRP or RAFT polymerization. The grafting-to method yielded active and thermally stable DNase I biohybrids.

We report the synthesis of new polymer-protein conjugates using a grafting-from method employing photoinduced electron transfer reversible addition-fragmentation chain transfer (PET-RAFT) polymerization. Lipase, an industrially important enzyme, was modified with a reversible addition-fragmentation (RAFT) chain transfer agent (CTA) using activated ester chemistry. To study the effects of polymer composition and molecular weight on the enzyme activity, a hydrophilic and hydrophobic polymer were grafted at three different polymer lengths. We found a significant impact of polymer composition on enzyme activity with hydrophobic polymer substantially increasing enzyme activity.

The synthesis of a sulfoxide-based water-soluble polymer, poly(2-(methylsulfinyl)ethyl acrylate) (polyMSEA), a polymeric analogue of DMSO, by atom transfer radical polymerization (ATRP) is reported. Well-defined linear polymers were synthesized using relatively low amounts of copper catalyst (1000 or 100 ppm). Two types of star polymers were synthesized by either an “arm-first” approach or a “core-first” approach using a biodegradable β-cyclodextrin core. The glass transition temperatures of both the linear polymer (16 °C) and star polymer (32 °C) were determined by differential scanning calorimetry (DSC). The lower critical solution temperature (LCST) of poly(MSEA) was estimated to be ca. 140 °C by extrapolating the LCST of a series of copolymers with NIPAM. Cytotoxicity tests revealed that both the linear and star polymers have low toxicity, even at concentrations up to 3 mg/mL.

Post-polymerization functionalization is a critical tool in preparing functional materials. The critical aspects of post-polymerization reactions are high yields, short reaction times, simple purification, near stoichiometric amounts of reactants, and facile reaction conditions. Polymeric epoxides (i.e. poly(glycidyl methacrylate)) represent an underutilized class of functionalized polymers due to long reaction times, high temperatures, and large excess of reactants to drive the reaction to high conversion in a reasonable time frame. In this manuscript we describe the use of a novel solvent trifluoroethanol (TFE) for the ring opening of amines with poly(glycidyl methacrylate). We demonstrate that TFE gives faster reaction times and higher yields than traditional solvents used for the ring opening reaction. We utilized TFE to prepare dual functionalized polymers that could be “clicked” using strain promoted azide alkyne cycloaddition and an amine-bearing drug ring opening of the polymeric backbone.

The modification of proteins with reactive handles has facilitated the use of these biomolecules in diverse fields including drug delivery, diagnostics, environmental remediation, and cell culture matrices. Recently, a new “click” type reaction, sulfur(VI) fluoride exchange (SuFEx), was reported between sulfuryl fluoride and various organic moieties (e.g. hydroxyl, amine) to yield a sulfamoyl fluoride ( NSO 2 F) or fluorosulfate ( OSO 2 F) moiety under biphasic conditions. The model protein bovine serum albumin (BSA) was reacted with SO 2 F 2 gas under biphasic conditions to yield SO 2 F functionalized protein. The resultant BSA-SO 2 F was characterized using gel electrophoresis, mass spectroscopy and Fourier transmission infrared spectroscopy to confirm the addition of SO 2 F functional group. SuFEx modification of BSA caused a marked change in the pH dependent size and zeta potential of the protein as well as increased the protein’s denaturation temperature. Crucially, BSA-SO 2 F was demonstrated to be biocompatible after 72 h incubation with A549 lung endothelial cells. Due to the unique and elective reactivity of the SO 2 F group with amines, BSA-SO 2 F could be self-condensed to form a biocompatible hydrogel that was used in co-culture with HEK 293 cells. In addition, polyethylene glycol (PEG) with reactive OSO 2 F groups at chain end was conjugated with BSA under various pH conditions through SuFEx chemistry. This communication, to our knowledge, is the first report of the application of the SuFEx in the field of bioconjugates.

Cationic hyperbranched polymers (HBP) were prepared by self-condensing vinyl polymerization of an atom transfer radical polymerization (ATRP) inimer containing a quaternary ammonium group. Two types of biocompatible shells, poly(oligoethylene glycol) methacrylate (polyOEGMA) and poly(2-(methylsulfinyl) ethyl methacrylate) (polyDMSO), were grafted respectively from HBP core to form core-shell structures with low molecular weight dispersity and high biocompatibility, polyOEGMA-HBP and polyDMSO-HBP. Both of the structures showed low cytotoxicity and good siRNA complexing ability. The efficacy of gene silencing against Runt-related transcription factor 2 ( Runx2) expression and the long-term assessment of mineralized nodule formation in osteoblast cultures were evaluated. The biocompatible core-shell structures were crucial to minimizing undesired cytotoxicity and nonspecific gene suppression. polyDMSO-HBP showed higher efficacy of forming polyplexes than polyOEGMA-HBP due to shell with lower steric hindrance. Overall, the gene silencing efficiency of both core-shell structures was comparable to commercial agent Lipofectamine, indicating long-term potential for gene silencing to treat heterotopic ossification (HO).

Post-polymerization modification of polymer chain-ends is a powerful tool for the preparation of functional materials. “Click” type reactions represent the most prevalent post-polymerization strategies. We applied the recently described sulfur(VI) fluoride exchange reaction to functionalize the chain-ends of four polymers prepared using activators generated by electron transfer atom transfer radical polymerization. Aryl-TBDMS terminated polymers were reacted with aryl sulfurofluoridate compounds with a near quantitative conversion. This research represents the application of a new click type reaction to prepare functional polymers.

α-Chymotrypsin, a commonly used protease, was modified with well-defined oligomers synthesized by RAFT. The well defined polymers were synthesized based on the monomers N,N-dimethylacrylamide (DMAm) or oligo(ethylene oxide) methyl ether acrylate (OEOA). The polymers were conjugated to free amine groups on chymotrypsin through an in-situ 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling approach. The protein–polymer conjugates retained enzymatic activity, and the higher molecular weight DMAm and OEOA polymer, created protein–polymer conjugates with significantly enhanced stability, presumably due to the high molecular weight polymer preventing autolysis of the α-chymotrypsin.

Combining modeling and experiment, we created multilayered gels where each layer was “stacked” on top of the other and covalently interconnected to form mechanically robust materials, which could integrate the properties of the individual layers. In this process, a solution of new initiator, monomer, and cross-linkers was introduced on top of the first gel, and these new components then underwent living (co)polymerization to form the subsequent layer. We simulated this process using dissipative particle dynamics (DPD) to isolate factors that affect the formation and binding of chemically identical gel as well as incompatible layers. Analysis indicates that the covalent bond formation between the different layers is primarily due to reactive chain-ends, rather than residual cross-linkers. In the complementary experiments, we synthesized multilayered gels using either free radical (FRP) or atom transfer radical polymerizations (ATRP) methods. Polymerization results demonstrated that chemically identical mat...

Hydrophilic polymers were attached to lysozyme by a combination of grafting-to and grafting-from approaches using RAFT polymerization. A hydrophilic oligomer was synthesized, and attached to the protein. The protein-oligomer hybrid contained the RAFT end group, enabling chain extension in solution. Lysozyme maintained activity throughout this process.

The field of RNA interference depends on the development of safe and efficient carriers for short interfering ribonucleic acid (siRNA) delivery. Conventional cationic monomers for siRNA delivery have utilized the nitrogen heteroatom to produce cationic charges. Here, we polymerized cationic sulfonium (meth)acrylate by activators regenerated by electron transfer (ARGET) atom transfer radical polymerization (ATRP) to form polymers with narrow molecular weight distributions for siRNA delivery. The tertiary sulfonium species was stable toward dealkylation in water but less stable in the polar aprotic solvent dimethyl sulfoxide. Block copolymers poly(ethylene oxide) with poly(meth)acrylate containing sulfonium moieties were prepared as an siRNA delivery platform. Results suggested block copolymers were biocompatible up to 50 μg/mL in vitro and formed polyplexes with siRNA. Additionally, block copolymers protected siRNAs against endonuclease digestion and facilitated knockdown of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) mRNA expression in murine calvarial preosteoblasts. The versatility, biocompatibility, and cationic nature of these tertiary sulfonium groups are expected to find widespread biological applications.

Phototunable supersoft elastomers were prepared by photo-cross-linking molecular bottlebrushes with poly(n-butyl acrylate) (PBA) side chains. Bottlebrushes were synthesized by atom transfer radical polymerization (ATRP) and Br-chain end functionalities were replaced by photo-cross-linkable coumarin units. Photo-cross-linking and photoscission of molecular bottlebrushes resulted in tunable elastomeric behavior quantified by dynamic mechanical analysis. The cell–substrate interactions, tuned by UV-light, depended on surface morphology and softness that could be as low as several kilopascals.

Covalent attachment of poly(ionic liquid)s (PILs) by click chemistry on glass or silicon (Si) surfaces was performed. Poly[1-(4-vinylbenzyl)-3-butylimidazolium bis(trifluoromethylsulfonyl)imide] (polyVBBI+Tf2N−), and copolymers of polyVBBI+Tf2N− with fluorescein O-methacrylate were synthesized by conducting an atom transfer radical polymerization (ATRP) from initiators containing azide or thioacetate groups. The azide- and thiol-terminated PILs were then successfully grafted onto alkyne and alkene modified glass/Si wafers by thermal azide–alkyne cycloaddition and photoinitiated thiol-ene click reactions, respectively. The modified surfaces were characterized by contact angle measurements and ellipsometry. The fluorescent PIL functionalized surfaces showed strong fluorescence under UV irradiation. This procedure of tethering PILs to substrates also provides an easy way to change the surface hydrophilicity by replacing the anions in the grafted PILs. The present approach could be readily applied for surface modifications with other types of PILs or their copolymers to achieve different functionalities on various surfaces.

Activators regeneration by electron transfer atom transfer radical polymerization (ARGET ATRP) of 2-aminoethyl methacrylate hydrochloride (AMA) was successfully performed for the first time. The polymerizations were conducted at 35 °C in isopropanol (IPA)/water mixtures or pure water, using CuBr2/TPMA (TPMA: tris(2-pyridylmethyl)amine) as a metal catalyst complex with slow feeding of ascorbic acid (AscA) to allow regeneration of the activator species. The reactions showed first-order kinetics with monomer conversion and high monomer conversion (>90%), and controlled polymers (Đ ≈ 1.3) were obtained using optimized conditions. For the first time, as a result of the PAMA’s high chain-end functionality, it was possible to extend the polymer and synthesize a well-defined block copolymer (PAMA-b-POEOMA) (OEOMA: oligo(ethylene oxide) methyl ether methacrylate) by ATRP techniques, using AMA as the first block. PAMA-based nanogels, with potential biomedical applications, were prepared by ARGET ATRP in inverse min...

Protein–polymer hybrids are an important class of biomaterials. Described is the preparation of a genetically incorporated a non-canonical amino acid (nCAA) containing an ester linked atom transfer radical polymerization (ATRP) initiator, followed by a controlled “grafting from” polymerization. A Methanococcus jannaschii tyrosyl-tRNA synthetase/tRNA CUA pair was selected to genetically encode p-bromoisobutyryloxymethyl- l -phenylalanine (biF) in response to an amber codon. This biF was directly incorporated into green fluorescent protein (GFP) at residue 134 generating biF-GFP. Activators regenerated by electron transfer (ARGET) ATRP was conducted under biologically relevant conditions to graft well-defined poly(oligo ethylene oxide methacrylate) from the biF-GFP. The biF-GFP retained its biofluorescence properties throughout the polymerization indicating the utility of ARGET ATRP for preparing protein–polymer hybrids. The presence of a base-labile ester bond in the initiator, allowed cleavage of the grafted polymer from the protein and directly analyze their molecular weight and molecular weight distribution using gel permeation chromatography (GPC). The cleaved final polymer had a M n = 27,000 and a molecular weight distribution of M w / M n = 1.27.

Activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) was successfully implemented in aqueous media for the first time. A well-controlled polymerization of oligo(ethylene oxide) methyl ether methacrylate (OEOMA) was conducted with 300 ppm or lower of a copper catalyst and tris(2-pyridylmethyl)amine (TPMA) ligand in the presence of an excess of halide salts. Ascorbic acid was continuously fed into the reaction mixture to regenerate the activator complex. The effects of the halide salt concentration, ligand concentration, feeding rate of the reducing agent, and copper concentration were systematically studied to identify conditions that provide both an acceptable rate of polymerization and good control over the polymer properties. The optimized polymerization conditions provided linear first-order kinetics, linear evolution of the molecular weight with conversion, and polymers with narrow molecular weight distributions (Mw/Mn < 1.3) at high monomer conversions (∼70%) ...

Initiators for continuous activator regeneration atom transfer radical polymerization (ICAR ATRP) with ppm amount of Cu catalyst was successfully developed in water. For the first time, Cu catalyst concentrations of 100 ppm and lower were used in aqueous media to prepare well-defined macromolecules. Polymers of oligo(ethylene oxide) methyl ether acrylate were synthesized with low dispersity (Mw/Mn = 1.15–1.28) using 20–100 ppm of an active CuBr/tris(pyridin-2-ylmethyl)amine-based catalyst in the presence of excess bromide anions. This technique was used to synthesize a thermoresponsive block copolymer of poly(oligo(ethylene oxide) methyl ether methacrylate)-b-poly(oligo(ethylene oxide) methyl ether acrylate). The methacrylic block had a lower critical solution temperature (LCST = 77 ± 2 °C) below that of the acrylic block. The hydrodynamic diameter of ca. 10 nm at temperatures below the LCST is consistent with free polymer chains in solution, and the diameter of ca. 30 nm above the LCST is consistent with...

Atom transfer radical polymerization (ATRP) methods were developed in water-based media, to grow polymers from proteins under biologically relevant conditions. These conditions gave good control over the resulting polymers, while still preserving the protein’s native structure. Several reaction parameters, such as ligand structure, halide species, and initiation mode were optimized in water and PBS buffer to yield well-defined polymers grown from bovine serum albumin (BSA), functionalized with cleavable ATRP initiators (I). The CuCl complex with ligand 2,2′-bipyridyne (bpy) provides the best conditions for the polymerization of oligo(ethylene oxide) methacrylate (OEOMA) in water at 30 °C under normal ATRP conditions (I/CuCl/CuCl2/bpy = 1/1/9/22), while the CuBr/bpy complex gave better performance in PBS. Activators generated by electron transfer (AGET) ATRP gave well-controlled polymerization of OEOMA at 30 °C with the ligand tris(2-pyridylmethyl)amine (TPMA), (I/CuBr2/TPMA = 1/10/11). The AGET ATRP react...

Fluorescein O-methacrylate was copolymerized with n-butyl acrylate by atom transfer radical polymerization in a “grafting from” reaction with a multifunctional linear macroinitiator to form pH responsive fluorescent bottlebrushes. The brush-like structure of the synthesized macromolecules was confirmed through molecular imaging by atomic force microscopy. NMR spectroscopy showed that the fluorescent units were successfully incorporated into the polymer. The synthesized bottlebrushes displayed highly fluorescent properties under basic conditions, yet showed no fluorescence under neutral or acidic conditions. The fluorescence could be turned on and off by changing the pH of the solution. These bottlebrush molecules could have potential applications in molecular imaging.

Poly(ethylene glycol) (PEG)-based star polymers with a cationic core were prepared by atom transfer radical polymerization (ATRP) for in vitro nucleic acid (NA) delivery. The star polymers were synthesized by ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and ethylene glycol dimethacrylate (EGDMA). Star polymers were characterized by gel permeation chromatography, zeta potential, and dynamic light scattering. These star polymers were combined with either plasmid DNA (pDNA) or short interfering RNA (siRNA) duplexes to form polyplexes for intracellular delivery. These polyplexes with either siRNA or pDNA were highly effective in NA delivery, particularly at relatively low star polymer weight or molar ratios, highlighting the importance of NA release in efficient delivery systems.

Cationic nanogels with site-selected functionality were designed for the delivery of nucleic acid payloads targeting numerous therapeutic applications. Functional cationic nanogels containing quaternized 2-(dimethylamino)ethyl methacrylate and a cross-linker with reducible disulfide moieties (qNG) were prepared by activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) in an inverse miniemulsion. Polyplex formation between the qNG and nucleic acid exemplified by plasmid DNA (pDNA) and short interfering RNA (siRNA duplexes) were evaluated. The delivery of polyplexes was optimized for the delivery of pDNA and siRNA to the Drosophila Schneider 2 (S2) cell-line. The qNG/nucleic acid (i.e., siRNA and pDNA) polyplexes were found to be highly effective in their capabilities to deliver their respective payloads.