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Bioconjugation techniques for biomolecule-polymer conjugation are numerous, however, slow kinetics and steric challenges generally necessitate excess reagents or long reactions times. Organometallic transformations are known to circumvent these issues; however, harsh reaction conditions, incompatibility in aqueous media and substrate promiscuity often limit their use in a biological context. This work reported herein, demonstrates a facile and benign organometallic Au(III) S-arylation approach that enables the synthesis of poly(ethylene glycol) monomethyl ether (mPEG)-protein conjugates with high efficiency. Isolable and bench-stable 2, 5, and 10 kDa mPEG-Au(III) reagents were synthesized via oxidative addition into terminal aryl iodide substituents installed on mPEG substrates with a (Me-DalPhos)Au(I)Cl precursor. Reaction of the isolable mPEG-Au(III) oxidative addition complexes with a cysteine thiol on a biomolecule resulted in facile and selective cysteine arylation chemistry forging covalent S-aryl linkages and affording the stable mPEG-biomolecule conjugates. Notably, low polymer reagent loadings were used to achieve near quantitative conversion at room temperature in one minute due to the rapid kinetics and high chemoselectivity of this Au-based bioconjugation approach. Therefore, this work represents an important addition to the protein-polymer conjugation chemical tool box.

Poly(styrenyl acetal trehalose) (pSAT), composed of trehalose side chains linked to a polystyrene backbone via acetals, stabilizes a variety of proteins and enzymes against fluctuations in temperature. A promising application of pSAT is conjugation of the polymer to therapeutic proteins to reduce renal clearance. To explore this possibility, the safety of the polymer was first studied. Investigation of acute toxicity of pSAT in mice showed that there were no adverse effects of the polymer at a high (10 mg/kg) concentration. The immune response (antipolymer antibody and cytokine production) in mice was also studied. No significant antipolymer IgG was detected for pSAT, and only a transient and low level of IgM was elicited. pSAT was also safe in terms of cytokine response. The polymer was then conjugated to a granulocyte colony stimulating factor (GCSF), a therapeutic protein that is approved by the Federal Drug Administration, in order to study the biodistribution of a pSAT conjugate. A site-selective, two-step synthesis approach was developed for efficient conjugate preparation for the biodistribution study resulting in 90% conjugation efficiency. The organ distribution of GCSF-pSAT was measured by positron emission tomography and compared to controls GCSF and GCSF-poly(ethylene glycol), which confirmed that the trehalose polymer conjugate improved the in vivo half-life of the protein by reducing renal clearance. These findings suggest that trehalose styrenyl polymers are promising for use in therapeutic protein-polymer conjugates for reduced renal clearance of the biomolecule.

Insulin, the oldest U.S. Food and Drug Administration (FDA)-approved recombinant protein and a World Health Organization (WHO) essential medicine for treating diabetes globally, faces challenges due to its storage instability. One approach to stabilize insulin is the addition of poly(trehalose methacrylate) (pTrMA) as an excipient. The polymer increases the stability of the peptide to heat and mechanical agitation and has a low viscosity suitable for injection and pumps. However, the safety and stabilizing mechanism of pTrMA is not yet known and is required to understand the potential suitability of pTrMA as an insulin excipient. Herein is reported the immune response, biodistribution, and insulin plasma lifetime in mice, as well as investigation into insulin stabilization. pTrMA alone or formulated with ovalbumin did not elicit an antibody response over 3 weeks in mice, and there was no observable cytokine production in response to pTrMA. Micropositron emission tomography/microcomputer tomography of

Enzyme nanogels (ENGs) offer a convenient method to protect therapeutic proteins from in vivo stressors. Current methodologies to prepare ENGs rely on either covalent modification of surface residues or the noncovalent assembly of monomers at the protein surface. In this study, we report a new method for the preparation of noncovalent ENGs that utilizes a heterobifunctional, photocleavable monomer as a hybrid approach. Initial covalent modification with this monomer established a polymerizable handle at the protein surface, followed by radical polymerization with poly(ethylene glycol) methacrylate monomer and ethylene glycol dimethacrylate crosslinker in solution. Final photoirradiation cleaved the linkage between the polymer and protein to afford the noncovalent ENGs. The enzyme phenylalanine ammonia lyase (PAL) was utilized as a model protein yielding well-defined nanogels 80 nm in size by dynamic light scattering (DLS) and 76 nm by atomic force microscopy. The stability of PAL after exposure to trypsin or low pH was assessed and was found to be more stable in the noncovalent nanogel compared to PAL alone. This approach may be useful for the stabilization of active enzymes.

Proherbicides increase the utilization efficiency of herbicides by reducing their off-site movement and premature degradation. Mesotrione is a prevalent herbicide used to control weeds in maize cro...

Antibodies and antigen binding fragments (FABs) are widely used as therapeutics and conjugated polymers can enhance the properties of these important biomolecules. However, limitations to the selectivity and stability of current conjugation methodologies can inhibit the exploration of new antibody-polymer conjugates. Herein, we describe a new strategy for the synthesis of these conjugates that forms a stable thioether bond and can be directly incorporated into an atom transfer radical polymerization (ATRP) initiator. Specifically, a bis-sulfone alkyl bromide initiator was synthesized and utilized in the activators generated by electron transfer (AGET) ATRP of ethylene glycol methacrylate and trehalose methacrylate to form the respective polymers. The trehalose polymer was then irreversibly inserted into the disulfide bonds of Herceptin and Herceptin FAB after mild reduction to form the conjugates with quantitative conversions as verified by Western Blot and mass spectrometry after cleavage of the polymer. The binding of the Herceptin and Herceptin Fab conjugates to the receptor was investigated by indirect ELISA (enzyme-linked immunosorbent assay) and the EC50’s were 0.90 and 2.74 nM, respectively, compared to Herceptin (0.26 nM) and the Fab (0.56 nM). The conjugates were subjected to heating studies at a constant 75 °C, the temperature determined in a heat ramp to be the threshold of stability for the antibody and FAB; the trehalose polymer was found to considerably increase the thermal stability of both Herceptin and Herceptin Fab. This work provides a new way to prepare polymer-antibody/Fab conjugates utilizing bis-sulfone end groups installed by atom transfer radical polymerization of the functionalized initiators and a way to stabilize these important molecules by conjugation to trehalose polymers.

Traceless self-immolative linkers are widely used for the reversible modification of proteins and peptides. This article describes a new class of traceless linkers based on

To improve the efficacy of antibody drug conjugates (ADCs), there has been significant focus on increasing the drug-to-antibody ratio (DAR) in order to deliver more payload. However, due to the hydrophobicity of many cytotoxics, highly-loaded conjugates often have lower physicochemical stability and poorer pharmacokinetic outcomes, requiring the development of new hydrophilic linkers. Herein, we report a platform for the preparation of functional, sequence-defined polymers for conjugation to antibodies. We demonstrate the successful synthesis of novel diazido macrocyclic sulfate monomers of varied size ranging from 4 to 7 ethylene glycol repeat units. These monomers were then successively ring-opened to produce sequence-defined polymers that contained either 4 or 6 azides for post-synthesis functionalization. Given the hydrophilic ethylene glycol backbone and chemically defined nature of the polymers, we envisioned this as a useful strategy in the preparation of highly-loaded ADCs. To demonstrate this, we prepared a model polymer-fluorophore scaffold composed of 4 coumarin molecules and conjugated it to Herceptin. We fully characterized the conjugate via mass spectrometry, which yielded a polymer-to-antibody ratio of 6.6, translating to a total of 26 fluorophores conjugated to the antibody at the inter-chain disulfides. We believe this technology to not only be a meaningful contribution to the field of sequence-defined polymers and conjugates, but also as a general and tunable platform for drug delivery.

Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR). The GHR antagonist, B2036, combines an amino acid substitution at 120 that confers GHR antagonist activity, with eight additional amino acid substitutions. Conjugation to polyethylene glycol (PEG) increases the serum half-life of these proteins due to reduced renal clearance. Recombinant forms of GH and its antagonists are mainly produced in prokaryotic expression systems, such as E. coli. However, efficient production in E. coli is problematic, as these proteins form aggregates as inclusion bodies resulting in poor solubility. In the present study, we demonstrate that N-terminal fusion to a thioredoxin (Trx) fusion partner improves soluble expression of codon-optimized B2036 in E. coli when expressed at 18 °C. Expression, purification and PEGylation protocols were established for three GHR antagonists: B2036, B20, and G120Rv. Following purification, these antagonists inhibited the proliferation of Ba/F3-GHR cells in a concentration-dependent manner. PEGylation with amine-reactive 5 kDa methoxy PEG succinimidyl propionate yielded a heterogeneous mixture of conjugates containing four to seven PEG moieties. PEGylation significantly reduced in vitro bioactivity of the conjugates. However, substitution of lysine to arginine at amino acid residue 120 in B2036 improved the in vitro activity of the PEGylated protein when compared to unmodified PEGylated B2036. Pharmacokinetic analysis demonstrated that the circulating half-life of PEGylated B20 was 15.2 h in mice. Taken together, we describe an effective strategy to produce biologically active PEGylated human GHR antagonists.

Polymers that stabilize biomolecules are important as excipients in protein formulation. Herein, we describe a class of degradable polymers that have tunable degradation rates depending on the polymer backbone and are able to stabilize proteins to aggregation. Specifically, zwitterion- and trehalose-substituted polycaprolactone, polyvalerolactone, polycarbonate, and polylactide were prepared and characterized with regards to their hydrolytic degradation and ability to stabilize insulin to mechanical agitation during heat. Ring-opening polymerization (ROP) of allyl-substituted monomers was carried out using organocatalysis, resulting in well-defined alkene-substituted polymers with good control over molecular weight and dispersity. The polymers were then modified using photocatalyzed thiol-ene reactions to install protein-stabilizing carboxybetaine and trehalose side chains. The resulting polymers were water-soluble and exhibited a wide range of half-lives, from 12 hours to more than 4 months. The polymers maintained the ability to stabilize the therapeutic protein insulin from activity loss due to aggregation, demonstrating their potential as degradable excipients for protein formulation.

Olefin metathesis has revolutionized synthetic approaches to carbon-carbon bond formation. With a rich history beginning in industrial settings through its advancement in academic laboratories leading to new and highly active metathesis catalysts, olefin metathesis has found use in the generation of complex natural products, the cyclization of bioactive materials, and in the polymerization of new and unique polymer architectures. Throughout this review, we will trace the deployment of olefin metathesis-based strategies for the modification of proteins, a process which has been facilitated by the extensive development of stable, isolable, and highly active transition-metal-based metathesis catalysts. We first begin by summarizing early works which detail peptide modification strategies that played a vital role in identifying stable metathesis catalysts. We then delve into protein modification using cross metathesis and finish with recent work on the generation of protein-polymer conjugates through ring-opening metathesis polymerization.

Hydrophilic amendments can enhance soil moisture content, which, in turn, can improve crop health under drought conditions. Understanding how different hydrogels interact with specific crops is necessary for optimal application. The soil conditioning abilities of a trehalose hydrogel and polyacrylate-based hydrogel were evaluated for tomatoes (Solanum lycopersicum) subjected to drought. Tomato plants were transplanted into individual pots with soil that contained trehalose hydrogel (0.4 wt%), polyacrylate-based hydrogel (0.4 wt%), or no hydrogel and subjected to a well-watered treatment or to pronounced soil drought, with or without rewatering. The health of tomato plants was monitored by measuring leaf total chlorophyll (a + b) concentration, leaf water potential (Ψleaf), stomatal conductance (gs) and relative growth rate (RGR). The polyacrylate-based hydrogel, but not the trehalose hydrogel, improved tomato plant function under drought conditions, as indicated by improved gs and RGR relative to the well-watered control. However, when subjected to a second drought, neither hydrogel was effective, and neither prolonged survival. The more hydrophilic polyacrylate-based hydrogel demonstrated promise in improving the growth of tomato plants under drought when included as a soil amendment at 0.4 wt%. This research is important for understanding the effects of these hydrogels as soil conditioners in drought prone systems.

Stimuli-responsive membranes that enable the smart control of membrane transport properties represent an interesting class of materials for a wide range of separation processes. In this work, surfa...

Multivalent protein-protein interactions serve central roles in many essential biological processes, ranging from cell signaling and adhesion to pathogen recognition. Uncovering the rules that govern these intricate interactions is important not only to basic biology and chemistry, but also to the applied sciences where researchers are interested in developing molecules to promote or inhibit these interactions. Here we report the synthesis and application of atomically precise inorganic cluster nanomolecules consisting of an inorganic core and a covalently linked densely-packed layer of saccharides. These hybrid agents are stable under biologically relevant conditions and exhibit multivalent binding capabilities, which enable us to study the complex interactions between glycosylated structures and a dendritic cell lectin receptor. Importantly, we find that subtle changes in the molecular structure lead to significant differences in the nanomolecule’s protein-binding properties. Furthermore, we demonstrate an example of using these hybrid nanomolecules to effectively inhibit protein-protein interactions in a human cell line. Ultimately, this work reveals an intricate interplay between the structural design of multivalent agents and their biological activities toward protein surfaces.

Functional handles appended to polymer chain ends are important tools often used as spectroscopic probes for determining polymer structure, affinity labels, and as reactive handles for the conjugation of functional payloads. An easily tunable molecular handle able to carry out multiple functions simultaneously would be of significant use at the polymer, materials, and biology interface. Here, we report the development of carborane-containing chain transfer agents (CTAs, commonly referred to as RAFT agents) which are used in reversible addition–fragmentation chain transfer (RAFT) polymerization. These carborane RAFT agents establish control over polymerization processes leading to monodisperse (Đ = 1.03–1.15) polymers made from N-isopropylacrylamide, styrene, 4-chlorostyrene, and methyl acrylate monomers. The tunable nature of the carborane-based scaffold appended on the polymer chain end serves as a general 1H NMR spectroscopic handle, which can be used to elucidate polymer molecular weight via end-group analysis. Isothermal titration calorimetry (ITC) measurements show that synthesized carborane terminated polymers exhibit strong binding to β-cyclodextrin with an affinity (Ka) of 9.37 × 104 M−1, thereby demonstrating its potential use as an affinity label. Additionally, we show that the free B–H vertices on the carborane RAFT agents exhibit a Raman vibrational signal at ∼2549 cm−1, a Raman-silent region for biological milieu, indicating its potential utility as an innate Raman active probe. The reported carborane RAFT agents bolster the expanding toolbox of molecular probes and serve as tunable platforms for incorporating additional and complementary handles for tailoring chain-end functionality and facilitating polymer analysis.

Growth hormone (GH) has been implicated in cancer progression andis a potential target for anticancer therapy. Currently, pegvisomant is the only GH receptor (GHR) antagonist approved for clinical use. Pegvisomant is a mutated GH molecule (B2036) which is PEGylated on amine groups to extend serum half-life. However, PEGylation significantly reduces the bioactivity of the antagonist in mice. To improve bioactivity, we generated a series of B2036 conjugates with the site-specific attachment of 20, 30, or 40 kDa methoxyPEG maleimide (mPEG maleimide) by introduction of a cysteine residue at amino acid 144 (S144C). Recombinant B2036-S144C was expressed in Escherichia coli, purified, and then PEGylated using cysteine-specific conjugation chemistry. To avoid issues with dimerization due to the introduced cysteine, B2036-S144C was PEGylated while immobilized on an Ni-nitrilotriacetic (Ni-NTA) acid column, which effectively reduced disulfide-mediated dimer formation and allowed efficient conjugation to mPEG maleimide. Following PEGylation, the IC50 values for the 20, 30, and 40 kDa mPEG maleimide B2036-S144C conjugates were 66.2 ± 3.8, 106.1 ± 7.1, and 127.4 ± 3.6 nM, respectively. The circulating half-life of the 40 kDa mPEG conjugate was 58.3 h in mice. Subcutaneous administration of the 40 kDa mPEG conjugate (10 mg/kg/day) reduced serum insulin-like growth factor I (IGF-I) concentrations by 50.6%. This in vivo reduction in serum IGF-I was at a considerably lower dose compared to the higher doses required to observe comparable activity in studies with pegvisomant. In conclusion, we have generated a novel PEGylated GHR antagonist by the solid-phase site-specific attachment of mPEG maleimide at an introduced cysteine residue, which effectively reduces serum IGF-I in vivo.

Regulation of human growth hormone (GH) signaling has important applications in the remediation of several diseases including acromegaly and cancer. Growth hormone receptor (GHR) antagonists currently provide the most effective means for suppression of GH signaling. However, these small 22 kDa recombinantly engineered GH analogues exhibit short plasma circulation times. To improve clinical viability, between four and six molecules of 5 kDa poly(ethylene glycol) (PEG) are nonspecifically conjugated to the nine amines of the GHR antagonist designated as B2036 in the FDA-approved therapeutic pegvisomant. PEGylation increases the molecular weight of B2036 and considerably extends its circulation time, but also dramatically reduces its bioactivity, contributing to high dosing requirements and increased cost. As an alternative to nonspecific PEGylation, we report the use of genetic code expansion technology to site-specifically incorporate the unnatural amino acid propargyl tyrosine (pglY) into B2036 with the goal of producing site-specific protein-polymer conjugates. Substitution of tyrosine 35 with pglY yielded a B2036 variant containing an alkyne functional group without compromising bioactivity, as verified by a cellular assay. Subsequent conjugation of 5, 10, and 20 kDa azide-containing PEGs via the copper-catalyzed click reaction yielded high purity, site-specific conjugates with89% conjugation efficiencies. Site-specific attachment of PEG to B2036 is associated with substantially improved in vitro bioactivity values compared to pegvisomant, with an inverse relationship between polymer size and activity observed. Notably, the B2036-20 kDa PEG conjugate has a molecular weight comparable to pegvisomant, while exhibiting a 12.5 fold improvement in half-maximal inhibitory concentration in GHR-expressing Ba/F3 cells (103.3 nM vs 1289 nM). We expect that this straightforward route to achieve site-specific GHR antagonists will be useful for GH signal regulation.

Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR). The GHR antagonist, B2036, combines an amino acid substitution at 120 that confers GHR antagonist activity, with eight additional amino acid substitutions. Conjugation to polyethylene glycol (PEG) increases the serum half-life of these proteins due to reduced renal clearance. Recombinant forms of GH and its antagonists are mainly produced in prokaryotic expression systems, such as

Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR), activating key signalling pathways including the JAK/STAT pathway. Excess GH secretion leads to acromegaly and tumoral expression has been implicated in cancer progression, suggesting that GH is also a potential target for anticancer therapy. Pegvisomant is the only GHR antagonist approved for clinical use. This antagonist is a PEGylated form of a mutated GH (B2036) that binds and blocks the receptor. Conjugation to polyethylene glycol (PEG) at multiple amine residues reduces in vitro bioactivity but extends the serum half-life resulting in improved in vivo bioactivity. We investigated whether we could generate a long-acting PEGylated GHR antagonist through site-specific conjugation of PEG. A codon optimised GHR antagonist, with an introduced free cysteine residue at amino acid site 144 (S144C), was generated by gene synthesis and recombinantly engineered by gene fusion with thioredoxin. Recombinant protein was expressed in E. coli and purified using a series of chromatographic methods. Antagonists were PEGylated using cysteine-specific conjugation chemistry. In vitro activity was determined using a Ba/F3-GHR viability assay, and in vivo pharmacokinetic and bioactivity was determined in mice. Fusion to thioredoxin was found to improve soluble protein expression at 30℃, resulting in dramatically increased yield. After a series of purification steps, including Ni-NTA, 3C protease cleavage and ion-exchange chromatography, a single band with a molecular mass of 22 kDa was observed by SDS-PAGE analysis. The recombinant antagonist was conjugated to 20 kDa or 30 kDa-PEG at amino acid site S144C. After purification, a single band with an effective molecular size of approximately 60 kDa (PEG-20kDa conjugate) or 70 kDa (PEG-30kDa conjugate) was observed by SDS-PAGE analysis. The unconjugated antagonist inhibited the proliferation of Ba/F3-GHR cells in a dose-dependent manner with a half maximal inhibitory concentration (IC50) of 10.1 ± 2.5 nM. Following PEGylation and purification, the PEG-20kDa and PEG-30kDa conjugates retained high in vitro bioactivity with an IC50 of 66.2 ± 3.8 nM and 106.1 ± 7.1 nM, respectively. Pharmacokinetic analysis demonstrated that PEGylation increased the serum half-life to approximately 15 hours in mice. Subcutaneous administration of the PEG-30kDa conjugate (10 mg/kg/day) reduced serum IGF-I levels in mice. In conclusion, we have generated a novel long-acting human GHR antagonist conjugate by introducing a free cysteine at a non-essential site of the antagonist and targeted attachment of PEG.

The Lewis acid–base adduct approach has been widely used to form uniform perovskite films, which has provided a methodological base for the development of high-performance perovskite solar cells. However, its incompatibility with formamidinium (FA)-based perovskites has impeded further enhancement of photovoltaic performance and stability. Here, we report an efficient and reproducible method to fabricate highly uniform FAPbI3 films via the adduct approach. Replacement of the typical Lewis base dimethyl sulfoxide (DMSO) with N-methyl-2-pyrrolidone (NMP) enabled the formation of a stable intermediate adduct phase, which can be converted into a uniform and pinhole-free FAPbI3 film. Infrared and computational analyses revealed a stronger interaction between NMP with the FA cation than DMSO, which facilitates the formation of a stable FAI·PbI2·NMP adduct. On the basis of the molecular interactions with different Lewis bases, we proposed criteria for selecting the Lewis bases. Owed to the high film quality, per...

Insulin is an important therapeutic protein for the treatment of diabetes, but it is unstable and aggregates upon exposure to environmental stressors encountered during storage and transport. To prevent degradation of the protein in this manner and retain as much in vivo bioactivity as possible, a well-defined insulin-trehalose glycopolymer conjugate was synthesized. To accomplish this, a strategy was employed to site-specifically modify insulin with a polymerization initiator at a particular conjugation site; this also facilitated purification and characterization. Lysine of the B chain was preferentially modified by conducting the reaction at high pH, taking advantage of its higher nucleophilicity than the N-terminal amines. Trehalose monomer was polymerized directly from this macroinitiator to form a well-defined conjugate. Bioactivity of the site-specific conjugate was shown to be higher compared to the non-specific conjugate and the same as the analogous site-specific polyethylene glycol (PEG) conjugate as confirmed by the insulin tolerance test (ITT) in mice. The conjugated trehalose glycopolymer also stabilized insulin to heat as measured by high-performance liquid chromatography (HPLC).

Proteins are an important class of therapeutics that have advantages including high target specificity, but challenges to their use include rapid clearance and low physical stability. Conjugation of synthetic polymers is an effective approach to address the drawbacks and enhance other properties such as solubility. In this review, we present various considerations in synthesizing protein-polymer conjugates for therapeutic applications with a focus on the choice of polymer, protein, and conjugation method, as well as characterization and evaluation of the resulting conjugate in order to maximize the therapeutic potential of the protein drug.

Fluorine-containing polymers have potential for use in medicine and other applications, but the synthesis of degradable fluorous polymers is underexplored. In this report, we present a facile route to degradable fluorinated polymers and characterize the effect of fluorous comonomer identity on the polymerization as well as the degradation kinetics of the resulting polymer. Copolymers of poly(ethylene glycol methyl ether methacrylate) (PEGMA), fluorous methacrylate (1H,1H,2H,2H-perfluorooctyl or 1H,1H,2H,2H,3H,3H-perfluoropentyl methacrylate), and cyclic ketene acetal 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) were synthesized via ruthenium-catalyzed living radical polymerization. It was observed that increasing the fluorous monomer content led to enhanced BMDO incorporation in the resulting polymer. Density functional theory calculations suggest that this is due to the decreased energy gap between the singly occupied molecular orbital (SOMO) of the methacrylate radical and the highest occupied molecular o...

An easy-to-implement science outreach demonstration featuring a mechanically and photochemically color-changing polymer is described. The active polymeric material is a filled poly(dimethylsiloxane) (PDMS) elastomer that is covalently functionalized with spiropyran (SP), which is both a photochemical and mechanochemical switch. The material can be reversibly changed from colorless to dark purple by exposing it to light from a blue laser pointer or providing a mechanical stimulus such as hitting the polymer with a hammer or dragging a blunt object across the surface. The keynote demonstration is a PDMS chemical-drawing board that allows children literally to “write without ink” using a laser pointer or a blunt stylus. Collectively, these demonstrations are suitable for various student groups and encompass concepts in polymer and materials chemistry, photochemistry, and mechanochemistry. This demonstration has been successfully employed dozens of times in multiple universities across North America.

There is considerable interest in the use of proteins as therapeutics and as chemical and biochemical reagents. However, many proteins are unstable and aggregate when exposed to stressors, including increased temperature, pH change, agitation, and desiccation. Polymers with side chain trehalose units were shown to be effective protein stabilizers, preventing aggregation and prolonging activity. Herein, we report the synthesis and characterization of four trehalose regioisomers containing a vinylbenzyl ether moiety at either the 2-O, 3-O, 4-O, or 6-O position. Computational analysis of these regioisomers suggested that they differ in their conformational flexibility, but all retained the native clam shell conformation of trehalose. Polymers were synthesized from the monomers separately via free radical polymerization and one polymer was prepared containing all of the regioisomers. The polymers were tested for their ability to stabilize insulin, and were found to prevent agitation-induced aggregation comparably. The results show that for insulin the effect of trehalose positional modification is minimal and suggest that the clam shell conformation itself may be more important than the polymer backbone attachment site for stabilization of proteins.

The majority of biomolecules are intrinsically atomically precise, an important characteristic that enables rational engineering of their recognition and binding properties. However, imparting a similar precision to hybrid nanoparticles has been challenging because of the inherent limitations of existing chemical methods and building blocks. Here we report a new approach to form atomically precise and highly tunable hybrid nanomolecules with well-defined three-dimensionality. Perfunctionalization of atomically precise clusters with pentafluoroaryl-terminated linkers produces size-tunable rigid cluster nanomolecules. These species are amenable to facile modification with a variety of thiol-containing molecules and macromolecules. Assembly proceeds at room temperature within hours under mild conditions, and the resulting nanomolecules exhibit high stabilities because of their full covalency. We further demonstrate how these nanomolecules grafted with saccharides can exhibit dramatically improved binding affinity towards a protein. Ultimately, the developed strategy allows the rapid generation of precise molecular assemblies to investigate multivalent interactions.

"Vaults" are ubiquitously expressed endogenous ribonucleoprotein nanoparticles with potential utility for targeted drug delivery. Here, we show that recombinant human vault nanoparticles are readily engulfed by certain key human peripheral blood mononuclear cells (PBMC), predominately dendritic cells, monocytes/macrophages, and activated T cells. As these cell types are the primary targets for human immunodeficiency virus type 1 (HIV-1) infection, we examined the utility of recombinant human vaults for targeted delivery of antiretroviral drugs. We chemically modified three different antiretroviral drugs, zidovudine, tenofovir, and elvitegravir, for direct conjugation to vaults. Tested in infection assays, drug-conjugated vaults inhibited HIV-1 infection of PBMC with equivalent activity to free drugs, indicating vault delivery and drug release in the cytoplasm of HIV-1-susceptible cells. The ability to deliver functional drugs via vault nanoparticle conjugates suggests their potential utility for targeted drug delivery against HIV-1.

Instability to storage and shipping conditions and injection administration remain major challenges in treating chronic conditions with biopharmaceuticals. Herein, formulations of poly(trehalose methacrylate) (pTrMA) were successfully optimized to stabilize insulin without appreciably increasing viscosity. Polymers were synthesized (2,400 – 29,200 Da), and added to insulin at different concentrations. pTrMA maintained >95% intact insulin against 250 rpm at 37 °C for 3 hours with at least 10 mol. eq. of 5.0 kDa, 7.5 mol. eq. of 9.4 kDa, 5 mol. eq. of 12.8 kDa, 1 mol. eq. of 19.8 kDa, and 0.5 mol. eq. of 29.2 kDa polymers, compared to 13.1% of insulin alone. The lowest pTrMA concentration formulations were more viscous than insulin alone, but the highest viscosity, U-600 with 10 mol. eq. of 5 kDa pTrMA, was only 1.43 cP at 25 °C. This data demonstrates that pTrMA is a promising low viscosity additive to stabilize the diabetes therapeutic insulin.

Heparin is a naturally occurring, highly sulfated polysaccharide that plays a critical role in a range of different biological processes. Therapeutically, it is mostly commonly used as an injectable solution as an anticoagulant for a variety of indications, although it has also been employed in other forms such as coatings on various biomedical devices. Due to the diverse functions of this polysaccharide in the body, including anticoagulation, tissue regeneration, anti-inflammation, and protein stabilization, and drawbacks of its use, analogous heparin-mimicking materials are also widely studied for therapeutic applications. This review focuses on one type of these materials, namely, synthetic heparin-mimicking polymers. Utilization of these polymers provides significant benefits compared to heparin, including enhancing therapeutic efficacy and reducing side effects as a result of fine-tuning heparin-binding motifs and other molecular characteristics. The major types of the various polymers are summarized, as well as their applications. Because development of a broader range of heparin-mimicking materials would further expand the impact of these polymers in the treatment of various diseases, future directions are also discussed.

Protein-polymer conjugates are unique constructs that combine the chemical properties of a synthetic polymer chain with the biological properties of a biomacromolecule. This often leads to improved stabilities, solubilities, and in vivo half-lives of the resulting conjugates, and expands the range of applications for the proteins. However, early chemical methods for protein-polymer conjugation often required multiple polymer modifications, which were tedious and low yielding. To solve these issues, work in our laboratory has focused on the development of controlled radical polymerization (CRP) techniques to improve synthesis of protein-polymer conjugates. Initial efforts focused on the one-step syntheses of protein-reactive polymers through the use of functionalized initiators and chain transfer agents. A variety of functional groups such as maleimide and pyridyl disulfide could be installed with high end-group retention, which could then react with protein functional groups through mild and biocompatible chemistries. While this grafting to method represented a significant advance in conjugation technique, purification and steric hindrance between large biomacromolecules and polymer chains often led to low conjugation yields. Therefore, a grafting from approach was developed, wherein a polymer chain is grown from an initiating site on a functionalized protein. These conjugates have demonstrated improved homogeneity, characterization, and easier purification, while maintaining protein activity. Much of this early work utilizing CRP techniques focused on polymers made up of biocompatible but nonfunctional monomer units, often containing oligoethylene glycol meth(acrylate) or N-isopropylacrylamide. These branched polymers have significant advantages compared to the historically used linear poly(ethylene glycols) including decreased viscosities and thermally responsive behavior, respectively. Recently, we were motivated to use CRP techniques to develop polymers with rationally designed and functional biological properties for conjugate preparation. Specifically, two families of saccharide-inspired polymers were developed for stabilization and activation of therapeutic biomolecules. A series of polymers with trehalose side-chains and vinyl backbones were prepared and used to stabilize proteins against heat and lyophilization stress as both conjugates and additives. These materials, which combine properties of osmolytes with nonionic surfactants, have significant potential for in vivo therapeutic use. Additionally, polymers that mimic the structure of the naturally occurring polysaccharide heparin were prepared. These polymers contained negatively charged sulfonate groups and imparted stabilization to a heparin-binding growth factor after conjugation. A screen of other sulfonated polymers led to the development of a polymer with improved heparin mimesis, enhancing both stability and activity of the protein to which it was attached. Chemical improvements over the past decade have enabled the preparation of a diverse set of protein-polymer conjugates by controlled polymerization techniques. Now, the field should thoroughly explore and expand both the range of polymer structures and also the applications available to protein-polymer conjugates. As we move beyond medicine toward broader applications, increased collaboration and interdisciplinary work will result in the further development of this exciting field.

Fibroblast growth factor 2 (FGF2) is a protein involved in cellular functions in applications such as wound healing and tissue regeneration. Stabilization of this protein is important for its use as a therapeutic since the native protein is unstable during storage and delivery. Additionally, the ability to increase the activity of FGF2 is important for its application, particularly in chronic wound healing and the treatment of various ischemic conditions. Here we report a heparin mimicking block copolymer, poly(styrenesulfonate-co-poly(ethylene glycol) methyl ether methacrylate)-b-vinyl sulfonate) (p(SS-co-PEGMA)-b-VS, that contains a segment that enhances the stability of FGF2 and one that binds to the FGF2 receptor. The FGF2 conjugate retained activity after exposure to refrigeration (4 °C) and room temperature (23 °C) for 7 days, while unmodified FGF2 was inactive after these standard storage conditions. A cell study performed with a cell line lacking native heparan sulfate proteoglycans indicated that the conjugated block copolymer facilitated binding of FGF2 to its receptor similar to the addition of heparin to FGF2. A receptor-based enzyme-linked immunosorbant assay (ELISA) confirmed the results. The conjugate also increased the migration of endothelial cells by 80% compared to FGF2 alone. Additionally, the FGF2-p(SS-co-PEGMA)-b-VS stimulated endothelial cell sprouting 250% better than FGF2 at low concentration. These data verify that this rationally designed protein-block copolymer conjugate enhances receptor binding, cellular processes such as migration and tube-like formation, and stability, and suggest that it may be useful for applications in biomaterials, tissue regeneration, and wound healing.

We present here the calculation of the mean time to capture of a tethered ligand to the receptor. This calculation is then used to determine the shift in the partitioning between (1) free, (2) singly bound, and (3) doubly bound ligands in chemical equilibrium as a function of the length of the tether. These calculations are used in the research article Fibroblast Growth Factor 2 Dimer with Superagonist in vitro Activity Improves Granulation Tissue Formation During Wound Healing (Decker et al., in press [1]) to explain quantitatively how changes in polymeric linker length in the ligand dimers modifies the efficacy of these molecules relative to that of free ligands. Keywords: Ligand binding

We report a novel storage technology of proteins with surface-perfluorinated poly(ethylene glycol) compartments in 2H,3H-perfluoropentane. The compartments were obtained from self-folding and self-assembly of an amphiphilic random copolymer bearing poly(ethylene glycol) and perfluoroalkyl pendants in the hydrofluorocarbon. Lysozyme and α-chymotrypsin as model proteins were efficiently encapsulated within the PEG compartments and quantitatively recovered therefrom with water. The recovered lysozyme maintained the original higher order structure without denaturation to show enzymatic activity for the hydrolysis of Micrococcus lysodeikticus as high as its original counterpart in water. The storage technology was further effective to inhibit inactivation of α-chymotrypsin.

Polymers with oligoethylene glycol side chains are promising in therapeutic protein-polymer conjugates as replacements for linear polyethylene glycol (PEG). Branched PEG polymers can confer additional stability and advantageous properties compared to linear PEGs. However, branched PEG polymers suffer from low conjugation yields to proteins, likely due to steric interactions between bulky side chains of the polymer and the protein. In an effort to increase yields, the linker length between the protein-reactive functional end-group of the polymer chain and branched PEG side chain was systematically increased. This was accomplished by synthesizing four well-defined poly(poly(ethylene glycol methyl ether) acrylates) (pPEGA) with pyridyl disulfide end-groups by reversible addition-fragmentation chain transfer (RAFT) polymerization mediated by chain transfer agents (CTAs) with different linker lengths. These, along with linear PEG and poly(N-isopropylacrylamide) (pNIPAAm), were conjugated to two model proteins, bovine serum albumin (BSA) and beta-lactoglobulin (βLG). The conjugation yields were determined by gel electrophoresis. The length of the linker affected conjugation yield for both proteins. For BSA, the conjugation yield step increased from 10% to 24% when the linker was altered from 1 ethylene glycol (EG) unit to 3, with no additional increase for 4 and 6 EG units. In the case of βLG, the yield gradually increased from 9% to the 33% when the linker length was increased from 1 to 6. PEG and pNIPAAm reacted with yields as high as 75% further emphasizing the effect of steric hindrance in lowering conjugation yields.

Tetrazine end-functionalized telechelic polymers were synthesized by controlled radical polymerization (CRP) and employed to generate T4 lysozyme homodimers. Mutant T4 lysozyme (V131C), containing a single surface-exposed cysteine, was modified with a protein-reactive trans-cyclooctene (T4L-TCO). Reversible addition–fragmentation chain transfer (RAFT) polymerization yielded poly(N-isopropylacrylamide) (pNIPAAm) with a number-average molecular weight (Mn by 1H NMR) of 2.0 kDa and a dispersity (Đ by GPC) of 1.05. pNIPAAm was then modified at both ends by postpolymerization with 6-methyltetrazine. For comparison, 2.0 kDa bis-tetrazine poly(ethylene glycol) (PEG) and 2.0 kDa bis-maleimide pNIPAAm were synthesized. Ligation of T4L-TCO to bis-tetrazine pNIPAAm or bis-tetrazine PEG resulted in protein homodimer in 38% yield and 37% yield, respectively, after only 1 h, whereas bis-maleimide pNIPAAm resulted in only 5% yield of dimer after 24 h. This work illustrates the advantage of employing tetrazine ligation o...

Biomolecule-polymer conjugates are constructs that take advantage of the functional or otherwise beneficial traits inherent to biomolecules and combine them with synthetic polymers possessing specially tailored properties. The rapid development of novel biomolecule-polymer conjugates based on proteins, peptides, or nucleic acids has ushered in a variety of unique materials, which exhibit functional attributes including thermo-responsiveness, exceptional stability, and specialized specificity. Key to the synthesis of new biomolecule-polymer hybrids is the use of controlled polymerization techniques coupled with either grafting-from, grafting-to, or grafting-through methodology, each of which exhibit distinct advantages and/or disadvantages. In this review, we present recent progress in the development of biomolecule-polymer conjugates with a focus on works that have detailed the use of grafting-from methods employing ATRP, RAFT, or ROMP.

Poly(ethylene glycols) (PEGs) with protein-reactive end-groups are widely utilized in bioconjugation reactions. Herein, we describe the use of ring-opening metathesis polymerization (ROMP) to synthesize unsaturated protein-reactive PEG analogs. These ROMP PEGs (rPEGs) contained terminal aldehyde functionality and ranged in molecular weight from 6 to 20 kDa. The polymers were readily conjugated to free amines on the protein hen egg-white lysozyme (Lyz). Biocompatibility of the unsaturated PEGs was assessed in vitro, revealing the polymers to be nontoxic up to concentrations of at least 1 mg/mL in human dermal fibroblasts (HDFs). The resulting unsaturated rPEG-lysozyme conjugates underwent metathesis-based depolymerization, resulting in decreased molecular weight of the conjugate.

There is a significant need for new biodegradable protein stabilizing polymers. Herein, the synthesis of a polymer with trehalose side chains and hydrolytically degradable backbone esters and its evaluation for protein stabilization and cytotoxicity are described. Specifically, an alkene-containing parent polymer is synthesized by reversible addition-fragmentation chain transfer polymerization, and thiolated trehalose is installed using a radical-initiated thiol-ene reaction. The stabilizing properties of the polymer are investigated by thermally stressing granulocyte colony-stimulating factor (G-CSF), which is expressed and purified using a custom-designed G-CSF fusion protein with a polyhistidine-tagged maltose binding protein. The degradable polymer is shown to stabilize G-CSF to 66% after heating at 40 °C. Poly(5,6-benzo-2-methylene-1,3-dioxepane (BMDO)-co-butyl methacrylate-trehalose) is degraded and its cellular compatibility is investigated. While the polymer is noncytotoxic, cytotoxic effects are observed from the degraded products in fibroblasts and murine myeloblasts. These data provide important information for future use of BMDO-containing trehalose glycopolymers for biomedical applications.

Heparin is a highly sulfated polysaccharide and is useful because of its diverse biological functions. However, because of batch-to-batch variability and other factors, there is significant interest in preparing biomimetics of heparin. To identify polymeric heparin mimetics, a cell-based screening assay was developed in cells that express fibroblast growth factor receptors (FGFRs) but not heparan sulfate proteoglycans. Various sulfated and sulfonated polymers were screened, and poly(vinyl sulfonate) (pVS) was identified as the strongest heparin-mimicking polymer in its ability to enhance binding of basic fibroblast growth factor (bFGF) to FGFR. The results were confirmed by an ELISA-based receptor-binding assay. Different molecular weights of pVS polymer were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers were able to facilitate dimerization of FGFRs leading to cell proliferation in FGFR-expressing cells, and no size dependence was observed. The data showed that pVS is comparable to heparin in these assays. In addition, pVS was not cytotoxic to fibroblast cells up to at least 1 mg/mL. Together this data indicates that pVS should be explored further as a replacement for heparin.

Small interfering ribonucleic acids (siRNAs) are important therapeutic agents and are challenging to deliver efficiently. To address this, covalent attachment of synthetic polymers to siRNA has become of great interest. In this report, we present a synthetic route to siRNA–polymer conjugates by the grafting from method, meaning that the polymerization of monomers occurs from an initiating site that is attached to siRNA. Specifically, a siRNA macroinitiator (siRNA-I) was prepared through disulfide exchange of 5′-thiol-modified siRNA with a pyridyl disulfide initiator. Activator generated by electron transfer atom transfer radical polymerization (AGET ATRP) of two monomers, poly(ethylene glycol) methyl ether methacrylate (PEGMA; Mn = 300) and di(ethylene glycol) methyl ether methacrylate (DEGMA), was undertaken to synthesize a series of siRNA–polymer conjugates. The resulting conjugates were characterized by mass spectrometry and 1H nuclear magnetic resonance spectroscopy and compared to siRNA–pDEGMA conjug...

A shortage of available organ donors has created a need for engineered tissues. In this context, polymer-based hydrogels that break down inside the body are often used as constructs for growth factors and cells. Herein, we report imine cross-linked gels where degradation is controllable by the introduction of mixed imine cross-links. Specifically, hydrazide-functionalized poly(ethylene glycol) (PEG) reacts with aldehyde-functionalized PEG (PEG-CHO) to form hydrazone linked hydrogels that degrade quickly in media. The time to degradation can be controlled by changing the structure of the hydrazide group or by introducing hydroxylamines to form non-reversible oxime linkages. Hydrogels containing adipohydrazide-functionalized PEG (PEG-ADH) and PEG-CHO were found to degrade more rapidly than gels formed from carbodihydrazide-functionalized PEG (PEG-CDH). Incorporating oxime linkages via aminooxy-functionalized PEG (PEG-AO) into the hydrazone cross-linked gels further stabilized the hydrogels. This imine crosslinking approach should be useful for modulating the degradation characteristics of 3D cell culture supports for controlled cell release.

Poly(ethylene glycol) (PEG)–protein therapeutics exhibit enhanced pharmacokinetics, but have drawbacks including decreased protein activities and polymer accumulation in the body. Therefore a major aim for second-generation polymer therapeutics is to introduce degradability into the backbone. Herein we describe the synthesis of poly(poly(ethylene glycol methyl ether methacrylate)) (pPEGMA) degradable polymers with protein-reactive end-groups via reversible addition–fragmentation chain transfer (RAFT) polymerization, and the subsequent covalent attachment to lysozyme through a reducible disulfide linkage. RAFT copolymerization of cyclic ketene acetal (CKA) monomer 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) with PEGMA yielded two polymers with number-average molecular weight (Mn) (GPC) of 10.9 and 20.9 kDa and molecular weight dispersities (Đ) of 1.34 and 1.71, respectively. Hydrolytic degradation of the polymers was analyzed by 1H NMR and GPC under basic and acidic conditions. The reversible covalent attachment of these polymers to lysozyme, as well as the hydrolytic and reductive cleavage of the polymer from the protein, was analyzed by gel electrophoresis and mass spectrometry. Following reductive cleavage of the polymer, an increase in activity was observed for both conjugates, with the released protein having full activity. This represents a method to prepare PEGylated proteins, where the polymer is readily cleaved from the protein and the main chain of the polymer is degradable.

Herein, amphiphilic/fluorous random copolymers bearing poly(ethylene glycol) (PEG) chains and perfluorinated alkane pendants were developed as novel non-cytotoxic polymers for protein conjugation. Three kinds of random copolymers with different initiating terminals (carboxylic acid, pyridyl disulfide, and N-hydroxysuccinimide ester) were prepared by reversible addition–fragmentation chain transfer (RAFT) copolymerization of a PEG methyl ether methacrylate and a perfluorinated alkane methacrylate with the corresponding functional chain transfer agents. All of the polymers were soluble in water to form nanostructures with perfluorinated compartments via fluorous interaction: large aggregates from the intermolecular multi-chain association and compact unimer micelles from the intramolecular single-chain folding. Such a PEGylated and perfluorinated random copolymer was non-cytotoxic to NIH 3T3 mouse embryonic fibroblast cells and human umbilical vein endothelial cells (HUVECs). Additionally, a random copolymer with a pyridyl disulfide terminal was also successfully conjugated with a thiolated lysozyme.

Effective delivery of therapeutic proteins is important for many biomedical applications. Yet, the stabilization of proteins during delivery and long-term storage remains a significant challenge. Herein, we report a trehalose-based hydrogel that stabilizes insulin to elevated temperatures prior to glucose-triggered release. The hydrogel is synthesized using a polymer with trehalose side chains and a phenylboronic acid end-functionalized 8-arm poly(ethylene glycol) (PEG). The hydroxyls of the trehalose side chains form boronate ester linkages with the PEG-boronic acid cross-linker to yield hydrogels without any further modification of the original trehalose polymer. Dissolution of the hydrogel is triggered upon addition of glucose as a stronger binder to boronic acid (K(b) = 2.57 M(−1) vs. 0.48 M(−1) for trehalose), allowing the insulin that was entrapped during gelation to be released in a glucose-responsive manner. Moreover, the trehalose hydrogel stabilizes the insulin as determined by immunobinding after heating up to 90 °C. After 30 min heating, 74% of insulin was detected by enzyme-linked immunosorbent assay (ELISA) in the presence of the trehalose hydrogel, whereas only 2% was detected without any additives.

Fibroblast growth factor-2 (FGF2) is a heparin binding protein that plays a role in a range of biological functions such as wound healing and bone regeneration. Heparin, a highly sulfated glycosaminoglycan, is required for FGF2 to bind to its receptor. Therefore, polymeric mimics of heparin are widely studied for their ability to manipulate FGF2-induced biological interactions. It is known that altering the degree of sulfonated monomer incorporation and size of heparin-mimicking polymers can affect protein-receptor binding. To elucidate the relationship between degree of sulfonation and receptor binding for the heparin-mimicking polymer, poly(styrene sulfonate-co-poly(ethylene glycol) methyl ether methacrylate) (p(SS-co-PEGMA)) a library was synthesized to contain nine polymers with degrees of sulfonation ranging from 0-100%. Kinetics of the polymerization was evaluated and reactivity ratios compared to literature results. These polymers were then tested for their ability to enhance FGF2 binding with its receptor as both covalent conjugates and as excipients. In a receptor based enzyme-linked immunosorbant assay (ELISA), as well as a cell-based study, the polymer with 81% SS incorporation enhanced receptor binding compared to FGF2 alone, and to a greater extent than the other polymers. Therefore, another library of polymers was prepared maintaining the degree of sulfonation at 81% and changing the size from 41 to 390 monomer repeat units. The polymers were again tested in receptor based ELISA and cell studies, and all of the different sizes performed similarly, except for degree of polymerization 295 and 390, which had reduced response in the cellular assay. These results provide important information for the use of pSS-co-PEGMA as a potential heparin-mimicking therapeutic.

Biocompatible polymers such as poly(ethylene glycol) (PEG) have been successfully conjugated to therapeutic proteins to enhance their pharmacokinetics. However, many of these polymers, including PEG, only improve the in vivo lifetimes and do not protect proteins against inactivation during storage and transportation. Herein, we report a polymer with trehalose side chains (PolyProtek) that is capable of improving both the external stability and the in vivo plasma half-life of a therapeutic protein. Insulin was employed as a model biologic, and high performance liquid chromatography and dynamic light scattering confirmed that addition of trehalose glycopolymer as an excipient or covalent conjugation prevented thermal or agitation-induced aggregation of insulin. The insulin-trehalose glycopolymer conjugate also showed significantly prolonged plasma circulation time in mice, similar to the analogous insulin-PEG conjugate. The insulin-trehalose glycopolymer conjugate was active as tested by insulin tolerance tests in mice and retained bioactivity even after exposure to high temperatures. The trehalose glycopolymer was shown to be non-toxic to mice up to at least 1.6 mg/kg dosage. These results together suggest that the trehalose glycopolymer should be further explored as an alternative to PEG for long circulating protein therapeutics.