Hydrophobic and Bulk Polymerizable Protein-Based ... · Hydrophobic and Bulk Polymerizable Protein-Based Elastomers Compatibilized with Surfactants W. Y. Chan,† E. J. King,‡ and

Hydrophobic and Bulk Polymerizable Protein-Based ElastomersCompatibilized with SurfactantsW. Y. Chan,† E. J. King,‡ and B. D. Olsen*,†

†Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge,Massachusetts 02139, United States‡Wellesley College, 106 Central Sreet, Wellesley, Massachusetts 02481, United States

*S Supporting Information

ABSTRACT: Proteins have great potential as biomass-derivedfeedstocks for material synthesis and can form strong materialsdue to their highly hydrogen-bonded nature. Elastomerscomprised of proteins and a synthetic rubbery polymer wereprepared by copolymerizing a methacrylated protein and a vinylmonomer, where proteins function as macro-cross-linkers andreinforcing fillers. Selecting a hydrophobic synthetic polymerblock partially mitigates the moisture absorption of protein-basedmaterials while maintaining desirable levels of mechanicalproperties. The use of a hydrophobic monomer is enabled bythe use of surfactants that function as compatibilizers, sinceproteins are generally insoluble in organic solvents and vinylmonomers. Surfactants also lower the softening temperature of proteins, allowing materials to be fabricated solvent free usingthermoplastic processing techniques. The preparation of a polyacrylate network toughened through incorporation of proteincross-linking domains is demonstrated using whey protein, the cationic surfactant benzalkonium chloride, and the hydrophobicmonomer n-butyl acrylate. The resulting materials are amorphous and disordered but have microphase-separated protein-richand polyacrylate-rich domains. All materials soften with increasing relative humidity, but the presence of a hydrophobicpolyacrylate decreases the material’s moisture absorption at high humidity levels when compared to pure protein and networkscomprised of a hydrophilic polyacrylate.

KEYWORDS: Protein thermoset, Surfactant, Plasticizer, Compatibilizer, Hydrophobic, Melt polymerization

■ INTRODUCTION

The use of biomass-based feedstocks in chemical and materialmanufacturing has been widely investigated as an approach toreduce dependence on fossil fuel-derived chemicals.1,2 Asproteins are abundant in biomass sources such as agriculturaland forestry feedstocks, plant- and animal-based agriculturalbyproducts, and municipal wastes, transformation of proteinsinto value-added products can contribute to materialsustainability.3,4 Proteins have many potential uses in plasticapplications due to their capability to form continuousmatrixes5 and the abundance of reactive functional groupsthat are amenable to chemical modification. However, in theabsence of protein modification or plasticizers, proteins areknown to be too brittle to handle and form.6

Plasticization is a commonly employed strategy thatincreases processability of protein-based materials and enablesthem to be thermoformed.7 The plasticizers reduce glasstransitions or softening temperatures of proteins by disruptingpolymer−polymer interactions and increasing the free volumeof protein chains. Various hydrophilic small moleculeplasticizers such as water, ethylene glycol, sorbitol, and glycerolhave typically been explored to allow proteins to be processedat lower temperatures that limit decomposition.7 In addition,

low melting point proteins and liquid crystalline polymers havebeen prepared by complexing charged proteins with ionicsurfactants.8−10 Protein melting points were lowered assurfactants disrupted electrostatic interactions between pro-teins, enabling proteins with catalytic activity to be liquefiedeven in the absence of solvent.8 In protein-based materials,plasticizers are crucial for ease of processing and molding, butthe increase in flexibility is typically achieved at the cost ofreduced mechanical strength.The mechanical properties of protein-based materials are

also usually strongly dependent on the humidity of theenvironment due to their hydrophilicity. This is oftencompounded by the use of hydrophilic plasticizers, whichgreatly limit material applications and versatility. As loweringthe hydrophilicity of protein-based materials, among otherdesirable properties, can greatly increase their utility, manyprevious studies have explored incorporating hydrophobicplasticizers such as lipids, long chain fatty acids, and waxes tolimit moisture absorption of protein films.11 Although these

Received: October 22, 2018Revised: February 20, 2019Published: April 29, 2019

Research Article

pubs.acs.org/journal/ascecgCite This: ACS Sustainable Chem. Eng. 2019, 7, 9103−9111

© 2019 American Chemical Society 9103 DOI: 10.1021/acssuschemeng.8b03557ACS Sustainable Chem. Eng. 2019, 7, 9103−9111

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molecules reduce material sensitivity to humidity, incompat-ibility between plasticizer and protein due to solubilitydifferences can lead to ineffective plasticization and poorsolvation of plasticizer in the polymer.12 The insolubility ofunmodified proteins in most organic solvents13 and commonmonomers14 in the absence of salts or other mediators alsopresents a major challenge for formulating blends andconjugates with non-water-soluble plasticizers or polymers.This issue is also encountered in protein biocatalysis, whereperforming reactions in organic solvents or even in solvent freeconditions can be of high value. Methods such as proteinchemical modification, immobilization on solid surfaces,colyophilization with protectants, and surfactant complexationhave been widely explored to address these solubilitychallenges.15,16

This work explores an approach to reduce the hydrophilicityof protein-based materials by covalently attaching proteins tohydrophobic polymer chains using whey protein isolate (WPI)as the model protein. The material synthesis requires mixingbetween the incompatible protein and water-insoluble vinylmonomers, which was achieved using an ionic surfactant ascompatibilizer. The surfactant of choice, benzalkoniumchloride (BAC), has a low melting point and is able toplasticize the protein. The dual role of surfactant as bothcompatibilizer and plasticizer critically enables solvent-freemelt polymerization of the protein-based copolymers and thepreparation of moldable thermosets. A copolymerizationstrategy described previously was employed to prepare strongand tough materials, where stiff protein domains werecovalently attached to flexible synthetic polymer segmentsthrough free radical copolymerization.17 n-Butyl acrylate wasselected as a model hydrophobic comonomer that forms therubbery polymer segments. The three components are blendedin the melt state and copolymerized to form a network ofprotein−surfactant complexes and polyacrylate. This syntheticstrategy enables partially renewable materials containingprotein reinforcing domains to be prepared using industriallyrelevant processes and can potentially be expanded in thefuture to incorporate vinyl monomers or rubbery polymersegments derived from biomass18−21 to produce fully biobasedplastics.

■ MATERIALS AND METHODSn-Butyl acrylate, poly(ethylene glycol) methyl ether acrylate (Mn480), and azobis(isobutyronitrile) were obtained from Sigma-Aldrich,while butanediol diacrylate and methacrylic anhydride were purchasedfrom Alfa Aesar. tert-Butyl peroxyacetate and benzalkonium chloridewere purchased from Acros Organics and MP Biomedicals,respectively. Whey protein isolate was purchased from Bipro USA.Protein−Surfactant Complex Preparation. Protein−surfactant

complexes were prepared by dissolving proteins in water to make 10%w/w protein solutions, followed by addition of benzalkonium chlorideat a surfactant to protein mass ratio of 1:1. The mixtures were stirreduntil homogeneous and lyophilized. Complexes with polymerizableproteins were also prepared in a similar manner. Proteins dissolved inaqueous solutions and modified with methacrylic anhydride, asdescribed in a previous work,17 were mixed with benzalkoniumchloride without intermediate purification steps.Copolymerization of Protein and Monomer. n-Butyl acrylate

and poly(ethylene glycol) methyl ether acrylate were passed overbasic alumina to remove inhibitors. To prepare the hydrophobiccopolymer, n-butyl acrylate was added to protein−surfactantcomplexes heated to 110 °C at a monomer to complex mass ratioof 1:1 and thoroughly mixed by manually stirring. Initiator tert-butylperoxyacetate (50 wt % in mineral oil) was added at an initiator to

monomer mole ratio of 1:80. The mixture was placed into a flat moldsandwiched with Teflon liners, transferred to a hydraulic press heatedto 120 °C, and polymerized under a pressure of 1000 psi for 30 min.After polymerization, the copolymers were cooled to room temper-ature under pressure, removed from the mold, and equilibrated atvarious relative humidity conditions for at least 72 h prior tomechanical characterization. The hydrohphilic material with poly-(ethylene glycol) methyl ether acrylate as the comonomer wasprepared using an analogous procedure, simply replacing n-butylacrylate with the hydrophilic monomer. A Memmert HPP 110 climatecontrol chamber was used to equilibrate samples at 23 °C and 20%,35%, 50%, or 80% relative humidity, while a desiccator with Drieritestored at room temperature was used for the 4% relative humiditycondition. A cross-linked poly(butyl acrylate) control was prepared bymixing n-butyl acrylate, butanediol diacrylate, and azobis-(isobutyronitrile) at a monomer to cross-linker to initiator moleratio of 300:1:3.75, pipetted in between two glass plates with a 1 mmspacer, and polymerized at 70 °C for 30 min.

Ternary Diagram Construction. Miscibility of whey protein,benzalkonium chloride, and n-butyl acrylate at various ratios wasdetermined by mixing the protein−surfactant complexes with butylacrylate at the desired ratios at 110 °C. After cooling, homogeneousor optically clear mixtures were designated as miscible, while mixturesthat were heterogeneous, opaque, or macrophase separated werelabeled immiscible. At low acrylate comonomer concentrations,mixtures appear more solid-like and were compression molded formiscibility determination. Mixtures that form optically transparentfilms were classified as miscible.

Mechanical Testing. Tensile testing specimens were cut using anASTM D1708 microtensile die from Pioneer Die-tecs. Five specimensper sample were stretched at a rate of 100% min−1 on a Zwick Z05machine. Averages and standard deviations of mechanical propertiesare reported.

Thermal Characterization. Moisture content in materials wasdetermined using thermogravimetric analysis (TGA) using a TAInstruments Discovery TGA instrument. Samples equilibrated underdifferent conditions were heated under air flow at a rate of 20 °Cmin−1. Total mass loss at 150 °C was attributed to moisture content.

Modulated differential scanning calorimetry (MDSC) wasperformed using a TA Instruments Discovery DSC instrument.Materials were subjected to two heating cycles and one cooling cyclewith a temperature ramp rate of 5 °C min−1 from −90 to 150 °C andtemperature modulation of 2 °C min−1 every 60 s. The secondheating ramp was used to determine reversible transition events.

Morphological Characterization. Small-angle and wide-angle X-ray scattering (SAXS and WAXS) data were acquired in transmissionmode using a Rigaku 002 microfocus X-ray source with Cu Kαradiation (0.154 nm) with a sample-to-detector distance of 109.1 mm.Data was acquired using a DECTRIS Pilatus 300 K hybrid panel arraywith an exposure time of 5 min for SAXS and 2 min for WAXS. Two-dimensional diffraction images were background corrected, azimu-thally averaged, and plotted as one-dimensional scattering profiles.

Copolymer morphologies were also studied using scanning electronmicroscopy (SEM). Flat samples were prepared by fracturing in liquidnitrogen and clamped vertically to observe the fractured surface.Materials were sputter coated with gold, and the microscope imageswere acquired at a voltage of 15 keV.

Microscope attenuated total reflection- infrared spectroscopy(ATR-FTIR) data were acquired on a Thermo Fisher ContinuumFourier Transform Infrared Microscope attached to an FTIR6700bench using a germanium crystal. Sixty-four scans were collected witha resolution of 4 cm−1. Samples were conditioned at 23 °C andvarious levels of relative humidity prior to testing, and measurementswere performed at ambient conditions. ATR and atmosphericcorrections were applied using the OMNIC software package, andall spectra were min−max normalized.

Swelling Ratio Quantification. Dried specimens were dehy-drated in a desiccator and weighed prior to submersion in dimethylsulfoxide (DMSO). Swollen samples were weighed again, and theswelling ratios were reported as the ratio of swollen mass over dry

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mass. Three specimens were tested for each material, and averagesand standard deviations are reported.

■ RESULTS AND DISCUSSIONCompatibilization of Protein and Hydrophobic

Monomer. This study demonstrates a versatile melt polymer-ization approach to prepare protein-based copolymers byconjugating stiff proteins to rubbery polymer segments in thepresence of surfactants as compatibilizers. Covalently bondedcopolymers of the protein and the polyacrylate combineadvantages of both components in terms of high strength andtoughness, as has been previously demonstrated in protein-based elastomers.17 Copolymers were prepared using polymer-izable proteins, where amine groups on the proteins werereacted with methacrylic anhydride (Scheme S1a) using apreviously described method.17,22 Proteins modified to havemore than one methacrylamide group can be conjugated tomore than one growing polyacrylate chain and therefore serveas macro-cross-linkers that produce cross-linked networksreinforced by stiff protein domains (Scheme S1b). As proteinsare typically hygroscopic, the non-water-soluble n-butylacrylate (n-BA) is selected as the comonomer, which formsthe low glass transition temperature polymer segments.n-Butyl acrylate has negligible water absorption and

therefore the potential to reduce the material’s overall moisturesensitivity but is immiscible with proteins at all temperaturesand lacks good common solvents. These constitute the mainchallenges when formulating materials, as copolymerizationstrategies are usually applied to both water-soluble proteinsand monomers or proteins that are directly soluble in themonomers.14,17 Drawing inspiration from studies that showthat protein−surfactant complexation imparts organic solventsolubility to proteins,23 a surfactant was selected as acompatibilizer to improve protein−monomer miscibility. Thisapproach is demonstrated using whey protein isolate (WPI),which was complexed with benzalkonium chloride (BAC), acationic surfactant. Protein−surfactant complexes were pre-pared by mixing the two in water followed by lyophilization.Upon addition of n-butyl acrylate, some compositions form

miscible or homogeneous dispersions and can be further meltpolymerized to form thermosets (Figure 1a).Combinations of WPI, BAC, and n-butyl acrylate that result

in miscible and melt polymerizable mixtures are shown in aternary diagram (Figure 1b). Binary mixtures of protein−acrylate are completely immiscible, while those of protein−surfactant and surfactant−acrylate are partially miscible.Miscible three-component mixtures formed viscous, trans-parent fluids or transparent films after either mixing orcompression molding at elevated temperatures. When thesurfactant to protein ratio is low (left side the ternary diagram),the complexes that are infusible in the absence of n-butylacrylate only form miscible mixtures when mixed with largeamounts of acrylate. On the other hand, at higher surfactant toprotein ratios (middle to right side of the ternary diagram),complexes are miscible with n-butyl acrylate primarily at lowacrylate compositions. These differences may be related to theratio of protein net charge and the amount of surfactant.Studies by Hanski et al. on complexes of synthetic cationicpolypeptide poly(L-lysine) and anionic surfactant dodecylben-zenesulfonic acid have shown that stoichiometric complexeswith one surfactant molecule for each lysine residue areinfusible even when heated.10 However, complexes thatcontain an excess of surfactant are thermoprocessable, whichparallels the miscibility observations of whey protein and BACcomplexes. Excess surfactant in the complexes serves asplasticizer but may lead to immiscibility at high acrylatecontent due to limited miscibility of the ionic surfactant in thenonpolar acrylate. A composition where the protein is fullycompatible with the acrylate monomer is selected forcopolymerization and for studies on material mechanicalproperties and structure. While the approach is demonstratedonly for one protein−surfactant combination, this strategy ofcompatibilizing protein and monomer using surfactants isgeneralizable across various other protein mixtures andmonomers.Besides functioning as a compatibilizer, the surfactant also

reduces the melting point of proteins and enables the proteinmixture to be thermoformed without the use of solvents.

Figure 1. (a) Surfactant, benzalkonium chloride (BAC), enables mixing between the otherwise incompatible whey protein isolate (WPI) and n-butyl acrylate (n-BA). Protein−surfactant complexes were copolymerized with n-BA in the melt state to produce copolymer sheets. (b) Ternarydiagram depicting ratios of WPI, BAC, and n-BA that result in miscible and immiscible mixtures.

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Thermal transitions of a desiccated protein−surfactant−polyacrylate copolymer and its constituents are measuredwith modulated differential scanning calorimetry (MDSC).Materials were dried prior to measurement to demonstrateproperties of whey protein, benzalkonium chloride, andacrylate during solvent-free blending and melt polymerization.Glass transitions of the amorphous materials manifest in stepchanges in their reversing heat capacity and peaks in the firstderivative of reversing heat capacity versus temperature(Figure 2). Dry whey protein does not undergo a glass

transition when heated up to 150 °C, while the surfactant,BAC, has a midpoint glass transition temperature (Tg) ataround −32 °C. A binary miscible mixture comprised of 50%protein and 50% surfactant has a single Tg at 68 °C, whichsuggests that the protein is plasticized. Tg’s of other proteinshave also previously been shown to be high when the protein isdry and are rapidly lowered in the presence of solvent orplasticizer24,25 as a result of disrupted hydrogen-bonding andelectrostatic interactions and increased protein chain mobility.The use of ionic surfactants in the synthesis of solvent-freeprotein melts has demonstrated that protein melting points canbe drastically lowered by complexing charged proteins withionic surfactants with long alkyl tails.8,9,26 This inspired theselection of BAC, a low melting point cationic surfactant, asthe plasticizer, since the main component of whey protein, β-lactoglobulin, has a small net negative charge at neutral pH(isoelectric point 5.3).27,28 The resulting WPI-BAC protein−surfactant complex not only has a low glass transitiontemperature but also softens considerably when heated andmixed with the liquid n-butyl acrylate. Above the blendingtemperature of 110 °C, the miscible three-component mixturescan appear as pastes or viscous fluids and are amenable tomolding and melt polymerization.Elastomeric protein−surfactant−polyacrylate copolymer

sheets were prepared by compression molding mixtures ofprotein−surfactant complexes and comonomer in the presenceof a thermal initiator (Figure 1a). During compression moldingat elevated temperatures, mixtures of protein, surfactant, and

acrylate monomers were softened and pressed into sheets,while the polymerization process cures the material. Moldingpressure does not have a significant impact on materialproperties, as shown in a comparison of copolymers moldedand polymerized at 250 and 1000 psi (Table S1). The lowvapor pressure surfactant does not participate in polymer-ization and is left in the material as a plasticizer. Since thecured material does not contain solvent or volatilecomponents, drying steps are eliminated. This is advantageousas materials can deform or fracture while drying,29 which canbe costly and limiting. The use of solvent also substantiallylimits the types of processes that may be used and shapes ofparts that may be fabricated.

Effect of Humidity on Mechanical Properties. Miscibleblends of proteins and highly hydrophobic monomers enablethe preparation of less hygroscopic protein copolymers. n-Butylacrylate was selected as a model hydrophobic monomer as itshomopolymer absorbs ∼0% moisture after incubation at 23 °Cand 80% relative humidity. In contrast, solution cast wheyprotein absorbs 13% moisture under the same conditions. Thedisparity in moisture absorption between proteins andhydrophobic synthetic polymers highlights the challenge offormulating protein-based materials with consistent propertiesregardless of environmental conditions, as water can softenmaterials by swelling or plasticizing polymers to reduce glasstransitions.30 Moisture absorption at 80% relative humidity fora protein−surfactant complex (mass ratio 1:1) and acopolymerized protein−surfactant−PBA (mass ratio: 1:1:2)is 11.6% and 7.2%, respectively, showing the positive effect ofn-butyl acrylate comonomer in decreasing the overall waterabsorption.To elucidate the role of monomers on moisture absorption

and provide a direct comparison to n-butyl acrylate, materialswere also prepared with a water-soluble monomer, poly-(ethylene glycol) methyl ether acrylate. Like butyl acrylate, thismonomer has a homopolymer Tg well below room temper-ature, which eliminates effects of Tg change with waterabsorption on mechanical property differences. While bothtypes of copolymers absorb the same amount of water afterdesiccation, differences in moisture content increase rapidlywith humidity levels (Figure 3a, Figure S1). At 80% relativehumidity, the hydrophilic copolymer absorbs nearly two timesmore moisture than the n-butyl acrylate-based copolymer.Across all conditions, the PEG-based hydrophilic copolymerperforms worse than the n-butyl acrylate-based copolymer.Major differences were observed for dried materials, eventhough their moisture content was similar. This may be due todifferences in monomer size, which has been observed in otherstudies where the larger monomer was hypothesized to formmore homopolymers than copolymers with the protein due tosterics.31 On the other hand, tensile properties of bothcopolymers exhibit similar trends with humidity (Figure 3b−d). Both materials soften considerably above 20% relativehumidity and have elongation at break and toughness that peakbetween 20% and 50% relative humidity. Even in highhumidity conditions, both copolymers maintain their structuralintegrity and toughness. An optimal relative humidity fortoughness is observed, as small amounts of water act asplasticizers and raise material extensibility at low humiditylevels. Above a certain water content, the markedly softermaterials fracture at much lower tensile strengths, resulting indecreased toughness. Similarities in the trends regardless of thepolyacrylate hydrophobicity suggest that water absorption and

Figure 2. (a) Reversing heat capacity thermograms and (b) first-orderderivative of reversing heat flow versus temperature from modulatedDSC runs on BAC, WPI, WPI-BAC complex, and WPI-BAC-polyBAcopolymer. Protein:surfactant mass ratio for the complex is 1:1, whilethe protein:surfactant:comonomer ratio for the copolymer is 1:1:2.Copolymer was prepared with protein with an average methacryla-mide functionality of 6. All materials were desiccated prior tomeasurements. Curves are offset for clarity.

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the resulting softening in protein reinforcing domainsdominate changes in mechanical properties.Material Morphology. Due to the nonspecific nature of

protein modification and the presence of diverse proteinspecies, the protein−surfactant−polyacrylate copolymers areexpected to exhibit microphase separation but not form well-ordered microstructures. The DSC thermogram of the WPI-BAC-poly(n-BA) copolymer exhibits features that support theexistence of distinct poly(n-BA) and WPI-BAC protein−surfactant transitions at around −52 and 57 °C, respectively(Figure 2, red curves). As these two Tg’s lie close to that ofpoly(n-BA) homopolymer and protein−surfactant complex,the hard and soft components are likely microphase separated.The surfactant is likely localized in protein domains, asindicated by the Tg and observations on the immiscibilitybetween surfactant and poly(n-BA) homopolymer. A repre-sentative small-angle X-ray scattering (SAXS) intensity curvefor a cross-linked copolymer is shown in Figure 4a. Scatteringintensity decreases monotonically with increasing wave-number, q, with no peaks and features that would be indicativeof a characteristic length scale. This is similar to previousobservations on copolymerized methacrylated whey proteinand hydroxypropyl acrylate17 and indicates a lack of long-rangeorder. Similar observations were made across varying materialcross-linking densities and equilibration humidity levels(Figure S2). Scanning electron microscope (SEM) images ofa fractured surface of the copolymer depict rough textures andno regular distinct phases (Figure 4b), which is consistent withobservations from SAXS.

Short-range ordering in the protein−surfactant complexesand protein−surfactant−polyacrylate blends and copolymerswas investigated using wide-angle X-ray scattering (WAXS) invacuum conditions to understand the structure in the absenceof water. Broad peaks observed confirm the amorphous natureof all of the blends and copolymers. The peak at around 14nm−1 observed in all materials including poly(n-butyl acrylate)homopolymer and benzalkonium chloride corresponds to a0.45 nm spacing and can be attributed to lateral alkyl tail−taildistances,32 protein backbone, and interstrand distances.33,34 Asharper peak at around 2.3 nm−1 (long period of 2.7 nm) ispresent in all surfactant-containing materials. These low-wavenumber scattering peaks for the complex, blend, andcopolymer were broader and shifted to lower wavenumberswhen compared to that of the surfactant (Table S2),suggesting the disruption of surfactant packing upon proteincomplexation. The amorphous protein−surfactant complexesand copolymers are isotropic when examined between crosspolarizers but are birefringent upon stretching (Figure S3).This is consistent with the SAXS results (Figure S4), where thecomplexes lack molecular ordering when unperturbed. Upon

Figure 3. (a) Moisture content of cross-linked WPI-BAC-polyacrylatecopolymers incubated at 23 °C and various relative humidity levels.Red and blue points indicate materials synthesized with n-butylacrylate and PEG methyl ether acrylate, respectively. (b, c, and d)Modulus, elongation at break, and toughness of copolymers as afunction of relative humidity. Protein:surfactant:comonomer ratio is1:1:2. Proteins were modified to have an average of 6 methacrylamidegroups per protein.

Figure 4. (a) Representative SAXS and WAXS curves for a cross-linked WPI-BAC-poly(n-BA) copolymer. WAXS curve offset to matchthe overlapping region with SAXS. (b) SEM image of a freeze-fractured copolymer surface. (c) Comparison of WAXS curves ofsurfactant (BAC), protein (WPI), poly(n-BA) homopolymer,protein−surfactant (WPI-BAC) complex (1:1 ratio), WPI-BAC-poly(n-BA) copolymer, and blend. Protein:surfactant:comonomerratio is 1:1:2 for all copolymers and blends studied.

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stretching, the complexes can be oriented, which has also beenpreviously observed in plasticized proteins and proteinblends.35

Structural changes of the protein as a result of surfactantcomplexation were observed using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy (Figure5). The intense amide I absorption of proteins (1600−1700

cm−1), which originates mainly from CO stretching withminor contribution from C−N stretching, is known to provideimportant structural information.36 Solution cast whey proteinhas a strong absorption peak at 1630 cm−1, consistent withlarge fractions of β-sheet structures in the protein mixturecomprised primarily of β-lactoglobulin.37,38 Upon complex-ation and high-temperature compression molding withbenzalkonium chloride, the amide I band appears to splitinto two main components: one at a lower frequency of 1626cm−1 and another at around 1655 cm−1. The 1630 cm−1 β-structure peak is affected by the strength of a hydrogen bondinvolving the amide carbonyl (CO), and the loweredfrequency suggested the presence of denatured and aggregatedproteins with strong intermolecular hydrogen bonds.36 Therelative intensity of the 1655 cm−1 band versus the 1626 cm−1

band is higher when whey protein was complexed withbenzalkonium chloride, indicating a larger presence of randomcoil or helix structures. These changes may suggest that theprocess of surfactant blending and compression molding led tothe unfolding of proteins and the formation of denaturedprotein aggregates with increased intermolecular hydrogen-

bonding interactions. When the protein−surfactant complexwas exposed to high humidity levels (80% relative humidity), afurther increase in the higher frequency component relative tothe 1626 cm−1 band was observed. No major differences wereobserved in the amide II region (1510−1580 cm−1) betweenthe protein and the protein−surfactant complexes. This regionhas previously been found to be less sensitive to proteinconformation39 but is affected by the hydrogen-bondinginteractions and environment of the N−H group.40−42 Thesimilarity between the protein and the protein−surfactantcomplex could therefore be attributed to the lack of functionalgroups that participate in hydrogen bonding in benzalkoniumchloride.

Material Mechanical Properties. Achieving high strengthand toughness in the material requires the presence of bothstiff protein domains for reinforcement and rubbery polymerdomains for extensibility. On its own, the protein−surfactantcomplex is stiff but has low elongation at break and toughness(Figure 6, Table S3), properties commonly observed for

plasticized protein films.7 These films were prepared bycompression molding, which softens and fuses the WPI-BACcomplexes to produce a continuous matrix. On the other hand,poly(n-butyl acrylate) is a liquid at room temperature, with aglass transition temperature of −55 °C. Unreinforced poly(n-BA) cross-linked with butanediol diacrylate at a cross-linker tomonomer mole ratio of 1:300 produces a soft rubber with lowmodulus and toughness. Solid protein-based copolymers wereprepared by polymerizing n-BA in the presence of the protein−surfactant complex, where methacrylamide groups on proteinsfunction as sites for the protein to be included in thepolyacrylate backbone (Scheme S1b). The copolymer whereproteins had an average of 6 methacrylamide groups perprotein is shown to possess both high mechanical strength andextensibility and is tougher than its constituents. Thissynergistic combination of a hard and soft component haspreviously been observed in other similar cross-linkedprotein−acrylate copolymers17 and is also typically regardedto be central in the design of high-performance engineeringplastics like polyurethanes.43,44

The capability of proteins to function as reinforcing blocksdemonstrates the potential use of these renewable biopolymersin manufacturing engineering plastics and reinforced elasto-mers, and proteins may also have unique characteristicsdistinct from those derived from fossil fuel-based feedstock.

Figure 5. (a) Microscope ATR-FTIR spectrum of water, surfactant(BAC), protein (WPI), and the protein−surfactant (WPI-BAC)complex at 1:1 mass ratio. All spectra were min−max normalized andshifted for clarity. (b) Comparison of the amide I and amide IIregions for WPI and WPI-BAC complex after incubation at variousrelative humidity levels.

Figure 6. Representative stress−strain curves of a cross-linked WPI-BAC-poly(n-BA) copolymer averaging 6 methacrylamide groups perprotein, protein−surfactant (WPI-BAC) complex, and a cross-linkedpoly(n-BA) at 1:300 cross-linker to monomer ratio. All materials wereconditioned at 23 °C and 50% relative humidity prior to testing.

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Proteins have a large number of reactive functional groups thatare amenable to further reactions or polymerization, andcopolymers can be prepared using straightforward approacheslike the free radical polymerization method demonstrated inthis work. As a filler, the availability of reactive groups onproteins also distinguishes them from inorganic fillers that aretypically employed to reinforce elastomers, such as silica,carbon black, and nanoparticles. Inorganic hard fillers provideadditional mechanical strength to polymers, and theireffectiveness of reinforcement has been shown to improvewhen filler−matrix interactions are strong.45 This is typicallyachieved by improving filler dispersion to achieve high surfacearea to volume ratios or by including coupling agents thatenable chemical bond formation between filler and thepolymer matrix. In the protein-based copolymers, covalentattachment between the protein and the rubbery polymer canprovide efficient transfer of external load between the twophases, and the filler−matrix interactions may be potentiallytuned in the form of cross-linking density.Modulating Cross-Linking Density in Copolymers.

The presence of methacrylamide groups in proteins as a resultof the reaction between whey protein and methacrylicanhydride heavily influences mechanical properties, as theyprovide sites for the protein to be covalently attached to therubbery polyacrylate chains during polymerization (SchemeS1b). The level of protein functionalization thereforemodulates cross-linking density. In addition to copolymers,blends were also prepared by polymerizing the acrylate in thepresence of unmodified proteins, where the proteins functiononly as filler. Characterization of the degree of methacrylationusing a model reaction with β-lactoglobulin was previouslydescribed.17 To verify that materials with increasing degrees ofprotein methacrylation have higher cross-linking densities,swelling experiments were performed in dimethyl sulfoxide(DMSO), a good solvent for swelling both the protein and thepolyacrylate phases. In the case of a blend, where the protein isunmodified, the swollen material lost structural integrity andwas nearly fully dissolved, whereas copolymers prepared withincreasing protein functionalization were observed to havedecreasing solvent uptake (Table S4), indicative of theexpected trend in cross-link densities. Mechanical propertycomparison between the blend and the copolymers showedthat the blend has higher modulus but lower elongation atbreak than all cross-linked materials with varying proteinmethacrylation levels (Figure 7). Both tensile strength andtoughness initially increase with increasing cross-linkingdensity but level off at high levels of methacrylation. Largedifferences between the blend and all copolymers of variouscross-linking densities could arise from structural differences,where proteins in the blend function only as fillers (SchemeS1c).

■ CONCLUSIONThis work presents a practical and scalable melt polymerizationapproach to prepare protein copolymers and demonstrates forthe first time that surfactants can be used as both plasticizersfor proteins and compatiblizers. The use of appropriatesurfactants enables mixing of proteins with monomers ofwide-ranging polarity and expands the accessible range ofmaterial properties for protein-based copolymers. Theplasticization capability of surfactants allows the copolymersto be thermoformed and melt polymerized, which is criticallyenabling for industrial processes such as injection and blow

molding. Materials were prepared by first complexing wheyprotein with a cationic surfactant benzalkonium chloride. Thecomplexes were shown to be partially miscible with thehydrophobic vinyl monomer n-butyl acrylate in the absence ofsolvent. Mixtures of protein, monomer, and surfactant can bethermoformed and cured, while shrinkage issues due topostprocessing solvent evaporation are eliminated. Copolymersconsisting of a protein−polyacrylate network and surfactant areproduced when methacrylamide functionalities are installedonto proteins. As proteins function as macro-cross-linkers, thecross-linking density is modulated by protein modificationlevel. Copolymers in general have lower stiffness but higherelongation at break, ultimate tensile strength, and toughnessthan un-cross-linked blends. The presence of both the stiffprotein and the flexible polyacrylate domains are crucial formechanical properties as individually these materials are eithertoo brittle or too soft. Copolymers may be microphaseseparated, as thermal transitions of both the polyacrylate phaseand the protein−surfactant phase are observed. However, theydo not have ordered microstructures.To assess the impact of incorporation of a hydrophobic

component into copolymers, a second material was preparedwith hydrophilic monomer poly(ethylene glycol) methyl etheracrylate. Moisture absorption of the two types of materials issimilar when materials were desiccated but increases with alarger magnitude for the hydrophilic copolymer at higherhumidities. Although n-butyl acrylate is effective at loweringthe copolymer’s overall moisture absorption, both materialsexhibit similar qualitative trends in material softening and havepeak toughness at 20−50% relative humidity. Therefore,protein−surfactant complexes represent an important technol-ogy for solvent-free processing of protein biomass intohydrophobic polymers, but despite a reduction in water

Figure 7. (a) Representative stress−strain curves, (b) modulus, (c)elongation at break, and (d) toughness of WPI-BAC-poly(n-BA)blend and copolymer with a range of methacrylation levels.Protein:surfactant:comonomer ratio is 1:1:2.

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uptake, challenges remain with managing humidity effects onthe protein domains.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acssusche-meng.8b03557.

Reaction scheme for protein methacrylation andcopolymerization; mechanical properties of materialspolymerized at different molding pressures; thermogra-vimetric analysis of protein−surfactant−polyacrylatecopolymer equilibrated at various humidity levels;comparison of small-angle X-ray scattering curvesbetween cross-linked protein−surfactant−polyacrylateat various relative humidity levels and between blendsand materials with different cross-linking densities;surfactant peak positions in wide-angle X-ray scatteringcurves; images of a protein−surfactant complex and aprotein−surfactant−polyacrylate under a cross polarizerat 0% and 50% strain; small-angle X-ray scattering curvefor the protein−surfactant complex; comparison ofmechanical properties between a cross-linked protein−surfactant−polyacrylate, protein−surfactant complex,and a cross-linked polyacrylate homopolymer control;swelling ratio of blend and copolymers with variouscross-linking densities (PDF)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. D. Olsen: 0000-0002-7272-7140Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported in part by the MRSEC Program ofthe National Science Foundation under Award DMR-1419807,the Massachusetts Institute of Technology (MIT) PortugalProgram, and the Masdar Institute of Science and Technology(MI) Flagship Project (Biorefinery: Integrated SustainableProcesses for Biomass Conversion to Biomaterials, Biofuels,and Fertilizer). The authors acknowledge the Institute forSoldier Nanotechnologies for providing the infrastructure forTGA and DSC. X-ray scattering experiments and FTIRexperiments were performed at the MIT Center for MaterialsScience and Engineering X-ray Diffraction Shared Experimen-tal Facilities and the Materials Analysis lab, supported in partby the MRSEC Program of the National Science Foundationunder award DMR-1419807. The authors acknowledge Dr.Angela Holmberg for insightful discussions.

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