Heather D. Maynard

Therapeutic protein-polymer conjugates: advancing beyond PEGylation.

Protein-polymer conjugates are widely used as therapeutics. All Food and Drug Administration (FDA)-approved protein conjugates are covalently linked to poly(ethylene glycol) (PEG). These PEGylated drugs have longer half-lives in the bloodstream, leading to less frequent dosing, which is a significant advantage for patients. However, there are some potential drawbacks to PEG that are driving the development of alternatives. Polymers that display enhanced pharmacokinetic properties along with additional advantages such as improved stability or degradability will be important to advance the field of protein therapeutics. This perspective presents a summary of protein-PEG conjugates for therapeutic use and alternative technologies in various stages of development as well as suggestions for future directions. Established methods of producing protein-PEG conjugates and new approaches utilizing controlled radical polymerization are also covered.

FDA-approved poly(ethylene glycol)– protein conjugate drugs

PEGylation or covalent attachment of poly(ethylene glycol) improves the pharmacokinetic properties of protein drugs. In vivo circulation lifetimes are increased and dosages are decreased, resulting in improved patient quality of life. PEG may be attached to proteins using a variety of different chemical reactions. This review discusses currently available FDA-approved PEGylated protein drugs, their intended use and target, and the PEG attachment chemistry utilized.

Synthesis of protein-polymer conjugates.

Protein-polymer conjugates are widely employed for applications in medicine, biotechnology and nanotechnology. Covalent attachment of synthetic polymers to proteins improves protein stability, solubility, and biocompatibility. Furthermore, synthetic polymers impart new properties such as self assembly and phase behavior. Polymer attachment at amino acid side-chains and at ligand binding sites is typically exploited. This Emerging Area focuses on synthetic methods to prepare protein-reactive polymers and also employing the protein itself as an initiator for polymerization.

Purification technique for the removal of ruthenium from olefin metathesis reaction products

Abstract Ring-closing metathesis (RCM) products of reactions utilizing RuCl2(CHPh)(PCy3)2 (1) as a catalyst were successfully purified of unwanted ruthenium using a water-soluble coordinating phosphine, tris(hydroxymethyl)phosphine, P(CH2OH)3. Several simple and efficient purification procedures were compared for the isolation of the product of the RCM of diethyl diallylmalonate. The efficiency of this procedure was demonstrated for the isolation of crown-ether 3.

Nanopatterning proteins and peptides

A variety of techniques have been developed to site-specifically immobilize biomolecules onto surfaces with resolutions below one micron. The ability to pattern proteins and peptides in particular has great potential for applications in biosensors, biomaterials, and tissue engineering. For example, immobilizing proteins at the nanoscale could lead to the development of diagnostic protein nanoarrays, while patterning peptides could lead to greater control over the cell/biomaterial interface. This review discusses the methods that have been reported for patterning proteins and peptides with submicron and nanometer resolutions.

Emerging synthetic approaches for protein–polymer conjugations

Protein-polymer conjugates are important in diverse fields including drug delivery, biotechnology, and nanotechnology. This feature article highlights recent advances in the synthesis and application of protein-polymer conjugates by controlled radical polymerization techniques. Special emphasis on new applications of the materials, particularly in biomedicine, is provided.

A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) plays a crucial role in diverse cellular functions from wound healing to bone regeneration. However, a major obstacle to the widespread application of bFGF is its inherent instability during storage and delivery. Herein, we describe stabilization of bFGF by covalent conjugation of a heparin-mimicking polymer, a copolymer consisting of styrene sulfonate units and methyl methacrylate units bearing poly(ethylene glycol) side chains. The bFGF conjugate of this polymer retained bioactivity after synthesis and was stable to a variety of environmentally and therapeutically relevant stressors such as heat, mild and harsh acidic conditions, storage, and proteolytic degradation, compared to native bFGF. After applied stress, the conjugate was also significantly more active than the control conjugate system where the styrene sulfonate units were omitted from the polymer structure. This research has important implications for the clinical use of bFGF and for stabilization of heparin-binding growth factors in general.

Biocompatible Hydrogels by Oxime Click Chemistry

Oxime Click chemistry was used to form hydrogels that support cell adhesion. Eight-armed aminooxy poly(ethylene glycol) (PEG) was mixed with glutaraldehyde to form oxime-linked hydrogels. The mechanical properties, gelation kinetics, and water swelling ratios were studied and found to be tunable. It was also shown that gels containing the integrin ligand arginine-glycine-aspartic acid (RGD) supported mesenchymal stem cell (MSC) incorporation. High cell viability and proliferation of the encapsulated cells demonstrated biocompatibility of the material.

Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors

Herein, we report the synthesis of trehalose side chain polymers for stabilization of protein conjugates to environmental stressors. The glycomonomer 4,6-O-(4-vinylbenzylidene)-α,α-trehalose was synthesized in 40% yield over two steps without the use of protecting group chemistry. Polymers containing the trehalose pendent groups were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization using two different thiol-reactive chain transfer agents (CTAs) for subsequent conjugation to proteins through disulfide linkages. The resulting glycopolymers were well-defined, and a range of molecular weights from 4200 to 49 500 Da was obtained. The polymers were conjugated to thiolated hen egg white lysozyme and purified. The glycopolymers when added or covalently attached to protein significantly increased stability toward lyophilization and heat relative to wild-type protein. Up to 100% retention of activity was observed when lysozyme was stressed ten times with lyophilization and 81% activity when the protein was heated at 90 °C for 1 h; this is in contrast to 16% and 18% retention of activity, respectively, for the wild-type protein alone. The glycopolymers were compared to equivalent concentrations of trehalose and poly(ethylene glycol) (PEG) and found to be superior at stabilizing the protein to lyophilization and heat. In addition, the protein-glycopolymer conjugates exhibited significant increases in lyophilization stability when compared to adding the same concentration of unconjugated polymer to the protein.

Protein-polymer conjugates: synthetic approaches by controlled radical polymerizations and interesting applications.

Protein-polymer conjugates are of interest to researchers in diverse fields. Attachment of polymers to proteins results in improved pharmacokinetics, which is important in medicine. From an engineering standpoint, conjugates are exciting because they exhibit properties of both the biomolecules and synthetic polymers. This allows the activity of the protein to be altered or tuned, anchoring to surfaces, and supramolecular self-assembly. Thus, there is broad interest in straightforward synthetic methods to prepare protein-polymer conjugates. Controlled radical polymerization (CRP) techniques have emerged as excellent strategies to make conjugates because the resulting polymers have narrow molecular weight distributions, targeted molecular weights, and attach to specific sites on proteins. Herein, recent advances in the synthesis and application of protein-polymer conjugates by CRP are highlighted.