Article pubs.acs.org/accounts
Controlled Radical Polymerization as an Enabling Approach for the Next Generation of Protein−Polymer Conjugates Emma M. Pelegri-O’Day and Heather D. Maynard* Department of Chemistry and Biochemistry and California Nanosystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095, United States CONSPECTUS: 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 graf ting 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 graf ting f rom 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.
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INTRODUCTION Covalent conjugation of synthetic polymers to bioactive proteins has led to interesting and novel types of biohybrids. By combining synthetically tunable chemical properties of polymers with well-defined and specific biological properties of proteins, a range of materials can be produced with precise control over molecular weight, biological activity, and function. These materials have applications as therapeutics, nanoreactors, and self-assembled structures, among others. The advent of controlled radical polymerization (CRP) techniques has expanded the scope of functional groups able to be incorporated into the polymer chains and has subsequently © XXXX American Chemical Society
enhanced the performance of protein conjugates. In this Account, we describe the development of CRP techniques for the preparation of protein−polymer conjugates and highlight examples where the use of functional and rationally designed polymers leads to next-generation conjugates. A variety of controlled polymerization techniques have been used to prepare protein−polymer conjugates. The two most commonly used CRP techniques are atom transfer radical polymerization (ATRP) and radical addition−fragmentation Received: May 27, 2016
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by activation of the resulting carboxylic acid with an Nhydroxysuccinimide (NHS) ester and conjugation with ε-amino group of lysines on proteins.20,21 A multistep approach is used for the preparation of all current FDA-approved protein− polymer conjugates.20 However, this approach suffers from inefficiency because of the multiple postpolymerization modifications required, as well as the difficulty of separating modified and unmodified polymer. For operational ease and to increase yield, our group and others sought to develop one-step syntheses of polymers containing protein-reactive groups by using CRP techniques.12−14 By synthesizing initiators or CTAs already containing the protein-reactive functionality, the desired polymers could be easily obtained in a single step. In our first example, we sought to develop an end-group to target cysteine residues in a protein of interest.12 An ATRP initiator containing the activated pyridyl disulfide group was prepared and used to polymerize 2-hydroxyethyl methacrylate (HEMA) with narrow dispersity (Đ ≤ 1.25) and high protein-reactive end-group retention (≥86%). Conjugation to bovine serum albumin (BSA) as a model protein was demonstrated and verified by gel electrophoresis and Ellman’s assay. Other early examples by Haddleton and others of protein-reactive endgroups prepared by CRP techniques utilized lysine-targeting functional groups. For instance, a NHS-terminated poly(ethylene glycol) methyl methacrylate (PEGMA) was prepared by ATRP and successfully conjugated to lysine residues on lysozyme.13 Analysis by gel electrophoresis showed that the conjugate was highly modified, with six to seven polymer chains attached per protein (lysozyme contains seven amines in total).13 PEGMA was also polymerized using an acetalprotected aldehyde initiator, and after acidic hydrolysis of the acetal, the aldehyde was conjugated to lysine residues in lysozyme via reductive amination.14 Following these early examples, an extensive library of protein-reactive ATRP initiators and RAFT CTAs has since been developed (see Figure 2 for some examples described in this Account). For instance, ATRP initiators have been synthesized with biotin22 as well as protected maleimide23 and aminooxy24 groups, in addition to the end-groups mentioned above. Azide-containing initiators have also been prepared and used for the polymerization of a methacryloxyethyl glucoside monomer.25 The resulting glycopolymers were conjugated to an azide-functionalized cowpea mosaic virus by using a dialkyne as a coupling agent.25 RAFT chain transfer agents (CTAs) have been synthesized with protected maleimide,26 pyridyl disulfide,27 and biotin28 end-groups, among others.19 Use of novel linkage chemistries enables the preservation of important protein structures. For instance, a dithiophenol maleimide-functionalized initiator was used for polymerization of PEGMA, and the resulting polymer was conjugated to salmon calcitonin through a disulfide bridging linkage.29 Additionally, more elaborate initiators and transfer agents have been synthesized for the preparation of conjugates with complex architectures such as dimers and star polymers. For instance, bifunctional and symmetric trithiocarbonate CTAs functionalized with pyridyl disulfide and protected aminooxy moieties were synthesized and used to polymerize poly(ethylene glycol) acrylate (PEGA) to a variety of molecular weights.30 The telechelic polymers could then be bis-functionalized with peptides through disulfide or oxime chemistry.30 More recently, we synthesized a bis-tetrazine poly(Nisopropropylacrylamide (pNIPAAm) polymer that underwent
chain transfer (RAFT) polymerization. ATRP utilizes a transition metal catalyst to control polymer growth from an alkyl halide initiator,1,2 whereas RAFT uses a thiocarbonylthiocontaining chain transfer agent (CTA) to control the reversible chain-transfer process.3 Controlled radical polymerizations can be carried out at both high and low temperatures, in a variety of polar and nonpolar solvents, and with vinyl monomers containing a range of functional groups. While CRP techniques are most commonly used in the synthesis of protein−polymer conjugates, other methods have been employed, including ring opening metathesis polymerization (ROMP),4,5 ring opening polymerization (ROP),6 and anionic polymerization, as in the case of the widely used biocompatible polymer poly(ethylene glycol) (PEG). The synthesis of protein−polymer conjugates by CRP techniques has been previously reviewed.7−11 Briefly, protein−polymer conjugates may be prepared using one of two general methods (Figure 1). In graf ting to, a protein and a
Figure 1. Graf ting to and grafting f rom approaches used to synthesize protein−polymer conjugates. The protein structure of streptavidin is from PDB entry 1N4J.
synthetic polymer are coupled through the installation of a protein-reactive group onto the polymer chain. The two macromolecules are linked using mild and biocompatible chemistries including reductive amination, disulfide exchange, or amidation.12−14 Some advantages of this method are easy characterization of the prefabricated synthetic polymer and the ability to use harsh or bioincompatible chemistries for the polymer chain synthesis. Graf ting f rom involves the growth of a polymer chain from an initiation site on the protein; this site may be installed through modification of the protein with a small-molecule ATRP initiator or RAFT CTA.15−18 This approach offers higher conjugation yields and easier purification and characterization of the protein-initiator complex with regard to placement of the polymer chains. In both cases, a variety of amino acids in the protein of interest may be targeted with careful selection of the protein-reactive group, and a recent review has detailed the variety of chemical modification methods currently available.19
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ONE-STEP SYNTHESES OF PROTEIN-REACTIVE POLYMERS First-generation protein−polymer conjugates were prepared by modifying an existing polymer to contain a protein-reactive group. The desired protein-reactive functionality could then be installed via postpolymerization modifications. For instance, the United States Food and Drug Administration (FDA)-approved conjugates Adagen and Oncaspar are both prepared through the reaction of hydroxyl-PEG with succinic anhydride, followed B
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Figure 2. Examples of protein-reactive ATRP initiators and RAFT CTAs and newer strategies toward protein-reactive polymers.
chains, difficulty in purifying molecules of similar molecular weight, as well as challenges involved with preparing conjugates with polymers of limited water solubility. Therefore, a graf ting f rom approach was developed, wherein a polymer chain is grown in situ from a protein modified with an ATRP initiator or RAFT CTA. We were the first to demonstrate the synthesis of a bioactive protein−polymer conjugate with this graf ting f rom technique by using a biotin-functionalized ATRP initiator to modify SAv and generate a macroinitiator.15 Because the interaction between SAv and biotin is very strong (Kd = 4 × 10−14 M),33 this enabled the very efficient preparation of conjugates where the modification site was known. Using a initiator-modified sacrificial resin to increase the concentration of initiating sites during the polymerization, pNIPAAm was successfully grown from the protein macronitiator, resulting in protein conjugates as visualized by gel electrophoresis and size exclusion chromatography.15 Next, we demonstrated a more general graf ting f rom approach by conjugating a pyridyl disulfide or maleimide initiator to BSA and to T4 lysozyme (T4L).18 pNIPAAm was grown from the proteins and the resulting conjugates displayed equivalent activities to the
a tetrazine-transcyclooctene click reaction with a transcyclooctene-functionalized lysozyme to yield protein dimers.31 The click reaction demonstrated higher efficiency of dimer formation compared to thiol-maleimide Michael additions.31 Alternatively, orthogonal functionalities can allow for the conjugation of two unique proteins to either end of the polymer. For instance, an α-biotin CTA was synthesized, the trithiocarbonate was radically exchanged with a protected maleimide azo initiator and the resulting polymer was used to prepare BSA-streptavidin (SAv) heterodimers.32 In vivo, proteins are often organized into dimers or multimers and their synthetic preparation is important both to study the properties of these constructs and to preorganize biomolecules into active conformations, as will be discussed further vide infra.
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GRAFTING FROM BIOMOLECULES The development of these initiators and CTAs represent significant advances in the technology for preparation of protein−polymer conjugates. However, downsides to this graf ting to technique are low conjugation yields due to steric hindrance between large biomacromolecules and bulky polymer C
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Figure 3. Protein-stabilizing trehalose glycopolymers synthesized by radical polymerization. (a) Polymerization and conjugation to lysozyme through disulfide chemistry. (b) Improved stabilization of lysozyme against lyophilization and heat by the trehalose glycopolymer conjugate. (c) Structures of an expanded library of trehalose glycopolymers. Panels (a) and (b) adapted with permission from ref 49. Copyright 2012 American Chemical Society. Panel (c) adapted with permission from ref 50. Copyright 2013 American Chemical Society. The protein structure of lysozyme is from PDB entry 4WLX.
difficult to prepare in aqueous conditions using grafting to approaches because of the vastly different solubility of hydrophobic polymers and hydrophilic proteins. By directly growing a polystyrene chain from a BSA or human serum albumin (HSA) macroinitiator, self-assembled amphiphilic nanocontainers were prepared with high yields in aqueous conditions.36 When horseradish peroxidase (HRP) was loaded into the containers, nanoreactors were prepared that retained the ability to catalyze 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation.36 Conjugates prepared by grafting f rom techniques have also been shown to retain favorable in vivo properties, suggesting that polymerization conditions do not affect protein activity and that the control over polymer conjugation site afforded by graf ting f rom conjugation will be significant in the future development of protein pharmaceuticals. Very recently, an interferon alpha (IFN-α)-pPEGMA conjugate using in situ graf ting f rom was shown to outperform the FDA-approved mPEG conjugate Pegasys in vivo.37 An alkyl halide was
unmodified proteins, showing that the polymerization conditions do not destroy protein activity or function.18 Other reports have demonstrated alternate graf ting f rom approaches. For instance, the lysine residues on chymotrypsin were targeted by treatment with 2-bromoisobutyryl bromide and ATRP of PEGMA was carried out on the resulting macroinitiator to yield the desired conjugate.16 RAFT techniques have also been used to carry out graf ting f rom approaches.17,34,35 An initial report used γ irradiation-initiated polymerization to grow pPEGA from a trithiocarbonate-modified BSA macroCTA.17 Azo initiators that dissociate at lower temperatures have also been successfully used to initiate growth of pNIPAAm and poly(hydroxyethyl acrylate) (pHEA) with modified BSA macroCTAs.34,35 The efficient graf ting f rom technique has consistently been shown to yield conjugates with improved homogeneity and greater ability to characterize and purify the resulting conjugates. This technique has also enabled the preparation of novel materials. For instance, amphiphilic conjugates are D
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conjugation.46,47 However, we became interested in using CRP techniques for the polymerization of the disaccharide trehalose. Trehalose is a naturally occurring nonreducing disaccharide that is extensively used in the food and cosmetic industries as a stabilizer and additive.48 Additionally, it has been widely reported as an important stabilizer of proteins against environmental stressors. Since efforts to maintain protein activity often significantly add to the cost of the protein drug, we were interested in developing improved protein stabilizers. Therefore, trehalose was modified to contain a polymerizable unit using transacetalization without having to use protecting group chemistry.49 By RAFT polymerization, well-defined polymers with protein-reactive pyridyl disulfide end-groups were prepared at a variety of molecular weights with good control and end-group retention.49 The polymers were conjugated to the model protein lysozyme through disulfide exchange, leading to glycopolymer conjugates (Figure 3a). The conjugates demonstrated stability against lyophilization and heat stress superior to PEG and also to free trehalose (Figure 3b).49 Further development led to a library of trehalose glycopolymers with varied linker and backbone chemistries (Figure 3a and c, Poly 1−4).50 Monomers were synthesized with both methacrylate and styrene backbones, as well as using ether, ester, and acetal linkages between the trehalose and polymerizable unit. The monomers were polymerized by free radical polymerization and the resulting polymers were used as additives to screen for their stabilizing potential. Three proteins were selected and subjected to heat and lyophilization to determine possible differences in stabilizing ability that may arise from the structural differences between the polymers. All polymers exhibited stabilization of HRP, β-galactosidase (βGal), and glucose oxidase (GOX), with slight differences observed at low polymer concentrations between styrene- and methacrylate-containing polymers depending on the identity of the protein. The broad stabilization observed supported the hypothesis that the mechanism is likely due to the combination of the osmolyte and nonionic surfactant character of the polymers. This combination presumably reduces water crystallization, exhibits the ability to chaperone refolding and prevents aggregation. In addition to linear polymers, hydrogels were also made from a mixture of mono-, di-, and trisubstituted trehalose units and shown to retain the full activity of the animal feed enzyme phytase after heating the gel to extreme heat.51 Both in vivo and in vitro testing have indicated that these materials are nontoxic and therefore have significant potential for in vivo therapeutic use.50,52 However, for long-term and generalizable use as protein stabilizers, it would be ideal to synthesize degradable analogs of trehalose glycopolymers. We have previously shown that degradable protein conjugates can be prepared using CRP techniques to include cyclic ketene acetal (CKA) monomers in the backbone of the growing vinyl polymer chain.53 Using a postpolymerization strategy of thiol− ene modification, we have also recently synthesized trehalose polymers containing 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), which undergo hydrolytic degradation as a result of the presence of esters in the backbone (Figure 3c, Poly 5).54 These polymers offer similar stabilization to previously reported styrene-based trehalose polymers and are good example of next-generation protein−polymer rational design. We have also synthesized stabilizing polymers designed to mimic natural polysaccharides. We were drawn to the heparin-
selectively installed at the C-terminus through enzymemediated ligation and aqueous ATRP carried out to yield a 66.5 kDa pPEGMA conjugate with similar pharmacokinetics as Pegasys. The IFN-α-pPEGMA conjugates displayed improved retention of bioactivity compared to Pegasys and subsequently significantly increased animal survival time in a mouse tumor model, likely due to the greater control over conjugation site. In another example, green fluorescent protein (GFP) fusion protein containing a C-terminal intein could also be modified with an ATRP initiator and pPEGMA grown from the initiating site to yield a fluorescent conjugate.38 pPEGMA-GFP was injected into tumor-laden mice and the conjugates displayed an increased tumor/blood ratio, suggesting that proteins with increased molecular weight may preferentially accumulate by the enhanced permeability and retention (EPR) effect.38 Recombinant human growth hormone (rh-GH) was modified nonspecifically with a NHS ester-modified ATRP initiator and graf ting f rom using PEGMA resulted in conjugates with drastically increased molecular weights.39 rh-GH conjugates exhibited stability against increased temperature, resistance to pepsin degradation, and in vivo studies in rats showed greater weight increase following biweekly injections compared to native protein.39
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BIOINSPIRED POLYMER DEVELOPMENT With the development of these advanced conjugation and polymerization techniques, the field has demonstrated the utility of CRP techniques in preparing protein conjugates. Building on these initial reports, we were then motivated to explore the preparation of bioinspired protein−polymer conjugates. Many protein conjugates utilize biologically compatible but functionally inert polymers. These may be linear polymers such as PEG, or polymers prepared by CRP techniques, such as pHEMA, or pPEGMA. These polymers may be easily prepared, can exhibit high conjugation efficiencies, and increase the size and proteolytic resistance of the resulting protein−polymer conjugate. However, there has been significant recent interest in using PEG alternatives to prepare protein conjugates. For instance, poly(oxazolines) have been widely used as PEG alternatives and have been shown to improve the pharmacokinetics of therapeutic proteins.40 Poly(glycerols) are also actively explored as both branched and linear alternatives.41 Degradable and biobased alternatives such as dextrin or polypeptides are also under active exploration and more examples have recently been reviewed by us and others.42,43 Conjugates made with these functional or substituted polymers often have improved biological benefits. For example, conjugates of α-chymotrypsin and polymers containing zwitterionic carboxybetaine moieties were shown to maintain greater protein activity upon exposure to elevated temperatures compared to the PEGylated versions.44 Conjugates of a cationic dendrimer and the potential celiac treatment proline-specific endopeptidase (PEP) have also been shown to help preserve protein activity against denaturation in the acidic environment of the stomach.45 In the remainder of this Account, we will focus on two example classes of bioinspired protein−polymer conjugates from our group that were enabled by developments in controlled radical polymerization. Synthetic glycopolymers are a common target for CRP due to their applicability in many biological processes. Our early reports in this area focused on the preparation of proteinreactive polymers of N-acetyl glucosamine and subsequent E
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Figure 4. Heparin-mimicking polymer synthesized by RAFT copolymerization. (a) Polymerization and conjugation to FGF2 through disulfide chemistry. (b) Stability of FGF2 conjugate against a range of environmental stressors. Adapted with permission from ref 59. Copyright 2013 Nature Publishing Group. The protein structure of FGF2 is from PDB entry 1BFG.
above physiological salt concentrations.58 Therefore, we were highly motivated to apply this material for the synthesis of FGF2-polymer conjugates. A well-controlled (Mn = 26.1 kDa, Đ = 1.16) protein-reactive polymer was synthesized by RAFT polymerization using a pyridyl disulfide-containing CTA and after removal of the trithiocarbonate, conjugated to one of the two free cysteines present in FGF2 (Figure 4a).59 The resulting conjugate was tested for resistance against heat, acidic conditions, and long-term storage and was found to exhibit remarkably enhanced stability compared to the native protein and the control conjugate pPEGMA-FGF2 (Figure 4b).59 For activation in vivo, two FGF2s must form a complex with two fibroblast growth factor receptors (FGFRs) facilitated by heparan sulfate, which is a glycan similar to heparin but attached on the cell surface. To test whether the pSS-coPEGMA polymers would successfully facilitate binding of FGF2 to its receptors in the absence of heparan sulfate, we used FR1C-11 BaF3 cells, an engineered line that expresses FGFRs but is lacking in cell-surface heparan sulfate.60 Interestingly, the conjugates exhibited minimal increased cell growth in the absence of excess heparin, indicating that the pSS-co-PEGMA
binding class of proteins, many of which are stabilized by the naturally occurring glycosaminoglycan heparin. Heparin is a highly sulfated polysaccharide which has been shown to strongly interact via electrostatic interactions with clusters of positively charged residues on heparin-binding growth factors, thus stabilizing the proteins.55 One such protein, fibroblast growth factor 2 (FGF2), is implicated in a variety of important cellular functions and has significant possibilities in wound healing treatment.56 However, FGF2 has demonstrated limited efficacy in clinical trials; one reason may be due to its instability and short half-life.57 Therefore, we were motivated to develop a synthetic heparin mimic using CRP techniques, which would enable us to effectively stabilize FGF2. Heparin contains both carboxylate and sulfate groups and it is thought that these negative charges are important for interaction with positively charged amino acids in the heparinbinding pocket. Therefore, we began with the synthesis of a copolymer containing negatively charged styrene sulfonate (SS) moieties.58 Surface plasmon resonance (SPR) measurements demonstrated that this SS-co-PEGMA copolymer exhibited significant dose-dependent binding to FGF2 that remained well F
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term storage. This sequential development of heparinmimicking polymers highlights the importance of rational design in the preparation of novel protein−polymer conjugates (Figure 6). In addition to their instability, certain growth factors also suffer from inefficiency at low concentrations due to their need to dimerize to trigger downstream signals. Previous reports have shown that predimerization of these growth factors can lead to improved protein activity.63,64 We recently demonstrated that the length of the tether connecting FGF2 dimers can have significant effects on bioactivity by using a series of homobifunctional linear PEG polymers.65 The PEG dimers were compared to native protein in vitro and were shown to have significantly improved activity, especially at low concentrations. In addition, the dimer formed a much more fertile wound bed in diabetic mice than FGF2 alone, suggesting the importance of predimerization for in vivo wound healing. Currently, we are working on preparing dimeric conjugates using the heparin-mimicking polymers as linkers. These nextgeneration protein polymer dimers should be synthetically available using our previously reported CRP-based dimerization strategies,31,66 and we expect that they should exhibit stability and activity potentially superior to PEG-based dimers.
polymer, at least at that molecular weight, did not bind to and facilitate FGF2-activation of the FGFRs. A series of other sulfated and sulfonated polymers were then screened to identify a heparin-mimicking polymer with the capacity to facilitate FGF2 binding and activation of FGFRs.61 Using the engineered cell line mentioned above (Figure 5a)
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CONCLUSIONS The field of protein−polymer conjugation has demonstrated significant progress in the past decade, beginning with the development of one-step graf ting to techniques and advancing to the ability to grow a protein chain directly from a protein macroinitiator. Further development led to the production of advanced architectures, with hetero- and homobifunctional chain transfer agents and initiators leading to defined protein dimers and multimers. The field has been also advanced by the advent of rationally designed polymers and their conjugation to therapeutically relevant and important proteins to enhance activity and or stability compared to the native protein. Future research should seek to further improve the design of these conjugates. New techniques and chemistries continue to progress the control over polymer sequence and architecture and protein conjugation sites. Despite these significant advances, there has been little variation in monomer identity. Many reports focus on PEG-based monomers such as PEGMA or PEGA. These materials should be newly evaluated, especially with concern over PEG toxicity, immunogenicity, and nondegradability becoming more prevalent. Novel materials should be considered and also rigorously evaluated using relevant and noncarcinogenic cell lines. The field should continue to look to biology, not only for polymer design, but also for well-designed and thorough biocompatibility, toxicity, immunogenicity, and
Figure 5. (a) Proliferation study in FR1C-11 BaF3 cells comparing the activating effects of heparin and pVS at various molecular weights. (b) ELISA-based FGF-FGFR binding enhanced by heparin and pVS. Adapted with permission from ref 61. Copyright 2015 American Chemical Society.
and an enzyme-linked imunosorbent assay (ELISA)-based receptor assay (Figure 5b), polymers were tested for their ability to effect dimerization and increase proliferation. A poly(vinyl sulfonate) (pVS) prepared by either free radical polymerization or RAFT polymerization was shown to achieve FGF2 receptor activation and cell proliferation comparable to heparin at both high and low concentrations. Since pSS-co-PEGMA stabilized the growth factor and pVS facilitated binding of FGF2 to the receptor, the polymer segments were then combined in a new block copolymer, pSSco-PEGMA-b-VS, to accomplish both goals.62 The block copolymer-FGF2 conjugate displayed similar proliferation to heparin and FGF2 in the cell line lacking heparin sulfate proteoglycans and higher activity in migration and angiogenesis assays, which are important events in wound healing. At the same time, the polymer increased the stability of FGF2 to long-
Figure 6. Evolution of heparin-mimicking polymers by combining stabilizing and activating properties of the natural polysaccharide heparin. G
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biodistribution experiments that will be necessary to further develop these materials for the clinic. Finally, protein−polymer conjugates should be increasingly expanded to a larger array of applications to solve major issues facing humanity today such as energy and food production. As we search to develop and improve the field of protein−polymer conjugates, it will be important to expand its interdisciplinary nature and seek expertise from scientists from a range of disciplines and to develop reproducible and large scale manufacturability for industrial use. This will serve to translate findings on CRPprepared polymer conjugates toward useful products for humankind.
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AUTHOR INFORMATION
Corresponding Author
*E-mail
[email protected]. Funding
The authors thank the National Science Foundation (CHE1507735) for funding. E.M.P.-O. thanks the Christopher S. Foote Graduate Research Fellowship in Organic Chemistry and the National Science Foundation for a Graduate Research Fellowship (DGE-1144087). Notes
The authors declare no competing financial interest. Biographies Emma M. Pelegri-O’Day received a B.A. from Williams College in 2012. She worked at the Universität Hamburg as a Fulbright Fellow before joining the Maynard group at UCLA as a graduate student in Chemistry in 2013. She currently studies biocompatible polymers for protein stabilization. Heather D. Maynard received a B.S. with Honors in Chemistry from the University of North Carolina at Chapel Hill in 1992 and a Ph.D. from the California Institute of Technology in 2000. She then moved to the Swiss Federal Institute of Technology in Zurich (ETH), where from 2000 to 2002 she was an American Cancer Society Postdoctoral Fellow. Dr. Maynard joined the UCLA faculty as an Assistant Professor in August 2002 as the first Howard Reiss Career Development Chair in the Department of Chemistry and Biochemistry and as a member of the California NanoSystems Institute. She is now a full Professor, Director of the Chemistry Biology Interface Training Program and Associate Director of Technology and Development for the California NanoSystems Institute.
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