Controlled Hydrophobic Biosurface of Bacterial Cellulose Nanofibers


Jun 6, 2017 - A novel, highly biocompatible bacterial cellulose (BC)-zein ... The surface hydrophobicity of the zein-modified BC nanofibers ... Partne...
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Controlled Hydrophobic Biosurface of Bacterial Cellulose Nanofibers through Self-Assembly of Natural Zein Protein Zhili Wan, Liying Wang, Lulu Ma, Yingen Sun, and Xiao-Quan Yang ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

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Controlled Hydrophobic Biosurface of Bacterial Cellulose Nanofibers through Self-Assembly of Natural Zein Protein Zhili Wan,1 Liying Wang,1 Lulu Ma,1 Yingen Sun,1 and Xiaoquan Yang*,1,2 1

Research and Development Center of Food Proteins, Department of Food Science and Technology,

South China University of Technology, 381 Wushan Road, Tianhe District, Guangzhou 510640, People's Republic of China 2

Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety,

South China University of Technology, 381 Wushan Road, Tianhe District, Guangzhou 510640, People's Republic of China

*

Corresponding author: Xiaoquan Yang

E-mail: [email protected]; [email protected]; Tel: (086) 20-87114262; Fax: (086) 20-87114263.

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ABSTRACT: A novel, highly biocompatible bacterial cellulose (BC)-zein composite nanofiber with controlled hydrophobic biosurface was successfully developed through a simple and green solution impregnation method, followed by evaporation-induced self-assembly (EISA) of adsorbed zein protein. The surface hydrophobicity of the zein-modified BC nanofibers could be controlled by simply changing the zein concentration, which is able to tune the morphology of self-assembled zein structures after EISA, thus affecting the surface roughness of composite membranes. Zein self-assembly at low concentrations (5 mg/mL) resulted in the formation of hierarchical zein structures (spheres and bicontinuous sponges) on BC surface, thus increasing the surface roughness and leading to the high hydrophobicity (water contact angle reached 110.5°). However, at high zein concentrations, these large zein spheres assembled into a flat zein film, which decreased the surface roughness and hydrophobicity of membranes. The homogenous incorporation of zein structures on BC surface by hydrogen bonding did not significantly change the internal structure and mechanical performance of BC nanofibers. In comparison with pure BC, the BC-zein nanofibers had a better biocompatibility, showing a significantly increased adhesion and proliferation of fibroblast cells. This is probably due to the rough surface structure of BC-zein nanofibers as well as the high biocompatibility of natural zein protein. The novel BC-zein composite nanofibers with controlled surface roughness and hydrophobicity could be of particular interest for the design of BC-based biomaterials and biodevices that require specific surface properties and adhesion. KEYWORDS: bacterial cellulose nanofibers, zein, surface modification, hydrophobic biointerface, controlled self-assembly, cell adhesion

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1. INTRODUCTION The biosurface between cell and materials is one of the most important considerations for designing and fabricating a specific tissue engineering scaffold or biomedical device, as the cell behaviors such as adhesion and proliferation are largely regulated by the interactions of cells with their surrounding microenvironment.1-3 As tissue engineering scaffolds, biomaterials are used to incorporate and support living cells to adhere and grow, contributing to new tissue formation both in vitro and in vivo.4 The characteristics of material surface, such as surface wettability,5,6 roughness,5,7 and topography,8 can directly affect the cellular response, which finally affect the growth and quality of new tissues. Therefore, the development of biomaterials and biodevices that show good mechanical performances and also have biointerfaces facilitating cell attachment is highly required in various biomedical fields. The use of multistructured nanofibers for fabricating biomaterials has gained much attention in biomedical applications to mimic the properties of extracellular matrix (ECM), facilitating the cell-biomaterial interactions and improving the biocompatibility.9,10 Bacterial cellulose (BC) produced by Acetobacter xylinum, is composed of three-dimensional natural nanofibers less than 100 nm wide, with many appealing properties that include biocompatibility, ultrafine network structure, high mechanical properties, and can be molded into different shapes and sizes.11-13 In recent years, due to its unique nanofibrillar structure, mechanical properties, and large-scale production approach,14 BC has attracted increasing interest as a versatile biomaterial for numerous biomedical purposes, such as wound-healing dressing,15,16 and scaffolds for regenerating soft and hard tissues (e.g. skin, blood vessels, bone and cartilage)13,16-18. Compared to the ECM collagen, BC nanofibers show a lower bioactivity and chemical activity mainly because of their highly hydrophilic nature, which could hinder to some extent the cells from interacting with BC nanofiber surface, resulting in

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the lack of cell attachment to BC nanofibers, and thus limit their use as a more amenable material in biomedical applications.19,20 Since the biomaterial interface plays a crucial role in mediating cell behaviors,1-4 surface modification of BC nanofibers is believed to be an effective way to enhance the cell-biomaterial interactions.20-24 For example, researchers have demonstrated that the surface of BC nanofibers could be successfully modified by physical adsorption of Arg-Gly-Asp (RGD) peptide or its bioconjugate with xyloglucan, improving endothelial cell adhesion and proliferation on BC.20-22 In a recent study, the BC nanofiber surface was modified via polysaccharide (chitosan and carboxymethyl cellulose) adsorption, to promote the adhesion and growth of retinal pigment epithelium.24 These studies indicate that surface modification of BC nanofibers by adsorption of natural or biological macromolecules can address their relatively low cell adhesion and proliferation. Considering the highly hydrophilic nature of BC nanofibers, it could be more feasible to enhance cell adhesion on BC surface through improvement of surface roughness and hydrophobicity, which have been recognized as major factors affecting the cell-biomaterial interactions in vitro or in vivo.5-7 However, most of the used natural biopolymers for surface improvements are hydrophilic, and thus they are hard to be used to make hydrophobic BC nanofiber surfaces. Also, such hydrophilic biopolymers may have weak points in tissue engineering applications due to their solubility in aqueous environment.25 In addition, although some studies have explored the surface hydrophobization of BC nanofibers by introducing functional groups, these surface treatments often involve the use of various organic chemicals, such as organic acids and anhydrides,26-28 which can be harmful to the environment and human health, limiting the use of BC as tissue engineering scaffolds due to safety concerns. Therefore, there is merit in the novel modification of BC nanofibers for producing hydrophobic biosurface by adsorption of a specific, natural and biocompatible biopolymer, which is expected to improve surface

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roughness and hydrophobicity of BC nanofibers. Herein, we utilize the natural biopolymer zein, the most abundant storage protein from maize, for the hydrophobic modification of BC nanofiber surface, to improve the cell-BC membrane interactions. Different from most of natural polymers, zein is a water-insoluble protein but can be dissolved in the ethanol-water mixture, because it contains over 50% hydrophobic amino acids.29 As an amphiphilic protein, zein is able to self-assemble into various microstructures upon solvent evaporation, such as spheres, packed spheres, and films, which were largely affected by zein concentration and ethanol-water ratio.30-32 In addition, zein has many advantages for its application in biomedical fields, such as good biodegradability and high biocompatibility.33,34 Therefore, based on the good biocompatibility and unique self-assembly behavior, the natural zein protein is highly potential for the hydrophobic modification of BC surfaces, and the composite BC-zein nanofibers with hydrophobic biosurface are also expected to have enhanced cell attachment and proliferation. In this work, we aimed to surface modify BC nanofibers with the hydrophobic zein protein, creating a novel BC-zein composite nanofiber membrane with hydrophobic biosurface to improve the cell-nanofiber interactions. To achieve our aim, we first prepared the hydrophobic BC-zein nanofibers through a simple and green route, and then investigated the influence of zein adsorption as well as the assembled zein microstructures on the surface roughness and hydrophobicity of BC nanofibers. We attempted to control the surface hydrophobicity of BC-zein composite nanofibers by utilizing the interesting zein self-assembly behavior, which could be tuned by protein concentration and the ethanol-water ratio of solvent during EISA, forming various zein structures and thus increasing the surface roughness of BC nanofibers. Finally, the mouse fibroblast cells were cultured to investigate the cell adhesion behavior on the modified BC nanofibers.

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2. EXPERIMENTAL SECTION 2.1. Materials. Bacterial cellulose (BC) pellicles were supplied by Hainan Guangyu Biotechnology Co., Ltd. (Haikou, China), and were further purified in 0.2 M NaOH at 80 °C for 1 h. After that, the BC was repeatedly washed with distilled water and then cut into small pieces of 4 cm × 4 cm. The membranes were sterilized by autoclaving at 121 °C and 0.1 MPa for 15 min and then stored refrigerated before use. Zein was obtained from Sigma Chemical Co. (St. Louis, USA). 2.2. Preparation of BC-Zein Composite Nanofiber Membranes. Zein solutions were prepared by dispersing zein in the ethanol-water mixture (80%, v/v), followed by ultrasonic treatment at 100 W for 5 min using a Sonic Ruptor 400 sonicator (OMNI, USA) to decrease particle size and improve distribution. To fabricate the composite nanofiber membrane, wet BC (4 cm × 4 cm) was first solvent exchanged from water to ethanol by immersing them in 80% ethanol-water solution for 24 h at 25 °C. This treatment is enough for the complete exchange of solvent in BC membranes from water to 80% ethanol. Subsequently, these membranes were immediately immersed in zein solutions for 2 h under light agitation, and then the obtained composite membranes were rinsed three times with 80% ethanol in order to completely remove the free zein on BC surface. After that, the composite membranes (4 cm × 4 cm) were placed under the hood in aluminum dishes (25 °C, 50% relative humidity) overnight (12 h) to allow for the EISA process of zein molecules. The preferential evaporation of ethanol results in an increase in the water content and thus the solvent hydrophilicity, driving the self-assembly of zein molecules by hydrophobic forces into various microstructures.30,31 The resulting evaporated samples were collected and further dried using a semi-automatic sheet former (Rapid-Köthen, Austria) at 90 °C and 0.1 MPa for 10 min. The preparation procedure of BC-zein composite nanofiber membranes was schematically illustrated in Figure 1. The composite

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membranes were coded as BC-zein 0.1%, BC-zein 0.25%, BC-zein 0.5%, BC-zein 1%, and BC-zein 2%, according to the concentrations of zein solution (1, 2.5, 5, 10, and 20 mg/mL zein in 80% ethanol, respectively). The unmodified pure BC membrane was coded as BC. 2.3. Characterization. 2.3.1. Thickness of Membranes. A Lorentzen & Wettre (L&W) Micrometer 250 (Sweden) was used to measure the thickness of membranes. The measurements were carried out at eight random points of each sample and the results were averaged. 2.3.2. Field-Emission Scanning Electronic Microscopy (FE-SEM). The membrane morphology was characterized using a Zeiss Merlin FE-SEM. The surface of dried membrane was sputter-coated with gold (JEOL JFC-1200 fine coater, Japan) and observed at 15 kV accelerating voltage. 2.3.3. Atomic Force Microscopy (AFM). A MultiMode 8 Scanning Probe Microscope (Bruker, USA) was used to investigate the topography and roughness of membrane surface. AFM images with 5 µm × 5 µm scan size were acquired in tapping mode using silicon nitride tips (Bruker, USA). The values of root-mean-square roughness (Rrms) and average roughness (Ra) were calculated using Nanoscope Analysis software. 2.3.4. Water Contact Angle (WCA). The surface hydrophobicity of membranes can be measured by WCA. The WCA of a hydrophilic surface is less than 90°, while for a hydrophobic surface the WCA is higher than 90°. WCA was obtained on a DataPhysics Instrument (OCA 20, Germany) equipped with a high-speed video camera. One drop of water (5 µL) was deposited on membrane surface using a microsyringe, and the drop image was captured by a video camera. The profile of the droplet was fitted and calculated according to Laplace-Young equation. Ten parallel measurements were carried out for each membrane. 2.3.5. Energy-Dispersive X-ray Spectroscopy (EDS). The elemental mapping for membranes was obtained on a S-3700N Hitachi SEM equipped with a EDS Bruker QUANTAX 400 detector. For

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nitrogen mapping process, the magnification and accelerating voltage were 3000 and 20 kV, respectively. Three different regions of each sample were analyzed. 2.3.6. Electron Probe Microanalyzer (EPMA). The presence and distribution of zein in the composite membranes were confirmed by locating nitrogen using a Shimadzu EPMA-1600 electron microprobe equipped with a Genesis EDS detector (EDAX, Japan). The sample surface is irradiated by a focused electron beam, resulting in the emission of characteristic X-ray of an element.35 The wavelength and intensity of the characteristic X-ray of nitrogen (green color) were analyzed to determine its content and distribution on membrane surface. 2.3.7. Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR). A Bruker Vertex 70 FTIR spectrometer coupling the ATR accessory was used to obtain the surface chemical structure of the membranes. The samples were scanned from 550 to 3700 cm−1 at 2 cm−1 resolution. 2.3.8. Wide Angle X-ray Diffraction (WAXD). WAXD measurements were carried out on a Bruker D8 Advance X-ray diffractometer. Cu Kα radiation (λ = 1.542 Å) was generated at 40 kV and 40 mA. The patterns were recorded in the region of angular range 2θ from 5° to 60° at a scan rate of 0.2°/s. 2.3.9. Mechanical Properties. The mechanical properties of the membranes were measured using an Instron-5565 tensile tester, equipped with a 100 N load cell, at a stretch rate of 1 mm/s. The samples were kept in the environment of 50% humidity and 25 °C for at least 48 h before tests. Force-displacement was recorded and the stress-strain curves were calculated to obtain the tensile parameters. Measurements were performed on at least eight specimens of each membrane. 2.4. Cell Culture. Mouse L929 fibroblasts were obtained from Laboratory Animal Center of Sun Yat-sen University. Cells were cultured at 37 °C in a 5% CO2 atmosphere in medium (DMEM, GIBCO), supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin/streptomycin

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(GIBCO). At 90% confluence, the flasks were washed three times using PBS (GIBCO). Then the cells were harvested using 0.25% trypsin (GIBCO) and resuspended in the same medium. The cells undergoing passages 2-4 were used for the experiments described below. 2.5. Cell Adhesion and Proliferation. To evaluate the cell adhesion on modified BC membranes, the membranes were exposed to ultraviolet radiation for 30 min before cell seeding. L929 cell suspensions were transferred into a 96-well plate with 1.0 × 104 cells per well. After incubation for 24 and 48 h, cell viability was measured by the MTT (tetrazolium dye) assay. MTT solution (5 mg/mL in PBS) was added to each well to reach a final concentration of 0.5 mg/mL. Cultures were continued for another 4 h, after which the MTT was gently removed using a syringe, and the crystals on the bottom of the wells were dissolved at 37 °C using DMSO (150 µL/well) as solvent. The absorbance was read at 570 nm using a microplate photometer (Multiskan FC, Thermo Scientific, USA). To assess the cell morphology, Hoechst staining was first used to visualize the cells attached on the membrane. The samples were rinsed three times in PBS and 0.1 mL of Hoechst 33342 in PBS (50 µg/mL) was then added into each well. After incubation for 15 min in the dark, the samples were washed with PBS for three times and observed using a Axioskop 40FL fluorescence microscope (Zeiss, Germany). For SEM observation, the membranes were first washed three times with PBS before fixing in 4% paraformaldehyde. Then, the SEM images of freeze-dried samples were captured using S-3700N SEM (Hitachi, Japan).

3. RESULTS AND DISCUSSION 3.1. Surface Morphology and Hydrophobicity of BC-Zein Composite Nanofibers. Figure S1 (see Supporting Information, SI) shows the images of the BC-zein composite nanofiber membranes

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after drying. As can be seen, all the membranes were very flexible and semitransparent, and could be folded easily without cracking. Moreover, with increasing zein concentration, the membrane thickness gradually increased (16.2-34.1 µm) (see Table 2), suggesting the successful incorporation of zein protein on the surface of BC nanofibers. This indicates that the present preparation procedure (Figure 1) could produce a relatively transparent and flexible BC-zein composite membrane. As previously mentioned, various self-assembled zein microstructures such as spheres, bicontinuous sponges, and films could be formed after EISA, which could be tuned by changing zein concentration.30,31 Thus, the pronounced changes on the zein microstructure as a function of zein concentration are expected to significantly affect the surface roughness and thus hydrophobicity of BC-zein composite nanofibers, and the results are described in the following sections. To gain more insight about the link between surface microstructure and hydrophobicity of BC, the surface morphology of unmodified BC or BC-zein composite nanofiber membranes was observed by using FE-SEM (Figure 2) and AFM (Figure 3). The results of FE-SEM images and WCA values of membrane surfaces were presented in Figure 2. As can be seen from Figure 2a, the unmodified BC membrane had a flat surface with a fibrillar network structure, which was consisted of BC nanofiber ribbons (Figure 3a-b). Accordingly, the BC membrane showed a low WCA value (below 30°), suggesting its highly hydrophilic nature. The surface structure and WCA value of BC membrane were not obviously affected by the ethanol treatment (see Figure S2, SI). In comparison with pure BC, the BC membranes modified by hydrophobic zein exhibited higher WCA values (see insets of Figure 2b-f), indicating a significant improvement in surface hydrophobicity. As expected, the surface hydrophobicity of BC-zein composite nanofibers appears to be affected by the changes of zein concentration. With increasing zein concentration from 0.1% to 0.5%, a significant increase in WCA values was observed, and the WCA reached the maximum value of 110.5° at 0.5% zein (inset

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of Figure 2d), showing the formation of a hydrophobic surface (> 90°). However, upon further increase in zein concentrations (1 and 2%), the WCA of the composite membranes started to decrease, suggesting the decreased surface hydrophobicity. In general, the hydrophobicity of material surface can be improved by increasing the surface roughness and/or lowering the surface energy, and the increase in surface roughness can enhance the hydrophobic properties of materials.32,36,37 Therefore, the observed differences on the hydrophobicity of composite membranes should be due to their surface roughness, which could be affected by the assembled zein structures on BC surface. As can be seen from Figure 2b-f, various zein structures appeared on the composite nanofiber surface, further confirming the successful incorporation of zein on BC surface (Figure S1, SI). Interestingly, the changes of zein structures seem to be dependent on the zein concentration, in line with the WCA results. With increasing zein concentration, we observed the microphase changes of zein aggregates on the membrane surface from large spheres (Figure 2b-c), bicontinuous sponges (Figure 2d-e), and then to zein particle films (Figure 2f and Figure S2). These observations were further confirmed by the 2D and 3D AFM images of membranes (Figure 3 and Figure S3). In comparison with the relatively smooth and flat zein films, the microstructures of large spheres and bicontinuous sponges (packed spheres) would endow the BC nanofiber with a higher surface roughness, thus contributing to improving surface hydrophobicity. This analysis is supported by the results of WCA (insets of Figure 2), which showed that the BC-zein 0.5% nanofiber membrane had the highest WCA value (110.5°). The contribution of surface roughness to the improvement of surface hydrophobicity of membranes will be further discussed in the following section. 3.2. Role of Surface Roughness on Improvement of the Hydrophobicity. It has been recognized that the hydrophobic properties of material surfaces can be enhanced by increasing surface roughness.37-39 Herein, the surface roughness of nanofiber membranes was quantified by analyzing

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their AFM images (5 × 5 µm scans, Figure 3 and Figure S3), to evaluate the role of surface roughness on surface hydrophobicity. Figure 4 shows the corresponding results of roughness parameters, the root-mean-square roughness (Rrms) and average roughness (Ra). As can be seen, compared to the unmodified BC nanofiber, the BC-zein composite nanofibers showed much higher Rrms values, mainly due to the presence of zein structures on BC surface (Figures 2 and 3, Figure S3). For the composite nanofibers, the Rrms values gradually increased with the increase of zein concentration from 0.1% to 0.5%, whereas further increase in zein concentration (1-2%, especially at 2%) led to a marked decrease in Rrms (Figure 4). Additionally, the changes of Ra presented a similar trend, which appears to be obviously affected by zein concentration. Thus, the BC-zein 0.5% nanofiber membrane had the highest values of Rrms and Ra, which were calculated to be 40.3 and 29.9 nm, respectively. Interestingly, it is noted that the general trends of these results are in good agreement with those observed in WCA data (Figure 2). This indicates that the surface roughness should be a key factor in determining the surface hydrophobicity of BC-zein membranes, and the increase in surface roughness could improve the hydrophobicity of membrane surfaces. It is known that the Wenzel’s model and Cassie’s model can be used to understand the wettability of a solid material surface. The state that water contacts the whole surface can be described by the Wenzel’s model, while the Cassie’s model generally analyzes the contact angle at a heterogeneous surface, which is composed of two different materials.38,39 The state transition from Cassie’s model to Wenzel’s model for the water droplet on the surface may occur after being placed on the surface for a short time due to the continuous wetting. For the BC-zein composite nanofibers, a vast amount of zein aggregates with various topographic structures (large spheres, bicontinuous sponges, and films) formed a rough surface on BC (Figures 2 and 3), and the space among the zein aggregates could trap air to improve the hydrophobicity.36,37,39 Therefore, the Cassie equation can be applied in this

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situation by assuming that the surface beneath the water droplet is composed of solid and air. Figure 5A shows the changes of WCA of nanofiber membranes as a function of time (10 s). Images of the water droplets at 1 and 10 s after landing on each membrane are presented in Figure 5B. As can be seen, for the BC-zein 0.5% membrane, no significant change in the WCA was observed after 4 s, suggesting the surface reached a balanced state. However, the WCA decreased from 110.5° to 101.8° after 10 s, suggesting that the state transition from Cassie to Wenzel for the water droplet may occur. In general, it is believed that when the surfaces have the same roughness, the WCA for the water in the Cassie state is higher than that in the Wenzel state. This is probably due to the controlled self-assembly behavior of zein and the formed zein structures on BC nanofiber surface. Similar changes were also found in other composite membranes (Figures 5A and 5B). For the BC-zein composite nanofibers, after the immersion process, zein molecules or small molecular aggregates were dispersed well in the BC network matrix (Figure 1). During EISA, the zein molecules or small aggregates self-assemble into large aggregates with different sizes and morphologies, such as nano/micro-sized spheres, packed spheres, and films,30,31 and these aggregates distributed evenly in the pores of BC nanofibers, resulting in the formation of a rough surface with various zein structures (Figures 2 and 3). At low zein concentrations (0.1 and 0.25%), the amount of zein aggregates (spheres) was not enough to fully cover the BC surface (Figures 2 and 3), leading to a relatively low surface roughness (Figure 4) and thereby low WCA value (Figure 5). Interestingly, the increasing zein concentration (0.5%) resulted in an increase in the amount and size of zein aggregates, forming a bicontinuous sponge (packed spheres) structure on the membrane surface (Figures 2 and 3), and consequently, the surface roughness and the corresponding WCA significantly increased (Figures 4 and 5). However, as the zein concentration further increased (2%), a smooth and flat zein film was formed due to the melting and fusion between zein spheres, which completely

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covered the BC nanofiber surface (Figures 2 and 3). In comparison with the separated and packed zein spheres, the flat zein film showed a lower surface roughness (Figure 4), and thus the hydrophobicity of composite nanofibers decreased (Figure 5). This indicates that the surface hydrophobicity of the novel BC-zein composite nanofiber membranes can be tuned by simply changing zein concentration, which could control the surface morphology of zein structures after EISA, thus affecting the surface roughness of final membranes. The controlled surface roughness and hydrophobicity of the BC-zein nanofibers could make them particularly interesting for the design and construction of biomaterials requiring specific surface and adhesion properties. 3.3. Surface Element Analysis of BC-Zein Composite Nanofibers. In this section, EDS was used to analyze the element composition and location of the nanofiber surface. Carbon (C), oxygen (O), nitrogen (N), and sulfur (S) were the possible elements involved during sample preparation and thus they were input into the EDS software. The amounts of these four elements on the surfaces of pure BC and BC-zein composite nanofibers were presented in Table 1. The EDS detected very small amount of sulfur in pure BC, which can be regarded as a measurement error. For the BC-zein nanofibers, as can be seen, with increasing zein concentration, a gradual increase in the amounts of nitrogen and sulfur was observed, which chemically confirmed that the zein protein was incorporated on BC nanofiber surface. Since nitrogen is present only in zein, the nitrogen map images of composite nanofibers were obtained to indicate the distribution of zein. As can be seen from Figure 6, nitrogen was represented with red spots in these images, suggesting that zein was evenly distributed onto the surface of BC-zein composite nanofibers. This is in good agreement with previous results (Figure S1, Figures 2 and 3). The presence and distribution of zein protein on BC nanofiber surface were further confirmed using the EPMA technique. Figure S4 (SI) shows the EPMA nitrogen map images, and the nitrogen was labelled as green color in the image. As can be seen, the homogenous

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distribution of zein on the nanofiber surface was again demonstrated, and the nitrogen signal increased with increasing zein concentration, in line with the EDS analysis (Table 1 and Figure 6). 3.4. Chemical Structure and Mechanical Properties of BC-Zein Composite Nanofibers. 3.4.1. ATR-FTIR and WAXD. The surface chemical structure of the BC nanofibers modified by zein adsorption was first analyzed using ATR-FTIR (Figure 7A). As can be seen, pure BC exhibited a typical spectrum of cellulose with some characteristic peaks at 3343 cm−1 (O-H stretching), 2895 cm-1 (C-H stretching of CH2/CH3 groups), 1642 cm−1 (carbonyl groups of the glucose), 1370 cm-1 (CH2 symmetric bending), 1054 cm−1 (C-O-C stretching), and 1030 cm-1 (C-O stretching). For the composite samples, the adsorption peaks at 1645 cm-1 and 1534 cm-1 were clearly observed, where were characteristic bands of amide I (C=O stretching) and amide II (N-H bending and C-N stretching) in protein, respectively. This further demonstrates the successful incorporation of zein on BC surface. Additionally, with increasing zein concentration, these typical peaks for BC gradually declined in the absorbance intensity or even disappeared (at 2% zein), suggesting that the BC nanofibers have been partly or fully covered by zein, in good agreement with the surface observations from FE-SEM and AFM (Figures 2 and 3). Moreover, the band at 3343 cm−1, assigned as the stretching vibrations of hydroxyl groups of BC, merged with that of zein and shifted to lower wavenumbers (1 and 2% zein), indicating a decrease in hydrogen bonding within BC.40 The decreased intensity of this band in the composites also suggests a reduced amount of free hydroxyl groups in BC surface, which is probably due to their involvement in the formation of hydrogen bonds with zein. This is further verified by the shrink of the peak at 2895 cm−1 due to the stretching of aliphatic CH groups of BC, indicating the formation of intermolecular hydrogen bonds between BC and zein. Figure 7B shows the WAXD spectra of BC and BC-zein composite nanofibers. As can be seen, for pure BC nanofiber the characteristic peaks at 2θ = 14.6°, 16.8° and 22.7° were observed, which

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correspond to the (1ī0), (110) and (220) planes of polymorph cellulose I, respectively.41 The characteristic peaks of ethanol-treated BC were almost identical to those of untreated BC (Figure S5, SI), indicating that the solvent exchange process (Figure 1) did not significantly affect the original crystallinity of BC nanofiber. In the cases of BC-zein composites, no significant changes in the WAXD patterns were observed as compared to that of pure BC, which indicates that the surface modification did not obviously affect the crystalline structure of BC nanofibers. This can be explained by the amorphous nature of zein, as well as the physical interactions (hydrogen bond) between zein and BC (Figure 7A). 3.4.2. Mechanical Properties. Since the physical stability of biomaterials is one of the leading factors for their successful biomedical applications, the effect of zein adsorption on the mechanical properties of BC membrane was further investigated. The tensile parameters of pure BC and BC-zein composite membranes, including Young’s modulus (YM), tensile strength (TS), and elongation at break (EAB), were shown in Table 2. When the zein concentration increased from 0.1% to 0.5%, a slight increase in the values of TS and EAB for the composite membranes was observed, suggesting the improvement in the overall tensile properties of BC membrane. This is probably due to the incorporation of zein structures (spheres and bicontinuous sponges, Figures 2 and 3), which might fill the pores of BC matrix and thus to some extent reinforce the nanofiber network. However, upon further increase of zein concentrations (1-2%), especially at 2% zein, the TS and EAB values of the composite decreased significantly, which can be explained by the formation of zein films on BC surface (Figures 2 and 3). Previous studies have reported that pure zein films are tough and resistant, but also brittle and easily broken, requiring the addition of plasticizers to improve film flexibility.42,43 Therefore, the brittle zein films on BC surface may weaken the tensile strength of the composite membranes, and make them less stretchable (Table 2).

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3.5. Cell Adhesion and Proliferation on the Modified BC Membrane. Figures 8 and 9 show the results of the adhesion and proliferation of mouse L929 fibroblast cells on the BC-zein membrane surfaces. As can be seen from Figure 8, all the membranes were noncytotoxic to fibroblast cells, showing good biocompatibility. The BC-zein composite membranes showed significantly higher fibroblast amounts after 24 and 48 h culture as compared to pure BC, indicating that the cell adhesion and growth were obviously enhanced. It is interesting to note that with increasing protein concentration from 0.1% to 0.5%, the cell amount on the BC-zein membranes increased significantly; however, there is no significant change in the cell amount when the zein concentration further increased (1-2%). Based on the previous results (Figures 2-5), we attribute the observed difference in cell adhesion on the composite membranes to the controlled self-assembly of hydrophobic zein protein, as well as its good biocompatibility. At low zein concentrations (0.1-0.5%), with increasing zein concentration, the surface roughness and hydrophobicity of composite membranes gradually increased (Figures 4 and 5), due to the coverage of spheres and bicontinuous sponge structure on BC surface (Figures 2 and 3). Accordingly, more surface area for cell attachment and growth could be provided by the rough surface structure of membrane (BC-zein 0.5%) (Figure 8). At high zein concentrations (1 and 2%), the formation of flat zein films led to the decrease in the surface roughness of membranes (Figures 2-4). However, the high biocompatibility of natural zein could make the zein film as a good substrate to improve the cell-substrate interactions,33,34 which could also provide a favorable attachment to the membrane surface (Figure 8). The fluorescent images of cells on the surfaces of pure BC and the BC-zein composite membranes after incubation for 24 and 48 h were displayed in Figure 9. As can be seen, cells were spread out on membrane surface, and the cell density after 48 h culture was higher than that after 24 h. The BC-zein composite membranes showed significantly higher cell densities as compared to pure BC,

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indicating the improved cell adhesion and proliferation, which is in good agreement with the data of MTT assays (Figure 8). This is further supported by the SEM images of L929 cells on these surfaces after 48 h culture (Figure S6). Interestingly, it is worth noting that the fibroblast cells appear to prefer to adhere and spread on the zein-modified BC regions as compared to pure BC nanofiber (marked by red arrows), which is probably due to the rough surface structure (Figure S6b, SI) and the good biocompatibility of zein protein (Figure S6d, SI), in line with the previous analysis (Figure 8). Therefore, these results suggest that the surface modification by the controlled self-assembly behavior of zein can effectively promote cell adhesion and proliferation on BC nanofiber, thus improving the overall biocompatibility of BC-based biomaterials.

4. CONCLUSIONS We have successfully fabricated a novel BC-zein composite nanofiber with controlled hydrophobic biosurface through a simple and green solution impregnation method followed by EISA of adsorbed zein. We demonstrated that the surface hydrophobicity of the zein-modified BC nanofibers could be controlled by simply changing zein concentration, which was found to be able to control the self-assembled zein microstructures after EISA, thus affecting the surface roughness of BC membranes. At low zein concentrations (0.5%), the formation of hierarchical zein structures (spheres and bicontinuous sponge) endowed the BC surface with a high roughness, thus increasing the surface hydrophobicity. At higher zein concentration (2%), the melting and fusion of large zein spheres led to the formation of flat zein films, which decreased the surface roughness and thus the hydrophobicity of the composite membrane. In addition, we showed that compared to pure BC, the zein-modified BC nanofiber membranes were more biocompatible, showing a significantly increased adhesion and proliferation of fibroblast cells. This should be attributed to the rough surface structure of BC-zein

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nanofibers as well as the high biocompatibility of natural zein. Moreover, the homogenous incorporation of zein on BC surface by hydrogen bonding was confirmed, and the internal structure and mechanical properties of BC nanofibers were not significantly affected by zein modification. Our results indicate that the novel BC-zein composite nanofibers with controlled surface roughness and hydrophobicity may be of particular interest for the design and construction of BC-based biomaterials or biodevices that require specific surface properties and adhesion.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Photographs of the prepared BC and BC-zein composite nanofiber membranes. Higher-magnification FE-SEM images of membrane surfaces. 2D AFM images and the corresponding section analysis for membrane surfaces. Elemental mapping of nitrogen by EPMA for membrane surfaces. WAXD spectra of the membranes of BC and BC with ethanol treatment. SEM images of L929 fibroblast cells on membrane surfaces.

ACKNOWLEDGMENTS Financial supports by the General Project of China Postdoctoral Science Foundation (2016M600655), the Fundamental Research Funds for the Central Universities (2017BQ101), and the National Natural Science Foundation of China (31371744) are kindly acknowledged.

REFERENCES (1) Stevens, M. M.; George, J. H. Exploring and Engineering the Cell Surface Interface. Science

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2005, 310, 1135-1138. (2) Guo, C. X.; Zheng, X. T.; Lu, Z. S.; Lou, X. W.; Li, C. M. Biointerface by Cell Growth on Layered Graphene–Artificial Peroxidase–Protein Nanostructure for in Situ Quantitative Molecular Detection. Adv. Mater. 2010, 22, 5164-5167. (3) Langer, R.; Tirrell, D. A. Designing Materials for Biology and Medicine. Nature 2004, 428, 487-492. (4) Boyan, B. D.; Hummert, T. W.; Dean, D. D.; Schwartz, Z. Role of Material Surfaces in Regulating Bone and Cartilage Cell Response. Biomaterials 1996, 17, 137-146. (5) Wilson, C. J.; Clegg, R. E.; Leavesley, D. I.; Pearcy, M. J. Mediation of Biomaterial-Cell Interactions by Adsorbed Proteins: a Review. Tissue Eng. 2005, 11, 1-18. (6) Wang, Y. W.; Wu, Q.; Chen, G. Q. Reduced Mouse Fibroblast Cell Growth by Increased Hydrophilicity of Microbial Polyhydroxyalkanoates via Hyaluronan Coating. Biomaterials 2003, 24, 4621-4629. (7) Wang, P. Y.; Clements, L. R.; Thissen, H.; Jane, A.; Tsai, W. B.; Voelcker, N. H. Screening Mesenchymal Stem Cell Attachment and Differentiation on Porous Silicon Gradients. Adv. Funct Mater. 2012, 22, 3414-3423. (8) Lim, J. Y.; Donahue, H. J. Cell Sensing and Response to Micro-and Nanostructured Surfaces Produced by Chemical and Topographic Patterning. Tissue Eng. 2007, 13, 1879-1891. (9) Ma, Z.; Kotaki, M.; Inai, R.; Ramakrishna, S. Potential of Nanofiber Matrix as Tissue-Engineering Scaffolds. Tissue Eng. 2005, 11, 101-109. (10) Shin, H.; Jo, S.; Mikos, A. G. Biomimetic Materials for Tissue Engineering. Biomaterials 2003, 24, 4353-4364. (11) Hestrin, S.; Schramm, M. Synthesis of Cellulose by Acetobacter Xylinum. 2. Preparation of

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Freeze-Dried Cells Capable of Polymerizing Glucose to Cellulose. Biochem. J. 1954, 58, 345. (12) Petersen, N.; Gatenholm, P. Bacterial Cellulose-Based Materials and Medical Devices: Current State and Perspectives. Appl. Microbiol. Biotechnol. 2011, 91, 1277-1286. (13) Svensson, A.; Nicklasson, E.; Harrah, T.; Panilaitis, B.; Kaplan, D.; Brittberg, M.; Gatenholm, P. Bacterial Cellulose as a Potential Scaffold for Tissue Engineering of Cartilage. Biomaterials 2005, 26, 419-431. (14) Ullah, M. W.; Ul-Islam, M.; Khan, S.; Kim, Y.; Park, J. K. Innovative Production of Bio-Cellulose Using a Cell-Free System Derived from a Single Cell Line. Carbohydr. Polym. 2015, 132, 286-294. (15) Czaja, W. K.; Young, D. J.; Kawecki, M.; Brown, R. M. The Future Prospects of Microbial Cellulose in Biomedical Applications. Biomacromolecules 2007, 8, 1-12. (16) Fu, L.; Zhang, J.; Yang, G. Present Status and Applications of Bacterial Cellulose-Based Materials for Skin Tissue Repair. Carbohydr. Polym. 2013, 92, 1432-1442. (17) Zahedmanesh, H.; Mackle, J.; Sellborn, A.; Drotz, K.; Bodin, A.; Gatenholm, P.; Lally, C. Bacterial Cellulose as a Potential Vascular Graft: Mechanical Characterization and Constitutive Model Development. J. Biomed. Mater. Res., Part B 2011, 97B, 105-113. (18) Zaborowska, M.; Bodin, A.; Bäckdahl, H.; Popp, J.; Goldstein, A.; Gatenholm, P. Microporous Bacterial Cellulose as a Potential Scaffold for Bone Regeneration. Acta Biomater. 2010, 6, 2540-2547. (19) Kuzmenko, V.; Sämfors, S.; Hägg, D.; Gatenholm, P. Universal Method for Protein Bioconjugation with Nanocellulose Scaffolds for Increased Cell Adhesion. Mater. Sci. Eng., C 2013, 33, 4599-4607. (20) Fink, H.; Ahrenstedt, L.; Bodin, A.; Brumer, H.; Gatenholm, P.; Krettek, A.; Risberg, B.

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Bacterial Cellulose Modified with Xyloglucan Bearing the Adhesion Peptide RGD Promotes Endothelial Cell Adhesion and Metabolism—A Promising Modification for Vascular Grafts. J. Tissue Eng. Regener. Med. 2011, 5, 454-463. (21) Bodin, A.; Ahrenstedt, L.; Fink, H.; Brumer, H.; Risberg, B.; Gatenholm, P. Modification of Nanocellulose with a Xyloglucan–RGD Conjugate Enhances Adhesion and Proliferation of Endothelial Cells: Implications for Tissue Engineering. Biomacromolecules 2007, 8, 3697-3704. (22) Andrade, F. K.; Costa, R.; Domingues, L.; Soares, R.; Gama, M. Improving Bacterial Cellulose for Blood Vessel Replacement: Functionalization with a Chimeric Protein Containing a Cellulose-Binding Module and an Adhesion Peptide. Acta Biomater. 2010, 6, 4034-4041. (23) Shi, Q.; Li, Y.; Sun, J.; Zhang, H.; Chen, L.; Chen, B.; Yang, H.; Wang, Z. The Osteogenesis of Bacterial Cellulose Scaffold Loaded with Bone Morphogenetic Protein-2. Biomaterials 2012, 33, 6644-6649. (24) Gonçalves, S.; Padrão, J.; Rodrigues, I. P.; Silva, J. P.; Sencadas, V.; Lanceros-Mendez, S.; Girão, H.; Dourado, F.; Rodrigues, L. R. Bacterial Cellulose as a Support for the Growth of Retinal Pigment Epithelium. Biomacromolecules 2015, 16, 1341-1351. (25) Cunha, A. G.; Gandini, A. Turning Polysaccharides into Hydrophobic Materials: a Critical Review. Part 1. Cellulose. Cellulose 2010, 17, 875-889. (26) Ifuku, S.; Nogi, M.; Abe, K.; Handa, K.; Nakatsubo, F.; Yano, H. Surface Modification of Bacterial Cellulose Nanofibers for Property Enhancement of Optically Transparent Composites: Dependence on Acetyl-Group DS. Biomacromolecules 2007, 8, 1973-1978. (27) Lee, K. Y.; Quero, F.; Blaker, J. J.; Hill, C. A.; Eichhorn, S. J.; Bismarck, A. Surface Only Modification of Bacterial Cellulose Nanofibres with Organic Acids. Cellulose 2011, 18, 595-605. (28) Tomé, L. C.; Freire, M. G.; Rebelo, L. P. N.; Silvestre, A. J.; Neto, C. P.; Marrucho, I. M.; Freire,

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C. S. Surface Hydrophobization of Bacterial and Vegetable Cellulose Fibers Using Ionic Liquids as Solvent Media and Catalysts. Green Chem. 2011, 13, 2464-2470. (29) Shukla, R.; Cheryan, M. Zein: the Industrial Protein from Corn. Ind. Crops Prod. 2001, 13, 171-192. (30) Wang, Y.; Padua, G. W. Formation of Zein Microphases in Ethanol−Water. Langmuir 2010, 26, 12897-12901. (31) Wang, Y.; Padua, G. W. Nanoscale Characterization of Zein Self-Assembly. Langmuir 2012, 28, 2429-2435. (32) Dong, F.; Padua, G. W.; Wang, Y. Controlled Formation of Hydrophobic Surfaces by Self-Assembly of an Amphiphilic Natural Protein from Aqueous Solutions. Soft Matter 2013, 9, 5933-5941. (33) Dong, J.; Sun, Q.; Wang, J. Y. Basic Study of Corn Protein, Zein, as a Biomaterial in Tissue Engineering, Surface Morphology and Biocompatibility. Biomaterials 2004, 25, 4691-4697. (34) Wang, H. J.; Lin, Z. X.; Liu, X. M.; Sheng, S. Y.; Wang, J. Y., Heparin-Loaded Zein Microsphere Film and Hemocompatibility. J. Controlled Release 2005, 105, 120-131. (35) Tianqing, L.; Chunfeng, M.; Xiangyu, S.; Songbai, X. Mechanism Study on Formation of Initial Condensate Droplets. AIChE J. 2007, 53, 1050-1055. (36) Sun, T.; Feng, L.; Gao, X.; Jiang, L. Bioinspired Surfaces with Special Wettability. Acc. Chem. Res 2005, 38, 644-652. (37) Yoshimitsu, Z.; Nakajima, A.; Watanabe, T.; Hashimoto, K. Effects of Surface Structure on the Hydrophobicity and Sliding Behavior of Water Droplets. Langmuir 2002, 18, 5818-5822. (38) Wenzel, R. N. Resistance of Solid Surfaces to Wetting by Water. Ind. Eng. Chem. 1936, 28, 988-994.

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(39) Cassie, A.; Baxter, S. Wettability of Porous Surfaces. Trans. Faraday Soc. 1944, 40, 546-551. (40) Lamboni, L.; Li, Y.; Liu, J.; Yang, G. Silk Sericin-Functionalized Bacterial Cellulose as a Potential Wound-Healing Biomaterial. Biomacromolecules 2016, 17, 3076-3084. (41) Qiu, K.; Netravali, A. N. Bacterial Cellulose-Based Membrane-Like Biodegradable Composites using Cross-Linked and Noncross-Linked Polyvinyl Alcohol. J. Mater. Sci. 2012, 47, 6066-6075. (42) Lai, H. M.; Padua, G. W.; Wei, L. S. Properties and Microstructure of Zein Sheets Plasticized with Palmitic and Stearic Acids. Cereal Chem. 1997, 74, 83-90. (43) Corradini, E.; Curti, P. S.; Meniqueti, A. B.; Martins, A. F.; Rubira, A. F.; Muniz, E. C. Recent Advances in Food-Packing, Pharmaceutical and Biomedical Applications of Zein and Zein-Based Materials. Int. J. Mol. Sci. 2014, 15, 22438-22470.

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Table 1. The elemental amounts of carbon (C), oxygen (O), nitrogen (N), and sulfur (S) on the surfaces of the BC and BC-zein composite nanofiber membranes, obtained by EDS analysis.a surface elemental composition (%) membranes

a

C

O

N

S

Total

BC

34.86 ± 0.41

65.12 ± 0.44

0.00 ± 0.00

0.02 ± 0.01

100

BC-zein 0.1%

30.93 ± 0.32

64.19 ± 0.29

4.73 ± 0.21

0.14 ± 0.02

100

BC-zein 0.25%

31.69 ± 0.29

60.39 ± 0.36

7.75 ± 0.13

0.18 ± 0.02

100

BC-zein 0.5%

32.74 ± 0.16

51.24 ± 0.30

15.76 ± 0.15

0.26 ± 0.03

100

BC-zein 1%

35.09 ± 0.35

44.96 ± 0.31

19.56 ± 0.12

0.40 ± 0.06

100

BC-zein 2%

36.37 ± 0.38

42.66 ± 0.42

20.30 ± 0.18

0.66 ± 0.05

100

Values are the mean and standard deviation.

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Table 2. Thickness and mechanical parameters of the BC and BC-zein composite nanofiber membranes.a membranes

a

thickness (µm)

YM (GPa)

TS (MPa)

EAB (%)

BC

16.2 ± 0.6

11.5 ± 2.9

151.6 ± 20.9

1.5 ± 0.4

BC-zein 0.1%

16.5 ± 0.5

12.4 ± 2.4

167.7 ± 35.5

1.8 ± 0.5

BC-zein 0.25%

18.9 ± 1.2

13.3 ± 2.8

169.3 ± 39.8

1.9 ± 0.7

BC-zein 0.5%

20.9 ± 1.5

13.4 ± 2.2

178.6 ± 22.6

2.3 ± 0.4

BC-zein 1%

23.3 ± 1.1

13.2 ± 1.5

145.5 ± 27.6

1.6 ± 0.6

BC-zein 2%

34.1 ± 1.5

13.1 ± 2.4

82.2 ± 19.8

1.0 ± 0.5

Values are the mean and standard deviation.

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Figure 1. Schematic illustration of the preparation of BC-zein composite nanofiber membranes. (1) Immersing in 80% ethanol solution for 24 h at 25 °C; (2) immersing in zein solutions with light agitation; (3) evaporation-induced self-assembly (EISA) of zein molecules at 25 °C; (4) hot-pressing at 90 °C and 0.1 MPa.

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Figure 2. FE-SEM images of surfaces for BC (a), BC-zein 0.1% (b), BC-zein 0.25% (c), BC-zein 0.5% (d), BC-zein 1% (e), and BC-zein 2% (f) composite membranes. Insets show the photographs of the WCA on each membrane surface: BC (27.2 ± 1.2°), BC-zein 0.1% (73.3 ± 1.8°), BC-zein 0.25% (96.3 ± 2.2°), BC-zein 0.5% (110.5 ± 2.1°), BC-zein 1% (103.5 ± 2.3°), and BC-zein 2% (88.9 ± 1.7°). Note: the formation of zein aggregates (large spheres and bicontinuous sponge) on BC surfaces (marked by red arrows, b-d).

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Figure 3. 2D (left) and 3D (right) AFM images of surfaces for BC (a, b), BC-zein 0.5% (c, d), BC-zein 1% (e, f), and BC-zein 2% (g, h) composite membranes.

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Figure 4. Rrms and Ra roughness values calculated from AFM images of the BC and BC-zein composite nanofiber membranes (values calculated from at least three 5 µm × 5 µm scans).

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Figure 5. (A) Time-dependent water contact angles of BC and BC-zein composite membranes. (B) Images of the water droplets at 1 and 10 s after landing on surfaces for BC (a), BC-zein 0.5% (b), BC-zein 1% (c), and BC-zein 2% (d) membranes.

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Figure 6. Elemental mapping of nitrogen (red spots) by EDS for the membrane surfaces of BC (a), BC-zein 0.1% (b), BC-zein 0.25% (c), BC-zein 0.5% (d), BC-zein 1% (e), and BC-zein 2% (f).

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Figure 7. ATR-FTIR (A) and WAXD spectra (B) of BC and BC-zein composite nanofiber membranes.

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Figure 8. MTT assays of L929 fibroblast cells on BC and BC-zein composite nanofiber membranes after 24 and 48 h culture.

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Figure 9. Fluorescent images (scale bar = 30 µm) of L929 fibroblast cells on BC, BC-zein 0.5%, BC-zein 1%, and BC-zein 2% membranes after 24 and 48 h culture. All membranes were subjected to Hoechst staining.

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Controlled Hydrophobic Biosurface of Bacterial Cellulose Nanofibers through Self-Assembly of Natural Zein Protein Zhili Wan, Liying Wang, Lulu Ma, Yingen Sun, and Xiaoquan Yang*

Table of Contents (TOC) Graphic

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