Dominik Konkolewicz

Well-Defined Macromolecules Using Horseradish Peroxidase as a RAFT Initiase.

Enzymatic catalysis and control over macromolecular architectures from reversible addition-fragmentation chain transfer polymerization (RAFT) are combined to give a new method of making polymers. Horseradish peroxidase (HRP) is used to catalytically generate radicals using hydrogen peroxide and acetylacetone as a mediator. RAFT is used to control the polymer structure. HRP catalyzed RAFT polymerization gives acrylate and acrylamide polymers with relatively narrow molecular weight distributions. The polymerization is rapid, typically exceeding 90% monomer conversion in 30 min. Complex macromolecular architectures including a block copolymer and a protein-polymer conjugate are synthesized using HRP to catalytically initiate RAFT polymerization.

Self-healing, malleable and creep limiting materials using both supramolecular and reversible covalent linkages

A self-healing material containing two reversible cross-linkers was made. Relatively rapidly exchanging hydrogen-bonded and slowly exchanging Diels–Alder based cross-linkers were incorporated. Two time scales allowed partial healing at room temperature, and near complete healing upon heating. Slow linkers limited creep at room temperature but allowed reshaping upon heating.

Polymer Structure and Conformation Alter the Antigenicity of Virus-like Particle–Polymer Conjugates

Covalent conjugation of water-soluble polymers to proteins is critical for evading immune surveillance in the field of biopharmaceuticals. The most common and long-standing polymer modification is the attachment of methoxypoly(ethylene glycol) (mPEG), termed PEGylation, which has led to several clinically approved pharmaceuticals. Recent data indicate that brush-type polymers significantly enhance in vitro and in vivo properties. Herein, the polymer conformation of poly(ethylene glycol) is detailed and compared with those of water-soluble polyacrylate and polynorbornene (PNB) when attached to icosahedral virus-like particles. Small-angle neutron scattering reveals vastly different polymer conformations of the multivalent conjugates. Immune recognition of conjugated particles was evaluated versus PEGylated particles, and PNB conjugation demonstrated the most effective shielding from antibody recognition.

Visible and sunlight driven RAFT photopolymerization accelerated by amines: kinetics and mechanism

Recently, photochemical polymerizations have received interest since they can be performed under mild conditions and they offer temporal control over the reaction. In this study, the kinetics of photochemical dithiobenzoate-mediated RAFT polymerization in the presence of triethylamine is explored. This system uses mild light sources such as visible light and sunlight, and does not require the use of expensive or rare earth catalysts. Instead triethylamine is combined with reagents used in RAFT polymerization. This study investigates the effects of light source, RAFT chain transfer agent concentration, and amine concentration, to understand the kinetic contributions of each component and possible mechanism of this process. Data suggests that there is electron transfer from the amine to the excited RAFT end-group, which is the major radical generation pathway. Radicals are also generated directly from the excited RAFT end-group. This method yields living polymers as evidenced by the synthesis of well-defined block copolymers.

3D printing of an interpenetrating network hydrogel material with tunable viscoelastic properties

Interpenetrating network (IPN) hydrogel materials are recognized for their unique mechanical properties. While IPN elasticity and toughness properties have been explored in previous studies, the factors that impact the time-dependent stress relaxation behavior of IPN materials are not well understood. Time-dependent (i.e. viscoelastic) mechanical behavior is a critical design parameter in the development of materials for a variety of applications, such as medical simulation devices, flexible substrate materials, cellular mechanobiology substrates, or regenerative medicine applications. This study reports a novel technique for 3D printing alginate-polyacrylamide IPN gels with tunable elastic and viscoelastic properties. The viscoelastic stress relaxation behavior of the 3D printed alginate-polyacrylamide IPN hydrogels was influenced most strongly by varying the concentration of the acrylamide cross-linker (MBAA), while the elastic modulus was affected most by varying the concentration of total monomer material. The material properties of our 3D printed IPN constructs were consistent with those reported in the biomechanics literature for soft tissues such as skeletal muscle, cardiac muscle, skin and subcutaneous tissue.

Dual stimuli responsive self-healing and malleable materials based on dynamic thiol-Michael chemistry

Thiol-maleimide adducts have been used as dynamic crosslinkers to form soft, elastic, and stimuli responsive polymeric materials. Thiol-Michael adducts can undergo dynamic exchange at elevated temperature or elevated pH values. Due to the dynamic behaviour of thiol-Michael adducts, crosslinked polymeric materials display significant healing after cutting into half, and malleability upon exposure to solutions of elevated pH. These materials are also thermally responsive, showing self-healing properties and malleability at high temperatures (90 °C). The self-healing properties of these polymer materials are significantly higher than materials with non-dynamic crosslinkers. In addition, in mechanical stability experiments, these materials showed creep resistance and complete creep recovery at room temperature and pressure. These results indicate that the thiol-Michael reaction is dynamic and reversible in response to thermal and pH stimuli. These stimuli responsive self-healing, elastic, malleable, and mechanically stable polymeric materials open the door to have potential utilization in different applications such as coatings or elastomers with extended lifetimes.

Grafting challenging monomers from proteins using aqueous ICAR ATRP under bio-relevant conditions

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.

Chapter Five – Strategies for Biophysical Characterization of Protein–Polymer Conjugates

Protein-polymer conjugates are increasingly viewed as promising avenues to producing industrial enzymes with high activity capable of withstanding potentially harsh reaction conditions, or to designing novel therapeutics with triggered release, controlled masking, or increased resistance to proteolytic degradation. Common among these applications are the desire to improve the stability of protein-polymer conjugates to unfolding by exposure to chemicals or thermal stress. Thus, assays that allow researchers to robustly and easily characterize protein-polymer conjugates by obtaining thermodynamic parameters for folding stand to play an important role in the development of improved protein-polymer conjugates. Herein, we describe two techniques, differential scanning fluorimetry and intrinsic tryptophan fluorescence, used in our laboratories to obtain thermodynamic parameters of unfolding that allow for direct comparison of protein-polymer conjugates and the myriad effects of variations in attachment site, polymer identity, and polymer length. These two experiments, which are easily amenable to parallelization, are presented as high-throughput replacements for more traditionally employed circular dichroism experiments and as complements to functional chemical stability or functional thermal stability experiments. Each assay is presented in a parallelized format that allows for rapid scaling and high-throughput analysis of protein-polymer conjugate libraries. Descriptions of the assays include a discussion of advantages and disadvantages alongside protocol details and approaches to data analysis.

Photolabile protecting groups: a strategy for making primary amine polymers by RAFT

A photolabile ortho-nitrobenzyl primary amine protecting group is incorporated into a methacrylic monomer (ONBAMA). RAFT gives well-defined polymers of ONBAMA of various sizes. The protecting group is sensitive to UV radiation, but stable against visible light, acid and base. This provides a facile pathway to creating well-defined primary amine polymers by RAFT.

Tuning thermoresponsive network materials through macromolecular architecture and dynamic thiol-Michael chemistry

This work reports synthesis of dynamic materials crosslinked with thiol-Michael linkages with distinct primary polymer architectures. RAFT polymerization allows control over degree of polymerization and macromolecular architecture. Well-defined branched and linear polythiol polymers were synthesized by RAFT and crosslinked using thiol-Michael chemistry. Branched and linear polymeric materials with different crosslink densities were evaluated by size exclusion chromatography, tensile testing, rheology, and differential scanning calorimetry. These materials are elastic and show dynamic behavior (e.g. healing ability and malleability) in response to thermal stimulus (90 °C) due to the presence of the stimulus responsive thiol-maleimide linkages as crosslinkers along the polymer backbone. The data suggest that materials synthesised by RAFT healed faster than materials of similar weight average chain length and crosslink density synthesized by conventional free radical polymerization. Healing ability and malleability properties of these dynamic materials are dramatically higher than materials crosslinked with static crosslinkers. Small molecular studies of thiol-maleimide adducts indicate the potential of using thiol-Michael linkages in dynamically crosslinked materials. In addition to significant re-healing and malleability properties, these materials showed mechanical stability in creep deformation, stress relaxation, and creep recovery experiments under ambient conditions due to their essentially static nature under these conditions.