Article pubs.acs.org/acsapm
Cite This: ACS Appl. Polym. Mater. XXXX, XXX, XXX−XXX
Mechanically Strong and Highly Tough Prolamin Protein Hydrogels Designed from Double-Cross-Linked Assembled Networks Jing Jing Wang,†,‡,∥ Yixiang Wang,†,§ Qiyang Wang,† Jingqi Yang,† Song-Qing Hu,‡ and Lingyun Chen*,† †
Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton T6G 2P5, Canada Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center), School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong 510641, China § Department of Food Science and Agricultural Chemistry, McGill University, Ste Anne de Bellevue H9X 3 V9, Canada ∥ Department of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
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‡
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ABSTRACT: This study introduced uniquely constructed double-cross-linked hordein/zein protein hydrogels with outstanding mechanical properties. Notably, the optimized hydrogels demonstrated a compressive stress of 1.90 MPa at a strain of 70%, excellent self-recovery after 40 cycles of loading−unloading treatments, and superior foldable properties. Further study of the hydrogel nanostructures and properties has revealed that the hordein highly participated in the formation of chemically cross-linked networks which maintained the elasticity of the hydrogels; whereas physical cross-linked domains that consisted of beadlike particles (diameter ∼80 nm) by hordein/zein assembly were evenly integrated inside the large chemical cross-linked framework and acted as “load carriers” to effectually absorb energy. Consequently, the intertwined spatial network structures and beadlike particles collectively and efficiently dispersed and absorbed energy to withstand large deformations throughout the chemically and physically cross-linked networks. Such a prolamin protein-based hydrogel has potential to be used in biobased load-bearing soft devices, which will diversify the use of zein and hordein as the byproducts of maize and barley. In addition, the generated knowledge may offer new opportunities to design and construct strong hydrogels from many other plant protein resources to unlock their potential as biopolymer and biocompatible materials. KEYWORDS: prolamin proteins, hydrogels, assembled structure, double cross-linking, mechanical properties
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INTRODUCTION Hydrogels are a unique form of soft and wet elastomers with a three-dimensional hydrophilic network, entrapping a large amount of water while maintaining their network structure integrity.1,2 During the past decades, hydrogels have been prominently used in pharmaceutical, biomedical, and personal care fields, such as drug delivery systems, tissue engineering scaffolds, and cosmetics.3,4 However, the traditional hydrogels are vulnerable to stress-induced deformation and propagation of cracks, which may lead to a dangerous loss in load-bearing capacity and limit their practical uses.5 Therefore, improving the mechanical properties and extending the practical applications of hydrogels have been significant topics in the field in recent years.2,3,6,7 Hydrogels are prepared from synthetic and/or natural polymers via covalent bonds or noncovalent interactions, such as hydrophobic interactions, ionic interactions, hydrogen bonding, or a combination of these interactions.2 The densely cross-linked network is the basis of superior mechanical © XXXX American Chemical Society
properties, including high modulus, high fracture strength, and high toughness.1 However, conventional chemically crosslinked hydrogels tend to display low mechanical strength and poor toughness, due to the inhomogeneity of the network and the lack of effective energy dissipation under deformation.8 Many attempts have been made to prepare hydrogels with enhanced mechanical properties, such as nanocomposite hydrogels,9 slide-ring hydrogels,10 hydrophobic modified hydrogels,11,12 and double-network hydrogels,2,13 etc. Among these, double-network hydrogels exhibit excellent strength and toughness. Double-network hydrogels are generally composed of a brittle and stiff network of energy-dissipating sacrificial linkages and a ductile and soft network of greatly extensible linkages, which are contributed by rigid covalent cross-linking and dynamic physical cross-linking, respectively.3,8 The Received: January 23, 2019 Accepted: May 7, 2019 Published: May 7, 2019 A
DOI: 10.1021/acsapm.9b00066 ACS Appl. Polym. Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Polymer Materials
purchased from Fisher Scientific (Markham, ON, Canada) and were used as received unless otherwise described. Fabrication of Double Cross-Linked Hordein/Zein Hydrogels. Zein and hordein with different weight ratios (Table 1) were
optimized double cross-linked cellulose hydrogels presented the compressive stress of 4.8 MPa at 74% fracture strain. Moreover, the fracture energy of the cellulose hydrogels reached a maximum value of 0.65 MJ m−3, which is 1150 times higher than that of the chemically cross-linked cellulose hydrogels.2 Meanwhile, the mechanical properties of chitin hydrogels have been enhanced using the same strategy.14 Prolamin proteins, such as zein and hordein, are largely available as the byproducts of the starch processing of maize and barley, which are the third and fourth most important cereal crops in the world.15 These proteins are mainly used as animal feed, and their applications in human foods have been limited due to their low solubility in aqueous solutions caused by a high content of nonpolar amino acids.15,16 Conversely, such molecular features make these proteins attractive to be converted into novel materials from renewable biomass. In addition, such protein-based materials have good biodegradability and biocompatibility. For example, zein has good film forming properties and has been used for coating and packaging in food and pharmaceutical industries,17 while hordein has been employed to build the micro/nano-delivery vehicles of nutraceuticals.18 However, strong and tough prolamin protein hydrogels have seldom been reported. Our previous research found that in acetic acid solution, hordein exhibited an extended flexible structure with a strong tendency to form noncovalent interactions with each other, while zein formed compact particles with a high α-helix conformation maintained in acetic acid. During electrospinning, these molecules were compressed to establish continuous ultrafine fibers with stronger mechanical properties by stronger intermolecular interactions, including hydrophobic interactions, intermolecular hydrogen bonds, etc. The fabricated strong electrospun fibers have the potential to be used as tissue engineering scaffold materials or natural delivery systems for biomedical applications.16,19 Inspired by the previous works, this research intended to fabricate novel hordein/zein hydrogels high in mechanical performances by combining the assembled protein structure and the double cross-linked networks, namely, (i) the chemical network formed with glutaraldehyde and (ii) the physical cross-linking through hydrophobic interaction and hydrogen bonding induced by the post-treatment of water. The structure and mechanical properties of the resultant hydrogels were subsequently investigated in detail. Approval of this new concept may lead to development of biopolymer hydrogels from plant proteins for new applications in high-technique areas, such as biobased soft and wet devices, tissue engineering scaffolds, artificial blood vessels, biopolymer plastic, catalyst supports, etc. Meanwhile this will develop high value applications from prolamin proteins as starch-processing byproducts of crops.
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Table 1. Compositions of Hordein/Zein Hydrogels code
hordein (%)
zein (%)
glutaraldehyde (%)
molar ratio of FGA:GA
z30-5 h30-5 hz30-3 hz30-5 hz30-7 hz20-2 hz20-3.3 hz20-5
0 30 15 15 15 10 10 10
30 0 15 15 15 10 10 10
5 5 3 5 7 2 3.3 5
1:1 1:1 2:1 1:1 1:2 2:1 1:1 1:2
dissolved in acetic acid by stirring at 20 °C (room temperature). Various amounts of glutaraldehyde were then added to obtain the desired molar ratios of the free amino group (FAG) of protein to the aldehyde group (AG) of glutaraldehyde (FAG:AG = 1:2, 1:1, and 2:1). The mixtures were transferred to a plastic tube and retained at 20 °C for 12 h to generate the chemical cross-linking. The obtained hydrogels were then washed by deionized water to remove acetic acid and glutaraldehyde and to simultaneously induce the physical crosslinking. Swelling Ratio and Water Content Measurements. The swollen hydrogels were weighed after the excess of water on the surface was absorbed with filter paper. The swelling ratio (Qe) was defined as follows, Qe = (WT − WD)/WD, where WT and WD represent the weights of swollen hydrogel and dry hydrogel, respectively. The equilibrium water content of the hydrogels was defined as WH2O = WS/WD, where WS and WD are the weights of the hydrogel before and after drying. Mechanical Properties. Compressive measurements were performed using an Instron 5967 universal testing machine (Instron Corp., MA, USA) at 20 °C, after the balanced swelling ratios of all hydrogels were reached. A cylindrical hydrogel with a diameter of 10 mm and a height of 6 mm was compressed using a 50 N load cell at a crosshead speed of 1 mm min−1. The cyclic loading−unloading tests, using the same samples at an equal test rate, were carried out by performing subsequent trials immediately after the initial loading. The compressive modulus (E) was calculated from the compressive stress−strain curves of hydrogels fabricated by different formula. Hydrogel Structure Characterization. Fourier transform infrared spectrometer (FTIR) spectra of hydrogel powders were recorded on a Nicolet 6700 spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). The freeze-dried hordein/zein hydrogels were mixed with KBr. Spectra were recorded as the average of 256 scans at 2 cm−1 resolution from 4000 to 400 cm−1. The morphology of the hydrogels was observed with scanning electron microscopy (SEM, Hitachi X650, Japan) at an acceleration voltage of 5−10 kV. The samples were freeze-dried, cleaved to expose their inner structure, and then sputtered with gold for 2 min. Contact Angle Measurement. The bubble contact angle was measured by a contact angle goniometer (Ramè-Hart Instrument Co., NJ, USA) using a cell of a quartz cuvette filled with deionized water. The air bubbles were placed under the surface of the double crosslinked hydrogels using a calibrated syringe. The contact angle was measured and given by the goniometer based on the shape of the captive bubbles. Protein Molecular Interactions of Hydrogels. To evaluate the molecular interactions involved in the formation of prolamin protein hydrogels, hz30-5 hydrogels with the height of 2 mm were soaked into sodium dodecyl sulfate (SDS, 1%, w/v), urea (8 M), and 2mercaptoehanol (2-ME, 0.2 M), which could disrupt hydrophobic interactions, hydrogen bonds, and disulfide bonds, respectively.21
EXPERIMENTAL SECTION
Materials. Zein (F4000, protein content of 92%, approximate molecular weight of 35 kDa, and total ash of 2% maximum) was purchased from Freeman Industries LLC (New York, USA) and used without further purification. Regular barley grains (Falcon) were kindly provided by Alberta Agricultural and Rural Development, Alberta. Barley protein content was 13.2 wt % (dry status), as determined by combustion with a nitrogen analyzer (FP-428, Leco Corporation, St. Joseph, MI). Hordein (total ash of 2% maximum) was extracted using the alcohol method according to our previous work,20 and the protein content (dry status) was 92 wt %, as determined by the same nitrogen analyzer. All chemical reagents were B
DOI: 10.1021/acsapm.9b00066 ACS Appl. Polym. Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Polymer Materials
Figure 1. Schematics of the preparation route for double-cross-linked prolamin protein hydrogels.
Figure 2. Compressive stress−strain curves of the double cross-linked hordein/zein hydrogels with a varying maximum compression (50, 60, and 70%). (A, B) The hydrogels of pure zein or hordein; (C−E) hz20-2, hz20-3.3, and hz20-5; (F−H) hz30-3, hz30-5, and hz30-7; (I) the calculated compressive modulus of hordein/zein hydrogels; (J) cyclic compressive loading−unloading tests of hz30-5 with 40 cycles. Frequency sweep analysis was subsequently conducted at the 1 mm gap using the DHR-3 rheometer (TA Instruments, DE, USA). Statistical Analysis. The experimental data were expressed as the mean ± standard deviation. One-way analysis of variance was used to compare the value differences (P < 0.05) using SPSS 17.0 (SPSS Inc., Chicago, IL).
structure of hydrogel networks. After water treatment, the physical cross-linking of hydrogels formed between beadlike particles of zein and extended hordein. Under stress, the chemical cross-linking as a large framework retained the elasticity and integrity of hydrogels, while the compact beadlike particles withstood large deformations of hydrogels to maintain the high strength. The mechanical properties of prolamin protein hydrogels with different molar ratios of FAG of protein to AG of glutaraldehyde (FAG-to-AG) were tested under loading− unloading compression. In Figure 2A, the stress of the z30-5 hydrogels (FAG:AG = 1:1) was 0.22 MPa at 50% strain, and then it increased to 0.28 and 0.40 MPa at strains of 60 and 70%, respectively. The stress decreased rapidly during unloading, and there was a large permanent deformation after compression, indicating that the zein hydrogels alone (z30-5) were lacking elasticity. In Figure 2B, the hordein
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RESULTS AND DISCUSSION Hydrogel Mechanical Properties. Figure 1illustrates the scheme to fabricate the strong and tough double-cross-linked prolamin protein hydrogels. Hordein and zein were dissolved in acetic acid to get a hybrid solution, and then cross-linker glutaraldehyde was added into the hordein/zein solution. Subsequently, the prolamin proteins, especially, the structurally flexible and extended hordeins, reacted with glutaraldehyde by chemical cross-linking between the amine groups and aldehyde groups to construct the hydrogel networks. Meanwhile, a high amount of compact zein molecules existed in the spatial C
DOI: 10.1021/acsapm.9b00066 ACS Appl. Polym. Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Polymer Materials
via chemical cross-linking by Kong et al.22 showed the compressive stress in kPa order by the compressive stress− strain test, which was significantly weaker than the compressive stress of the hordein/zein hydrogels in MPa order. The double chemically and physically cross-linked chitosan hydrogels by Azevedo et al.24 exhibited weaker compressive stress (
