Keratin-Based Biocomposites Reinforced and Cross-Linked with Dual

Research Article pubs.acs.org/journal/ascecg

Keratin-Based Biocomposites Reinforced and Cross-Linked with Dual-Functional Cellulose Nanocrystals Kaili Song,†,‡ Helan Xu,‡ Kongliang Xie,† and Yiqi Yang*,‡,§,∥ †

Key Laboratory of Science and Technology of Eco-Textiles, Ministry of Education, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China ‡ Department of Textiles, Merchandising and Fashion Design, §Department of Biological Systems Engineering, and ∥Nebraska Center for Materials and Nanoscience, University of Nebraska-Lincoln, 234 HECO Building, Lincoln, Nebraska 68583-0802, United States ABSTRACT: Green composites from naturally derived polymers have gained more and more research interests recently due to their sustainable and environmentally friendly nature. In this work, biocomposite films based on keratin reinforced by surface-functionalized cellulose nanocrystals are prepared for the first time. Dialdehyde groups were successfully introduced onto the surface of cellulose nanocrystals with their reinforcing and cross-linking effects on the properties of keratin biocomposites systematically studied. Ouali and Halpin−Kardos models were applied to elucidate reinforcing mechanism of the nanofillers. The functionalized nanofillers which could improve interfacial interactions facilitated not only formation of percolating nanofiller network in keratin matrix but also effective interfacial stress transfer, leading to significantly improved mechanical properties of the biocomposites. This reinforcing as well as cross-linking strategy by functionalized cellulose nanocrystals could enrich the exploration of high-performance composites and extend exploration of the applications of keratin materials in the fields of tissue engineering or drug delivery. KEYWORDS: Biocomposites, Cellulose nanocrystals, Keratin, Surface functionalization, Reinforcement

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INTRODUCTION Petroleum-derived polymers have been extensively applied in many fields and accompanied the development of society since the dawn of civilization until today.1−3 Consumption of the petroleum-based polymers has been witnessed in an evergrowing trend due to their intrinsic versatility, outstanding physiochemical properties and excellent processability. However, considering the finite and unsustainable nature of petroleum resources, it is urgent to exploit renewable and naturally derived alternatives. In addition, after the service life of those petroleum-derived polymers, it usually takes several hundreds of years for them to degrade in a natural environment, leading to serious impact on environment and ecosystem.4,5 To address this challenge, numerous attempts have been made to reduce the dependence on and consumption of petroleum-based polymers. Use of renewable, environmentally friendly, biodegradable and biocompatible polymers (e.g., cellulose, chitin, starch, pectin, alginate, xanthum, soy protein, and guar gum) to replace petroleumderived polymers is becoming promising and critical for better environmental protection and sustainable development.6−9 Research interest has especially been intensified in exploring biopolymers from waste resources or industrial byproducts. Keratin, a promising natural protein that could be extracted from waste resources such as chicken feathers, a poultry © 2017 American Chemical Society

byproduct, or unspinnable poor-quality raw wool from the textile industry, is an abundant, hidden biopolymer to be exploited.10,11 Keratin, being the major component of feathers, wools, nails, hair, and horns, is an abundant nonfood protein featuring excellent biocompatible and biodegradable properties.12 Moreover, keratin could be obtained from butchery wastes such as feathers, horns/nails, and poor quality wools or used fabrics from the textile industry, produced worldwide every year. From the molecular level view, keratin is distinguished from other proteins due to the high content of cysteine (8−20% of total amino acid residues) endowing it with water stability higher than that of others such as soy protein or gelatin. Because of its excellent biocompatibility, biodegradability, and nontoxicity, keratin, in a variety of forms, has been applied in tissue engineering, drug delivery, wound healing, personal care products, and so on. However, the poor mechanical properties of keratin materials hinder its further application at large scale. To address this drawback, numerous attempts had been witnessed to improve the mechanical property of keratin materials, such as chemical cross-linking and blending with Received: January 9, 2017 Revised: May 28, 2017 Published: June 19, 2017 5669

DOI: 10.1021/acssuschemeng.7b00085 ACS Sustainable Chem. Eng. 2017, 5, 5669−5678

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ACS Sustainable Chemistry & Engineering other synthetic polymers.13,14 Chemical cross-linking uses toxic, or even carcinogenic, cross-linking agents, such as formaldehyde or glutaraldehyde;15,16 thus, is not suitable for largescale applications. In contrast, blending with other synthetic polymers usually led to loss of inherited biocompatibility and biodegradability of keratin products. Recently, biocomposites based on biopolymer reinforced by different kinds of reinforcing agents have received considerable interests due to the impressive reinforcing effect and processing feasibility.17−19 Recently, cellulose nanocrystals (CNCs) have attracted increasing attention as nanoscale fibers. In nature, cellulose exhibits excellent biodegradable, renewable, and biocompatible properties. CNCs, which combined natural abundance and excellent mechanical properties, are a promising candidate for polymer reinforcement agents.20−23 It is known that interfacial interaction and filler dispersion are the most crucial factors that influence the final mechanical properties of nanocomposites.2,24 For better reinforcing effect, the nanofillers should be homogeneously dispersed in the polymer matrix to form a percolation network. However, the strong interfacial interaction between nanofillers and the matrix would better facilitate stress transfer from matrix to filler network.25 Thus, some research has been carried out either to improve the dispersion of CNCs in the polymer matrix or to enhance the interfacial interaction between nanofiller and polymer matrix. To improve the dispersion of CNCs in polymer matrix, surfactant could be used to modify the surface of CNCs.26,27 However, the interfacial interaction between CNCs and polymer matrix would be decreased by surfactant. Another strategy was to enhance the interfacial interaction between CNCs and polymer matrix by establishing covalent bonds between the two phases. For example, aldehyde-functionalized CNCs could be obtained via chemical modification to introduce a covalent bond between nanofiller and polymer matrix.25,28−31 Thus, we introduced aldehyde groups on CNC surface via a facile oxidative process and incorporated the modified CNC into keratin film to obtain biocomposites. Furthermore, we studied the effect of these dual-functional CNCs on the properties of keratin matrix. In the present study, we developed a naturally derived, environmentally friendly, and strong biopolymer film based on keratin reinforced by surface-modified CNCs. Covalent crosslinks were formed between the amine groups of keratin molecules and the aldehyde groups of CNCs via nucleophilic addition. The benefits of this study were the following: (1) The reinforcing and cross-linking effect was achieved by incorporating of aldehyde functionalized CNCs in the composites. (2) The cross-linking between CNCs and keratin molecules significantly enhanced the interfacial interactions which effectively transferred stress from soft matrix to rigid CNC skeletons in the composites. (3) Both tensile strength and elongation of the nanocomposites were greatly improved as the incorporation of this dual-functional CNCs. This novel crosslinking and reinforcing strategy via functionalized CNCs would enrich the fabrication and exploration of high-performance CNC-based nanocomposites. The keratin composite films developed in this study showed an impressive improvement in terms of mechanical properties, which could also open a door for practical applications of keratin-based materials.

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were purchased from VWR international, Bristol, CT. Cysteine was purchased from EMD Chemicals Inc. Gibbstown, NJ. Urea was purchased from Oak Chemical, Inc. West Columbia, SC. All chemicals were analytical-grade and used directly as received. Preparation of Dialdehyde Cellulose Nanocrystal (DCNC). CNC was obtained by sulfuric acid (65 wt %) hydrolysis of cotton fabric at 45 °C for 45 min at a cotton/sulfuric acid ratio of 1:30 according to the reported method with minor modification.32 DCNC was obtained by oxidation of CNC using sodium periodate at the weight ratio of 1:1 at 30 °C for 4 h according to the reported method with little modification.33 The reaction system was covered using aluminum foil to prevent the decomposition of sodium periodate induced by light. The product was dialyzed against distilled water for 2 days to remove unreacted chemicals. The final concentration of CNC and DCNC suspension was measured by dry weight method. Determination of Aldehyde Group Content. The aldehyde group content of DCNC was determined by nitrogen elemental analysis of its oxime derivative. Hydroxylamine hydrochloride (0.2 M) was dissolved in 0.1 M acetate buffer (by mixing 84.7 mL of 0.1 M acetic acid and 15.3 mL of 0.1 M sodium acetate). Then, 10 mg of DCNC was added into 10 mL of the prepared hydroxylamine hydrochloride solution and stirred at room temperature overnight. The DCNC derivative was dialyzed against distilled water and ovendried before elemental analysis. Preparation of Keratin Biocomposites. Keratin solution was prepared according to the procedure developed previously by our group with little modification.34 Briefly, 100 g of chicken feathers was immersed in 1700 mL of a solution containing 8 M urea and 10 g of cysteine at pH 10.5. The feather dispersion solution was stirred vigorously at 70 °C for 10 h. Keratin solution was collected by centrifuge which removed the undissolved feather. The keratin powder was obtained by precipitation of keratin solution at its isoelectric point (pH 4.6) using hydrochloride acid (0.5 N) and oven-drying. A 6 wt % keratin solution was prepared by dissolving the obtained powder in 80/20 (v/v) acetic acid solution. Then, a series of concentrations of DCNCs or CNCs (1, 2.5, 5, and 10 wt % based on the weight of keratin) were added into keratin solution, and the solution was sonicated in ice bath using an ultrasonic processor (VCX 500:500 W, Sonics & Materials, Newton, CT) for 10 min. DCNC-reinforced keratin nanocomposites were obtained by solution casting and crosslinking at 70 °C for 1 h. The thicknesses for the obtained DCNC/ keratin nanocomposites were 0.21, 0.19, 0.21, and 0.18 mm with the increasing loading of DCNCs. The thickness for the obtained CNC/ keratin nanocomposites were 0.20, 0.18, 0.21, and 0.20 mm with the increasing loading of CNCs, respectively. Characterization of DCNC and CNC Nanoparticles. The structures of DCNC and CNC were characterized by Fourier transform infrared spectroscopy (FT-IR, Nicolet Analytical Instruments, Madison, WI) under a scan resolution of 6 cm−1, and measurement was performed using KBr pellets. Zeta-potential of the DCNC and CNC suspension was measured using a Zeta sizer NanoZS90 (Malvern, U.K.). The morphology of the obtained DCNC and CNC was observed using field-emission scanning electron microscopy (FE-SEM, S3000N, Hitachi Inc., Schaumburg, IL). The DCNC and CNC powders were freeze-dried and sputter-coated with gold/ palladium and observed at a voltage of 10 kV. Observation was also done using transmission electron microscopy (TEM, Hitachi H7500, Hitachi Inc., Parlin, NJ) after placing the DCNC solution on copper mesh and drying. Characterization of the Biocomposites. The composite films were soaked in liquid nitrogen for 10 min before being fractured. The morphology of the fracture surface was observed by FE-SEM. Mechanical properties of the composites were tested according to ASTM standard D-3822 by Instron tensile testing machine (Model 4400, Norwood, MA). According to the standard, films with size of 8 cm × 1.5 cm were prepared and conditioned for 3 days at 21 °C and 65% relative humidity (RH) before mechanical testing. The testing gauge length was 1 in., and crosshead speed was 15 mm min−1. For each sample, 10 specimens were measured. Water uptake property of the composites was measured by immersing the composites in 1000

EXPERIMENTAL SECTION

Materials. White chicken feathers were kindly provided by Feather Fiber Corp., Nixa, MO. Cotton fabrics were first cut into pieces with dimensions of 20 mm × 20 mm. Sulfuric acid and sodium periodate 5670


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Figure 1. Preparation of chemically modified CNC, and FTIR spectra of CNCs and DCNCs. mL of distilled water at room temperature. The composites were weighed (W0) before and after being taken out (Wt, removing the excess water using filter water) at a certain time. The water uptake ratio was calculated using eq 1:

Water uptake (g g −1) =

Wt − W0 W0

Swelling ratio % =

(1)

ηsp =

M1 − M 2 × 100% M2

m w − md × 100% mw

(6)

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(3)

where M1 was film weight before drying and M2 was film weight after drying for 24 h. Relative weight loss of the composites were mesured by immering the film in distilled water for 72 h and calculated by eq 4 and swelling ratio of composite film was calculated by eq 5: Relative weight loss % =

(t − t0)/t0 C

where t was the time for the diluted solution to pass through Ubbelohde capillary viscometer, t0 was the time for the solvent to pass through Ubbelohde capillary viscometer, and C was the concentration of the diluted solution. 13 C Nuclear Magnetic Resonance Spectroscopy (13C NMR). To confirm cross-linking was established between DCNC and keratin, 13 C NMR was carried out, the nanocomposite with 5 wt % of DCNC is selected as an example. Keratin and 5 wt % CNC/keratin were selected as comparison. NMR spectra were obtained on a 400 MHz NMR spectrometer (Ascend 400, Bruker Company, Switzerland). All samples were measured as solutions in the deuterated trifluoroacetic acid and LiCl (about 20 mg sample in l mL of deuterated trifluoroacetic acid with 1 mg of LiCl). Chemical shifts were reported in ppm and the coupling constant was given in Hz.

(2)

where M1 (g) was the weight of the chamber before placed into the humidity chamber, M2 (g) was the weight of the chamber after placed in the humidity chamber for certain time. L (m) was mean thickness of the film. A (m2) was the area of inside round circle of the chamber, t (s) was the total testing time, and ΔP was the partial water vapor pressure difference (Pa) across the films. Moisture contents of keratin films were measured by drying oven method. The keratin films (30 mm × 30 mm) were dried at 105 °C for 24 h in an oven to remove all the adsorbed water. The MC was calculated using eq 3: Moisture content =

(5)

where md was the oven-dried weight of the film after immersing in water, mc was the weight of the film after immersing in water (remove the excess water using a filter water), and mw was the weight of the film after being conditioned for 3 days at 21 °C and 65% RH. Intrinsic viscosities of the keratin, CNC/keratin and DCNC crosslinked keratin composite solutions were determined according to the reported method.35 An Ubbelohde capillary viscometer with the capillary diameter of 0.5−0.6 mm was used (Shanghai Liangjing Glassware Company, China). Formic acid was used as solvent to dissolve keratin and cross-linked keratin composites. For each sample, at least five different diluted solution was determined. The intricsic viscosity was calculated by plotting the concentration of diluted solution against its specific viscosity (ηsp).

Water vapor permeability (WVP) of the films was measured gravimetrically according to the standard method of ASTM E96−95. A squared chamber with an inside diameter of 4.5 cm and an average depth of 3.0 cm was used in this study. Films were cut into a round shape with diameter of 4.8 cm and placed on the top of the chamber. Then, chamber was sealed tightly using screws after placing the rim on top of films. Then the chamber was put into a chamber at designated temperature and relative humidity. The weight loss of the squared chamber was recorded at a certain time interval. The WVP (g·m·m−2· S·Pa) of the films was calculated by eq 2: (M1 − M 2)L WVP = At ΔP

mc − m w × 100% mw

RESULTS AND DISCUSSION Characterization of the Functionalized Cellulose Nanocrystals. Cellulose nanocrystals were oxidated by periodate, resulting in the formation of aldehyde groups at C2 and C3 of the glucopyranose unit of cellulose as shown in Figure 1. The functionalized CNCs (DCNCs) could act as both cross-linking and reinforcing agent when incorporated into

(4) 5671


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Figure 2. Morphology of DCNCs. (A) FE-SEM micrograph, (B) TEM micrograph, and (C) particle size distribution determined by DLS (inset: digital graph of DCNCs suspension).

keratin matrix. The successful functionalization of CNCs were confirmed by FTIR spectra as shown in Figure 1. The broad band near 3400 cm−1 was assigned to O−H stretching vibrations, and the peak at 2900 cm−1 was assigned to C−H stretching vibrations. The absorption peaks at 1050 and 1163 cm−1 were associated to the stretching vibrations of C−O in cellulose. However, those peaks became a broad band together with the peak at 1025 cm−1 as the result of partial oxidative breakage of C2−C3 linkage of the glucose units on cellulose. In the spectrum of DCNCs, the new band at 1732 cm−1, which was attributed to the stretching vibration of aldehyde groups was observed, indicating that the functionalization was successfully carried out. The content of accessible aldehyde groups on DCNCs was 2.1 mmol g−1 determined from elemental analysis of its oxime derivative. The amorphous region of native cellulose is more susceptible and accessible to acid than the crystalline domain, resulting in formation of CNCs by acid hydrolysis. Figure 2A,B shows the morphology of the prepared DCNCs. The obtained DCNCs exhibited rodlike morphology with a mean length of around 200 nm and width of 7 nm, as shown in TEM image. In addition, the FE-SEM image in Figure 2A confirms the existence of some point-to-point connections between individual nanoparticles which were formed during the drying process of the suspension. The obtained DCNCs showed a narrow size distribution in the range of 70−245 nm as depicted in Figure 2C. The inset indicated that the DCNCs was stabilized in the suspension due to electrostatic repulsion of the sulfate ester groups introduced during the acid hydrolysis. The sulfur content of CNCs before and after periodate oxidation was 0.54 and 0.51%, respectively, determined by elemental analysis. Benefits of this chemical modification include the following: (1) The introduced aldehyde groups on CNCs could react with amino groups on keratin to form a cross-linking network within keratin molecules. (2) CNCs could work as the reinforcing phase in soft keratin matrix, yielding a nanoparticle reinforced binary composites. Characterizations of Nanocomposite Films. Figure 3 shows the 13C NMR spectra of keratin, CNC/keratin, and DCNC cross-linked keratin composites. Comparing with the spectrum of keratin and CNC/keratin, there was a new peak appeared at DCNC cross-linked keratin composite at 154.5 ppm as indicated in the spectrum of DCNC/keratin, which was attributed to the −CN− bond in DCNC/keratin composites. This could confirm that the cross-linking reaction occurred between DCNC and keratin.36 The peak around 176 ppm was related to the amide carbonyl carbons of the keratin, and the

Figure 3. 13C NMR spectra of keratin, CNC/keratin, and DCNC/ keratin nanocomposite.

peak around 127 ppm indicated the presence of aromatic group containing amino acid sequences in keratin. The peaks at about 53 and 37 ppm were attributed to the α- and β-carbon present in leucine and cysteine residues, respectively. Those peaks at 15−30 ppm were associated with the carbon resonance of the alkyl groups of keratin side chains as also reported by other researchers.37 Those strong peaks at 110−120 and 164 ppm were attributed to the carbon resonance of trifluoroacetic acid. Compared with keratin, there was no new peak appearing on the spectrum of CNC/keratin composite film. Molecular weight of keratin could be increased after crosslinking. Thus, to demonstrate molecular weight changes, the intrinsic viscosities of pure keratin, CNC/keratin, and DCNC cross-linked keratin nanocomposite solutions were determined and listed in Table 1. Comparing with keratin solution and CNC/keratin solution, the increased intrinsic viscosity of the DCNC cross-linked keratin composite solution (shown in Table 1) could be attributed to the increased molecular weight Table 1. Intrinsic Viscosity of Keratin, CNC/keratin, and DCNCs Cross-Linked Keratin Solution intrinsic viscosity (dL g−1) keratin solution 5 wt % CNC/keratin solution 5 wt % DCNCs cross-linked keratin solution 5672

0.323 ± 0.18 0.392 ± 0.23 0.976 ± 0.19


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Figure 4. Stress−strain curves of (A) DCNCs reinforced keratin nanocomposites and (B) CNCs reinforced keratin nanocomposites.

in many fields such as food packaging. The WVP of the composite films under a series of relative humidities was illustrated in Figure 5A. The relative weight loss, moisture content, and swelling ratio of nanocomposite films at 65% RH were summarized in Table 2. At 100−30 RH%, pure keratin film showed the highest WVP of 10.9 × 10−10 g·m·m−2·s·Pa compared to other nanocomposite films because of the intrinsic hydrophilicity of protein materials. With a loading of 5 wt % DCNCs, the WVP of the nanocomposite film decreased by 89% to 1.2 × 10−10 g·m·m−2·s·Pa, which is clear evidence that the barrier property of keratin composite films improved significantly. The nanocomposite films showed a decrease in barrier property at higher external relative humidity environment. This might be ascribed to the increase of the size of those nanoporous inside structure of the film as a result of swelling behavior of protein films under high humidity. The WVP of the nanocomposites showed first a decrease and then an increase trend after the loading of DCNCs exceeds 5 wt %. The increase of WVP after corporation of 5 wt % DCNCs was mainly attributed to poor dispersion of the nanofiller in keratin matrix. The aggregation of DCNCs lead to more defects in the nanocomposites which allowed water vapor to permeate. Water uptake behavior of DCNCs/keratin and CNCs/keratin composite films was shown in Figure 5B,C. The water uptake by the composite films decreased after incorporation of nanofillers compared with that of pure keratin film. The incorporation of high crystalline DCNCs or CNCs into keratin film matrix resulted the formation of tortuous pathway for the migration of water molecule from the surface of composite films to the inner side of the films, thus resulting in the decrease of water uptake by the composite films. However, the water uptake by CNCs/keratin composite films was higher than that of DCNCs/keratin films under the same loading of nanofillers. This was mainly due to the formation of cross-linked network within DCNCs/keratin films which resulted in the reduction of free volume within films. Those results contributed to the improvement of the barrier property against water vapor as well as water stability of keratin composite films, which would be beneficial for its application as engineering materials. Reinforcing Mechanism of DCNC/keratin Composite Films. The mechanical property of nanofiller-reinforced composites was mainly depended on the dispersion state of the reinforcing agents as well as the interfacial adhesion between the filler phase and polymer matrix. Thus, to better understand the distinctive reinforcing effect of DCNCs in keratin matrix, the fracture surface morphology of nano-

of keratin as a result of cross-linking reaction. No significant increase of the intrinsic viscosity of CNC/keratin composite comparing with that of pure keratin. Thus, together with the results from Figure 3, it can be further confirmed that the crosslinking reaction only occurred between DCNC and keratin. Figure 4 shows the stress−strain curves of DCNCs/keratin and CNCs/keratin composite films with different loading of nanofillers. Both CNCs and DCNCs showed effective reinforcement in the composite films as can be seen from the gradually increased of the tensile strength and Young’s modulus with the increasing of nanofiller content. The highest Young’s modulus and tensile strength of the composite films were 451 and 26.2 MPa, respectively, achieved by incorporating of 5 wt % DCNCs, which were 9.1 and 5.8 times of those of pure keratin film. DCNC/keratin composite films showed a significantly increased of elongation values as well as possessed the highest elongation at break. The significant increase of Young’s modulus resulted from the reinforcing effect of rigid DCNCs network within keratin matrix. However, the elongation of CNC/keratin composite films decreased gradually as the addition of CNC increased from 2.5 to 10 wt %, which is a common phenomenon for nanoparticle reinforced composites. Incorporation of reinforcing building blocks usually resulted in the decrease of elongation. Compared with CNCs, the DCNCs exhibited superior reinforcing effect in keratin matrix. The elongation of the DCNC/keratin films was significantly improved which is opposite to the general trend of fiber reinforced composites. This might be ascribed to the following reasons: (1) The aldehyde groups on DCNCs could react with amino groups on keratin and lead to formation of cross-linked network within keratin molecules. (2) The enhanced interfacial interactions between DCNCs and keratin matrix as a result of the crosslinking bonds effectively bridged the cracks at the fracture interface and avoided the propagation of those cracks. (3) DCNCs could act as plasticizer by the formation of hydrogen bonds with keratin molecules and rupture the intramolecular hydrogen bonds of keratin molecules, thus leading to easier slippage of keratin molecular chains and increase of elongation of the composites. The elongation of the DCNC reinforced composite films showed a decreasing trend when the loading was higher than 5%, which might result from the aggregation of DCNCs in keratin matrix acted as defects with relatively high nanofiller loading. WVP and water uptake behavior are crucial parameters that determine the feasibility of the application of protein-based film 5673


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Figure 6. FE-SEM micrographs of the fracture surface of (a) neat keratin film, (b) 1 wt % DCNCs/keratin film, (c) 2.5 wt % DCNCs/ keratin film, (d) 5 wt % DCNCs/keratin film, and (e) 10 wt % DCNCs/keratin film.

indicated by red circle and arrows in Figure 6b−e. Surprisingly, the nanocomposites exhibited homogeneously fractured surface without aggregated microfibers or voids at any nanofiller loading level. The absence of aggregated fibers and holes on the fracture surface firmly demonstrated that the DCNCs were well-separated into individual particles and homogeneously distributed in keratin matrix. The general trend for fiberreinforced composites is the increase of tensile strength and modulus with the reduction of elongation, which was mainly due to the poor filler−polymer interfacial bonding. The observed improvement of elongation of the composites could be contributed to the strong interfacial interaction resulted from chemical bonding between aldehyde and amine groups. To further elucidate the reinforcing mechanism of nanofillerreinforced keratin composite films, the Ouali and the Halpin− Kardos models were applied to simulate the Young’s modulus of DCNCs/keratin films. The Ouali model is a further extension of the classical phenomenological series−parallel model developed by Takayanagi with employment of the percolation theory. This model predicts the modulus of composites by considering three phases: nonpercolating nanofiller phase, percolating nanofiller network, and polymer matrix. The modulus of composites can be calculated using the following equation: Ec = Figure 5. (A) Water vapor permeability of DCNCs/keratin composite films at different relative humidity. Water uptake behavior of (B) DCNCs/keratin and (C) CNCs/keratin composite films.

Ψ=0

keratin

1% DCNC

2.5% DCNC

5% DCNC

10% DCNC

30.5

27.7

25.3

20.2

19.9

16.2 5.2

11.6 3.2

9.5 2.7

7.6 1.5

6.4 1.2

(7)

where vR is the volume fraction of nanofiller, Em and Ef are the modulus of the matrix and nanofiller, respectively, and Ψ is the adjustable parameter, which is given by

Table 2. Relative Weight Loss, Moisture Content, and Swelling Ratio of Nanocomposite Films at 65% RH

relative weight loss (%) moisture content swelling ratio

(1 − 2Ψ + ΨvR )EmEf + (1 − vR )ΨEf 2 (1 − vR )Ef + (vR − Ψ)Em

when vR < vRc

⎛ v − vRc ⎞ Ψ = vR ⎜ R ⎟ when vR > vRc ⎝ 1 − vRc ⎠

(8)

The Ouali model is well-established to describe the stiffness of nanocomposites in which the rigid nanofiller is homogeneously dispersed throughout the polymer matrix to form a percolation network above the percolation threshold (vRc, the critical filler volume fraction needed for percolation). The percolation threshold vRc can be calculated by following equation: 0.7 vRc = (9) A

composites was observed and shown in Figure 6. The neat keratin film exhibited typical sign of plastic deformation with rough and irregular fracture surface as can be seen from Figure 6a. Incorporating of DCNCs generated distinctive surface morphologies in which extensive microsize fibers appeared as 5674


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ACS Sustainable Chemistry & Engineering where A is the aspect ratio of DCNCs; a value of 20 was adopted based on DLS and TEM analysis. The Halpin−Kardos model is a semiempirical model for simulating the modulus of nanofiber reinforced composites. Considering the DCNCs/keratin films as randomly aligned and discontinuous fiber lamina, its Young’s modulus can be calculated from the following equation: Ec = 0.184E + 0.816E⊥

(10)

where E = Em

E⊥ = Em

η =

η⊥ =

Ef Em Ef Em Ef Em Ef Em

1 + vR ξη 1 − vR η

1 + 2vR η⊥ 1 − vR η⊥ −1 +ξ −1 +2

where E⊥ and E∥ are the transverse and longitudinal Young’s modulus of the composite, Em is the Young’s modulus of the matrix, and Ef is the modulus of the nanofiber. ξ is the shape factor dependent on fiber morphology. The equation ξ = 2L/w is applied for relatively short fibers such as DCNCs. The modulus of keratin (Em) was taken as 60 MPa based on mechanical property measurement. The modulus of DCNCs varies in range of 50 to 200 GPa depending on the types of cellulose nanocrystal structure. On the basis of the origin of cellulose used in this study, a value of 150 GPa was used for modeling. The experimental and simulation results using two models for DCNCs/keratin nanocomposites and CNCs/keratin nanocomposites are shown in Figure 7. The Ouali model gave a closer prediction of modulus for DCNCs reinforced keratin composites compared with that of Halpin−Kardos model, considering the percolation network as shown in Figure 7A. The Halpin−Kardos model is based on the assumption that single filler was encased in a cylindrical shell of matrix to form a self-consistent network. Filler−filler interactions are not considered in this model. However, since the concentration of DCNCs exceeded its percolation threshold (2.4 wt %, calculated using eq 9), the interactions between neighboring DCNCs could not be ignored. On the contrary, the Halpin− Kardos model gave a close prediction of the modulus of CNCs/ keratin nanocomposites. That means the interaction between CNC fillers was not significant enough due to the absence of percolation network within the nanocomposites. This result highlighted the following: (1) the important role of filler−filler interactions in improving the properties of nanocomposites reinforced by fibers, (2) the well-formed DCNCs percolation network that were cemented together by physical and chemical interactions with keratin molecules also contributed to the high modulus of the composites, and (3) the enhanced interfacial interaction between filler and matrix, resulting from chemical bonding between DCNCs and keratin molecules, that also played an important role in improving the

Figure 7. Young’s modulus as a function of nanofiller content: Experimental values and predicted values using two models for (A) DCNCs/keratin nanocomposites and (B) CNCs/keratin nanocomposites.

performance of the nanocomposites, leading to a more effective stress transfer at the interface. In this study, the sufficient loading transfer from matrix to filler network could be attributed to the chemical bonding between DCNCs and keratin. These results also have important implications in fabricating high performance composites by enhancing the interfacial attractions between the two phase. Therefore, in pursuing of the right filler for reinforcement, surface modification of filler should also be taken into consideration. On the basis of the above discussion, the distinctive reinforcing mechanism for DCNCs/keratin composites was reasonably proposed and schematically illustrated in Figure 8. The surface modification of CNCs not only promoted the formation of percolation network due to the improved dispersion state in keratin matrix but also enhanced their interfacial interaction with keratin molecules as indicated from the mechanical experimental/theoretical prediction results and the fracture surface morphology observations. Such enhanced interfacial adhesion most likely resulted from the chemical bonding between DCNCs and keratin molecules at the DCNCs/keratin interfaces. Consequently, when an external force was exerted on the DCNCs/keratin composites, the enhanced percolation network and interfacial bonding facilitated a more effective stress transfer from the soft keratin matrix 5675


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Figure 8. Schematic illustration of the failure mode and reinforcing mechanism of DCNCs in keratin matrix.

Figure 9. Comparison of tensile strength (A) and WVP (B) of keratin films.

mechanical and barrier property have not been reported until now.

to the rigid DCNCs network, leading to the superior mechanical performance effect. Comparison of Mechanical and WVP Properties of Keratin-Based Films. In Figure 9, tensile strength and barrier property of keratin-based films are compared with those of others work.38−41 Figure 9A clearly shows that keratin composite films prepared by DCNCs reinforcing and crosslinking exhibited tensile strength superior to that of films prepared by plasticization or chemical cross-linking. Furthermore, the nanocomposite films prepared in this work showed a higher tensile strength than that of others even at a low nanofiller incorporation, which suggested that DCNCs could serve as a feasible and cost-effective reinforcing agent for fabricating composites possess excellent performance. The relatively poor water vapor barrier property hindered the widely application of keratin films, thus, it is imperative to reduce water sensitivity of keratin materials. Figure 9B depicted the WVP of keratin film obtained in this study and others. We can see that the nanocomposite films prepared in this work possessed a reasonable barrier property as did some films prepared by chemical cross-linking. The nanocomposite film blending with 5% DCNCs exhibited the best barrier property among all the other films as highlighted with the bright green circle in Figure 9B. However, it is worth mentioning that the cross-linking agents currently in use are usually toxic. To the best of our knowledge, keratin films with relatively high

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CONCLUSIONS Biocomposite keratin films reinforced by surface-functionalized CNCs were successfully prepared. Surface functionalization of CNCs resulted in the introduction of aldehyde groups on its backbone which endowed it with dual-functional effects as both reinforcing and cross-linking agents. Mechanical properties, WVP, and water stability of the biocomposites were systematically studied. Morphology of the fracture surface of the biocomposites were observed by FE-SEM. The biocomposites exhibited significantly improved mechanical properties and excellent water stability. Furthermore, Ouali and Halpin− Kardos models were applied to elucidate the reinforcing mechanism of the nanofiller. It was found that the formation of percolating nanofiller network in keratin matrix and improved interfacial adhesions were responsible for the imperative reinforcing effect of the biocomposites.

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AUTHOR INFORMATION

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*Tel.: +001 402 472 5197. Fax: +001 402 472 0640. E-mail: [email protected]. 5676


Research Article

ACS Sustainable Chemistry & Engineering ORCID

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Kongliang Xie: 0000-0002-4676-3762 Yiqi Yang: 0000-0001-8153-4159 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by USDA-National Institute of Food and Agriculture (Multi-State Project S1054 (NEB 37-037)), USDA Hatch Act, and the Agricultural Research Division at the University of Nebraska-Lincoln. We are grateful to the China Scholarship Council for providing financial support to K.S.

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Keratin-Based Biocomposites Reinforced and Cross-Linked with Dual

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