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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.

Polyether ether ketone (PEEK, 1) is an important material for the fabrication of implants employed in spinal fusion surgery. Although its radiolucency and favorable elastic modulus have made PEEK an attractive choice for interbody fusion devices, its poor osseointegrative properties prevent the formation of a strong union between implant and surrounding bone structures and remain a major liability. Recent advancements in PEEK surface technology have resulted in improved osseointegration; however, the identification of an ideal implant material has proven challenging. In this manuscript, we describe our preliminary investigation into the realm of PEEK-based fusion devices that has culminated in the discovery of a mild, solution-based process for the preparation of covalently surface modified PEEK biomaterials that display enhanced osteoconductive properties. Surface modification occurred under mild reaction conditions via the acid-mediated addition of various commercially available hydrophilic oxyamine and...

The targeted delivery of polymers to neurons is a challenging yet important goal for polymer based drug delivery. We prepared a fentanyl based atom transfer radical polymerization (ATRP) initiator to target the Mu opioid receptor (MOR) for neuronal targeting. We incorporated our recently discovered rigid acrylate linking group into the initiator to retain a high degree of binding to the MOR and grafted random or block copolymers of poly(oligo(ethylene oxide) methacrylate)-block-(glycidyl methacrylate). Trifluoroethanol promoted amine ring opening of the glycidyl methacrylate was used for post-polymerization modification of the fentanyl initiated polymers to attach a near-infrared fluorescent dye (ADS790WS) or to build a targeted siRNA delivery system via modification with secondary amines. We examined the biocompatibility, cellular internalization, and siRNA binding properties of our polymer library in a green fluorescent protein expressing SY SH5Y neuroblastoma cell-line.

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.

The synthesis of novel fentanyl acrylate derivatives via bromo-fentanyl using Heck coupling is described. The synthesis is concise and represents an efficient and useful method for functionalizing fentanyl for medicinal chemistry investigations. The agonistic and analgesic activities are evaluated by Mu opioid receptor activation and hot plate assays in mice.

Aqueous initiators for continuous activator regeneration atom transfer radical polymerization (ICAR ATRP) was applied to graft well defined acrylamide, N,N-dimethylacrylamide and N-vinylimidazole homo and block copolymers from a model protein initiator (bovine serum albumin (BSA)) under bio-relevant conditions. Using N-vinylimidazole as a ligand catalytic biohybrid nanoparticles were prepared by loading palladium into a block copolymer of poly(N-vinylimidazole)-b-poly(oligo(ethylene oxide) acrylate) grafted from BSA. The protein–polymer biohybrid catalyst successfully catalyzes Suzuki–Miyaura couplings in aqueous media under aerobic conditions.

Background Polyetheretherketone (PEEK), and more recently titanium-coated PEEK, have been given serious consideration as biomaterial design choices for spinal interbody implants. Shortcomings in these materials necessitate further innovation into materials research, for example, on polyetherketoneketone (PEKK). Common complications such as surface delamination (as with titanium coating) and lack of bone apposition (as with PEEK) indicate the need for a new material that inherently displays preferable bone growth characteristics without sacrificing structural integrity. Purpose To compare three biomaterials with respect to their osseointegrative capacity. Study Design Evaluate the in vivo material characteristics of three separate biomaterials in an ovine bone defect model: PEEK, titanium-coated PEEK (Ti-coated PEEK), and 3D printed PEKK. Biomechanical, histological and radiographic testing was the basis for evaluation and material characterization. Methods Eight ovine specimens were implanted with one of each of the three types of biomaterials tested in both left and right epicondyles using a femoral bone defect model, and were sacrificed at 8 and 16 weeks. Implants were then analyzed using a push-out method, histological staining, and various radiographic tests. Industry funding was provided for the completion of this research study, followed by an independent third party review of all relevant data for publication. Results PEKK implants demonstrated bone ingrowth, no radiographic interference, no fibrotic tissue membrane formation, significant increase in bony apposition over time, and significantly higher push-out strength compared to standard PEEK. The PEKK implant displayed bone growth characteristics comparable to Ti-coated PEEK with significant improvements in implant integrity and radiographic properties. Conclusion This study found that PEKK displayed preferable characteristics when compared to PEEK and Ti-coated PEEK, and is therefore a potential alternative to their use.

Treating burn-related injuries remains challenging, because of donor site morbidity, bacterial infection, and major scarring. Recent advances have led to the development of promising natural and synthetic polymer scaffolds, the objective of which is to regenerate skin using a “top-down” approach, in which the top layers of the skin populated by fibroblasts and keratinocytes are regenerated first. An often-overlooked component of the integumentary system is the subcutaneous adipose tissue, which has been implicated in a variety of activities, including wound healing. We have created a novel coating composed of collagen VI (col VI) and heparin sulfate (HS) that promotes adipocyte attachment and adipogenesis at a rate significantly faster at both early and late time points (5 and 23 days), compared to a noncoated control. In addition, Vicryl, which is a biodegradable surgical implant mesh, was coated with the optimized col VI–HS ratio. Coated Vicryl had an increased number of attached adipocytes with more-pr...

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.

Novel water-dispersible hybrid iron oxide nanoparticles grafted with a polymeric analogue of dimethyl sulfoxide (DMSO) were prepared. Superparamagnetic iron oxide nanoparticles with immobilized atom-transfer radical polymerization (ATRP) initiators were prepared via an in situ method using 12-(2-bromoisobutyramido)dodecanoic acid as a surface ligand/initiator. The initiator-functionalized particles were employed in a surface-initiated initiator for continuous activator regeneration ATRP to graft poly(2-(methylsulfinyl)ethyl acrylate) (a polyacrylate analogue of DMSO) from the surface. The resulting hybrid nanoparticles showed a high magnetic relaxivity ratio (r2/r1) of 600 at 7 T in fetal bovine serum, and a good biocompatibility up to 1000 mg L–1.

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.

Bright signal outputs are needed for fluorescence detection of biomolecules at their native expression levels. Increasing the number of labels on a probe often results in crowding-induced self-quenching of chromophores, and maintaining the function of the targeting moiety (e.g., an antibody) is a concern. Here we demonstrate a simple method to accommodate thousands of fluorescent dye molecules on a single antibody probe while avoiding the negative effects of self-quenching. We use a bottlebrush polymer from which extend hundreds of duplex DNA strands that can accommodate hundreds of covalently attached and/or thousands of noncovalently intercalated fluorescent dyes. This polymer–DNA assembly sequesters the intercalated fluorophores against dissociation and can be tethered through DNA hybridization to an IgG antibody. The resulting fluorescent nanotag can detect protein targets in flow cytometry, confocal fluorescence microscopy, and dot blots with an exceptionally bright signal that compares favorably to commercially available antibodies labeled with organic dyes or quantum dots., Here we demonstrate a simple method to accommodate thousands of fluorescent dye molecules on a single antibody probe while avoiding the negative effects of self-quenching. We use a bottlebrush polymer from which extend hundreds of duplex DNA strands that can accommodate hundreds of covalently attached and/or thousands of noncovalently intercalated fluorescent dyes. The fluorescent bottlebrush polymer is then attached to an antibody via DNA hybridization, resulting in an exceptionally bright immunofluorescent label.

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.

The use of reversible deactivation radical polymerization (RDRP) methods has significantly expanded the field of bioconjugate synthesis. RDRP procedures have allowed the preparation of a broad range of functional materials that could not be realized using prior art poly(ethylene glycol) functionalization. The review of procedures for synthesis of biomaterials is presented with a special focus on the use of RDRP to prepare biohybrids with proteins, DNA and RNA.

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...

The combination of polymers with nucleic acids leads to materials with significantly advanced properties. To obviate the necessity and complexity of conjugating two macromolecules, a polymer initiator is described that can be directly covalently linked to DNA during solid-phase synthesis. Polymer can then be grown from the DNA bound initiator, both in solution after the DNA-initiator is released from the solid support as well as directly on the solid support, simplifying purification. The resulting polymer-DNA hybrids were examined by chromatography and fluorescence methods that attested to the integrity of hybrids and the DNA. The ability to use DNA-based supports expands the range of readily available molecules that can be used with the initiator, as exemplified by direct synthesis of a biotin polymer hybrid on solid-support. This method expands the accessibility and range of advanced polymer biohybrid materials.

A facile approach was developed to prepare crosslinked ionic polymer hydrogel films by photo-crosslinking utilizing p-vinylbenzyl trimethylammonium chloride (VBTMACl) or p-vinylbenzyl trimethylammonium hydroxide (VBTMAOH) as the monomer and poly(ethylene oxide) dimethacrylate (PEODMA, Mn = 750) as the crosslinker. The films with different crosslinking degrees (20%, 40%, 60%, 80%, and 100%) were prepared and characterized by swelling measurements, scanning electron microscopy (SEM), UV-visible spectroscopy, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, dynamic mechanical analysis (DMA), thermogravimetric analysis (TGA), and small-angle X-ray scattering (SAXS). It was found that the mechanical and thermal properties of the films were largely influenced by the contents of the crosslinker in the films. By ion-exchange of the anions in the films with various other anions, the hydrophobicity/hydrophilicity of the films was changed. In addition, fluorescent films were prepared by treatment with fluorescein, and paramagnetic films with FeCl4− as a counter anion showed catalytic activity for Friedel–Crafts alkylation. The ionic films with quaternary ammonium chloride groups displayed antimicrobial activity against Escherichia coli (E. coli) with almost 100% killing efficiency. Multifunctional films with various tunable properties have significant potential for a wide range of applications.

Supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP) of the cationic monomer (3-acrylamidopropyl)trimethylammonium chloride (AMPTMA) was successfully performed for the first time. The polymerizations were performed in water or ethanol–water mixtures at room temperature in the presence of Cu(0), using relatively low concentrations of soluble copper catalyst and an excess of ligand (Me6TREN). The reaction conditions were optimized to give the best control over the polymerization under environmentally friendly conditions. The polymerization data showed good control over the molecular weights with narrow molecular weight distributions for the entire polymerization. The preservation of the chain-end functionality was confirmed by self-chain extension and the synthesis of a block copolymer containing AMPTMA and oligo(ethylene oxide) methyl ether acrylate (OEOA). SARA ATRP was also extended to the synthesis of alkyne-terminated poly-AMPTMA (PAMPTMA), which was subsequently functionalized, using copper(I) catalyzed azide–alkyne cycloaddition, with an azido-functionalized coumarin derivative.

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...

The synthesis of a water/plasma soluble, noncytotoxic, "clicked" sugar-derivative of curcumin with amplified bioefficacy in modulating amyloid-β and tau peptide aggregation is presented. Curcumin inhibits amyloid-β and tau peptide aggregation at micromolar concentrations; the sugar-curcumin conjugate inhibits Aβ and tau peptide aggregation at concentrations as low as 8 nM and 0.1 nM, respectively. In comparison to curcumin, this conveniently synthesized Alzheimer's drug candidate is a more powerful antioxidant.

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.

Azide-terminated poly(tert-butyl acrylate) was synthesized via atom transfer radical polymerization (ATRP). Subsequent deprotection was performed to yield poly(acrylic acid) (PAA) possessing a reactive chain-end. A one-pot sequential amidation of the PAA with the amine derivatives of a near-infrared fluorescent dye (ADS832WS) and glucose produced NIRF dye-incorporated water-soluble copolymers. End-group modifications were performed to produce alkyne/biotin-terminated copolymers which were further employed to generate dye-incorporated polymer-protein hybrids via the biotin-avidin interaction with avidin or "click" bioconjugation with azide-modified BSA. We have overcome two fundamental limitations in the synthesis of bioconjugates: (a) the basic restriction in the diversity of copolymers which can be synthesized for producing bioconjugates, (b) the limitation in the number of dyes/drug molecules that can be attached per protein molecule. The copolymers possessed enhanced optical properties compared to the dye due to increased solubility in water. Potential utility of these copolymers and conjugates in multiwell plate based assays, cell surface imaging and in vivo animal imaging were explored.

This invention pertains to methods for producing homogeneous recombinant proteins that contain polymer initiators at defined sites. The unnatural amino acid, 4-(2′-bromoisobutyramido)phenylalanine of formula 1, was designed and synthesized as a molecule comprising a functional group further comprising an initiator for an atom-transfer radical polymerization (‘ATRP”) that additionally would provide a stable linkage between the protein and growing polymer. We evolved a Methanococcus jannaschii (Mj) tyrosyl-tRNA synthetase/tRNACUA pair to genetically encode this unnatural amino acid in response to an amber codon. To demonstrate the utility of this functional amino acid, we produced Green Fluorescent Protein with the unnatural amino acid initiator of formula 1 site-specifically incorporated on its surface (GFP-1). Purified GFP-1 was then used as an initiator under standard ATRP conditions with oligo(ethylene oxide)monomethyl ether methacrylate, efficiently producing a polymer-GFP bioconjugate wherein the polymer is connected at a specifically selected site on GFP.

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.

Curcumin, which is derived from the plant Curcuma longa, has received considerable attention as a possible anti-cancer agent. In cell culture, curcumin is capable of inducing apoptosis in cancer cells at concentrations that do not affect normal cells. One draw-back holding curcumin back from being an effective anti-cancer agent in humans is that it is almost completely insoluble in water and therefore has poor absorption and subsequently poor bioavailability. Here we have generated a number of curcumin derivatives (tetrahydro-curcumin, curcumin mono-carboxylic acid, curcumin mono-galactose, curcumin mono-alkyne and dendrimer-curcumin conjugate) to test whether any of them display both cytotoxicity and water solubility. Of those tested only dendrimer-curcumin conjugate exhibited both water solubility and cytotoxicity against SKBr3 and BT549 breast cancer cells. When compared to curcumin dissolved in DMSO, dendrimer-curcumin conjugate dissolved in water was significantly more effective in inducing cytotoxicity, as measured by the MTT assay and effectively induced cellular apoptosis measured by caspase-3 activation. Since dendrimer-curcumin conjugate is water soluble and capable of inducing potent cytotoxic effects on breast cancer cell lines, it may prove to be an effective anti-cancer therapy to be used in humans.

Protein–polymer hybrids (PPHs) represent an important and rapidly expanding class of biomaterials. Typically in these hybrids the linkage between the protein and the polymer is covalent. Here we describe a straightforward approach to a noncovalent PPH that is mediated by DNA. Although noncovalent, the DNA-mediated approach affords the highly specific pairing and assembly properties of DNA. To obtain the protein–DNA conjugate for assembly of the PPH, we report here the first direct copper catalyzed azide–alkyne cycloaddition-based protein–DNA conjugation. This significantly simplifies access to protein–DNA conjugates. The protein–DNA conjugate and partner polymer–DNA conjugate are readily assembled through annealing of the cDNA strands to obtain the PPH, the assembly of which was confirmed via dynamic light scattering and fluorescence spectroscopy.

Polymer biomolecule hybrids represent a powerful class of highly customizable nanomaterials. Here, we report star-polymer conjugates with DNA using a "ligandless" Cu(I) promoted azide-alkyne cycloaddition click reaction. The multivalency of the star-polymer architecture allows for the concomitant conjugation of other molecules along with the DNA, and the conjugation method provides control over the DNA orientation. The star-polymer DNA nanoparticles are shown to assemble into higher-order nanoassemblies through hybridization. Further, we show that the DNA strands can be utilized in controlled disassembly of the nanostructures.

Curcumin, the primary active ingredient in the spice turmeric, was converted to reactive monofunctional derivatives (carboxylic acid/azide/alkyne). The derivatives were employed to produce a 3 + 2 azide-alkyne "clicked" curcumin dimer and a poly(amidoamine) (PAMAM) dendrimer-curcumin conjugate. The monofunctional curcumin derivatives retain biological activity and are efficient for labeling and dissolving amyloid fibrils. The curcumin dimer selectively destroys human neurotumor cells. The synthetic methodology developed affords a general strategy for attaching curcumin to various macromolecular scaffolds.