Article pubs.acs.org/Langmuir
Changes in Cell Adhesiveness and Physicochemical Properties of Cross-Linked Albumin Films after Ultraviolet Irradiation Hironori Yamazoe,*,† Hisashi Nakanishi,†,‡ Yukiyasu Kashiwagi,§ Masami Nakamoto,§ Akira Tachibana,‡ Yoshihisa Hagihara,† and Toshizumi Tanabe‡ †
National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan § Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan ‡
S Supporting Information *
ABSTRACT: We discovered the unique cell adhesive properties of ultraviolet (UV)-irradiated albumin films. Albumin films prepared using a cross-linking reagent with epoxy groups maintained native albumin properties, such as resistance to cell adhesion. Interestingly, the cell adhesive properties of films varied depending upon the UV irradiation time; specifically, cell adhesiveness increased until 2 h of UV irradiation, when the cell number attached to the film was similar to that of culture dishes, and then cell adhesiveness decreased until 20 h of UV irradiation, after which the surface returned to the initial nonadhesive state. To elucidate the molecular mechanisms underlying this phenomenon, we examined the effect of UV irradiation on albumin film properties. The following changes occurred in response to UV irradiation: decreased α-helical structure, cleavage of albumin peptide bonds, and increased hydrophilicity and oxygen content of the albumin film surface. In addition, we found a positive correlation between the degree of cell adhesion and the amount of fibronectin adsorbed on the film. Taken together, UV-induced changes in films highly affect the amount of cell adhesion proteins adsorbed on the films depending upon the irradiation time, which determines cell adhesion behavior.
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INTRODUCTION
Cell adhesion is thought to occur via cell adhesion proteins, such as fibronectin, vitronectin, etc., contained in culture media, which are adsorbed on the substrate in advance. Integrins, which are cell surface receptors, interact with specific amino acid sequences of adsorbed cell adhesion proteins, such as arginine−glycine−aspartate (RGD).15 A widely used method for preventing cell adhesion is to coat the substrate surface with serum albumin, conferring resistance to cell adhesion protein adsorption on the substrate surface.16 Cells do not adhere directly to albumin, owing to the lack of the cell adhesion amino acid sequence.17 We previously prepared a waterinsoluble cross-linked albumin film that maintains native albumin properties, such as resistance to cell adhesion and drug-binding ability.18 Additionally, we found that the nonadhesive surface of the albumin film could be altered to allow for cell adhesion by exposing the film to ultraviolet (UV) light. With the use of this UV-convertible cell adhesive property, cellular micropatterns can be created by exposing films to UV light through a mask with a desired pattern, followed by the selective adhesion of cells to the UV-irradiated region.19 The advantage of our method is that the required reagents,
The regulation of cell adhesive behavior on the surface of materials is a critical factor for the development of biomaterials. Scaffolds for tissue engineering applications need to have the ability to support cell adhesion, spreading, and proliferation.1,2 Materials that are resistant to protein adsorption and cell adhesion have great potential for blood-contacting medical devices because plasma protein adsorption and platelet adhesion can lead to thrombus formation and subsequent device dysfunction.3−5 Recent attempts to guide the alignment of myoblasts with the aim of recapitulating muscle tissue have shown that excess surface modifications to promote cell adhesion limit cell movement after attachment, leading to poor cell alignment.6 In these cases, materials that induce moderate cell attachment might be desirable to achieve both the initial cell attachment and subsequent reorientation of cells. Furthermore, the area-specific regulation of cell adhesiveness has been demonstrated for the fabrication of cellular micropatterns on substrates using photolithography or microcontact printing techniques.7−10 Applications of cellular patterning techniques for the creation of cell-based materials, such as cell arrays and biosensors, and organized structures for use in tissue engineering have been demonstrated in some studies.11−14 © XXXX American Chemical Society
Received: October 27, 2015
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Figure 1. Effect of the UV irradiation time on cell adhesiveness of albumin films. Phase-contrast microscopic observations of L929 cells at 5 h after inoculation on the albumin films (A) before UV irradiation and (B−H) at 1−24 h after UV irradiation. Scale bar = 100 μm. (I) Cell adhesion on the albumin films before and after UV irradiation or control culture dish. UV irradiation was conducted for 2, 14, and 24 h. Data are represented by the means ± SD (n = 6). Experiments were performed 3 times. Two samples were examined in each experiment. (∗) p < 0.03 and (∗∗) p < 0.0001 versus culture dish.
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including serum albumin and a cross-linking reagent with epoxy groups, are inexpensive, and the preparation of the cross-linked albumin film involves a simple, one-step cross-linking reaction at room temperature. Although several photoresponsive materials have also been developed for the regulation of cell adhesive behavior and applied to the creation of cellular micropatterns on a substrate,20−22 their implementation is limited, owing to the complicated process required to synthesize the photoresponsive materials. To gain insight into the effect of UV irradiation on albumin film properties, we performed additional investigations and found that the cell adhesiveness of the albumin film varies depending upon the UV irradiation time. Specifically, cell adhesiveness gradually increased until 2 h of irradiation and then gradually decreased until 20 h of irradiation, after which cell adhesiveness was lost. Although the underlying mechanisms remain unclear, these unique features of albumin films could be useful for the development of materials with desired cell adhesive properties and intricately patterned substrates containing multiple regions with different degrees of cell adhesiveness. Thus, our present study aims to understand the molecular mechanisms underlying UV-induced cell adhesiveness of albumin films. Cell adhesive behavior on albumin films is thought to be determined by the cell adhesion proteins adsorbed on the film, whose amounts and orientation are highly dependent upon the surface properties of the film. Hence, we examined the changes in surface chemical composition, surface wettability, and protein structure of the albumin film. Moreover, protein adsorption behavior on albumin films was evaluated using peroxidase-labeled fibronectin. On the basis of these results, the relationships among film properties, protein adsorption, and cell adhesion are discussed.
MATERIALS AND METHODS
Cross-Linking of Albumin. Cross-linking of albumin was carried out as described previously.16 Briefly, bovine serum albumin (Sigma, St. Louis, MO) was dissolved in phosphate-buffered saline (PBS, pH 7.4) to yield a 3% solution that was then reacted with 200 mM ethylene glycol diglycidyl ether (EGDE, Wako, Osaka, Japan) with vigorous stirring for 24 h at room temperature. The reaction mixture was dialyzed for 3 days at room temperature against Milli-Q water using cellulose tubing (molecular weight cutoff = 14 kDa; Wako) to remove unreacted EGDE. Then, the albumin concentration was adjusted to 2% by the addition of Milli-Q water and filtered through a 0.22 μm filter for sterilization. Cell Adhesion to the UV-Irradiated Albumin Film. Crosslinked albumin solution was poured onto a 3.5 cm cell culture dish and dried overnight at room temperature to coat the dish with cross-linked albumin film. Then, the film-coated dishes were exposed to UV light (DNA-FIX DF-254, 254 nm, ATTO, Tokyo, Japan) for a predetermined time. Mouse L929 fibroblast cells were grown on the culture dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma), 100 mg/mL streptomycin, 100 units/mL penicillin, and 292 μg/mL glutamine. L929 cells were collected by the addition of a 0.05% trypsin/EDTA solution and resuspended in DMEM-based culture medium. The cells were plated on the albumin film-coated dishes with or without UV treatment at a density of 4.8 × 104 cells/cm2. At 5 h after incubation in a humidified 5% CO2 incubator at 37 °C, the cells were washed with PBS 3 times to remove unattached cells. Attached cells were collected by trypsinization, and the cell number was counted. Cell counting and cell observations were performed using a hemocytometer and a phasecontrast microscope (CKX41, Olympus, Tokyo, Japan), respectively. Spectroscopic Investigation. Cross-linked albumin solution was poured onto a quartz plate (3 × 3 × 0.03 cm, Vidrex Co., Ltd., Fukuoka, Japan). After 30 min of incubation, plates were placed on the rotor of a spin coater (1H-DX2, MIKASA Co., Ltd., Tokyo, Japan), rotated at 1000 rpm for 5 s, and then dried overnight at room temperature. Albumin film-coated plates were exposed to UV light as described above and used for the circular dichroism (CD) and UV B
DOI: 10.1021/acs.langmuir.5b03958 Langmuir XXXX, XXX, XXX−XXX
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Figure 2. Phase-contrast micrographs of L929 cells adhered on the (A) culture dish or albumin films for (B) 2 h and (C) 14 h of UV irradiation after a 1 day culture. Scale bar = 50 μm.
Figure 3. (A) CD spectra of albumin films before and after UV irradiation. UV irradiation was conducted for 2, 14, and 24 h. (B) Residual α-helix content (expressed as a percentage) of albumin films after UV irradiation for 2, 14, and 24 h. Data are represented by the means ± SD (n = 6). Experiments were performed 3 times. Two samples were examined in each experiment. (∗) p < 0.0001. absorption analyses. The CD spectra of albumin films before and after UV irradiation were measured with a CD spectrophotometer (J-820, JASCO Co., Ltd., Tokyo, Japan). Each spectrum was corrected for baseline by subtracting the spectral contribution of the quartz plate. The α-helix content of albumin molecules in a film was determined from the ellipticity at 222 nm,23,24 and the residual α-helix content after UV irradiation was estimated using the following equation: residual α-helix content (%) = (ellipticity of albumin films after UV irradiation at 222 nm/ellipticity of albumin film before UV irradiation at 222 nm) × 100. UV absorption spectra of the films before and after UV irradiation were also measured using an UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). Contact Angle Measurement. The water contact angle was measured using a contact angle meter (model DMe-201, Kyowa Interface Science Co., Ltd., Saitama, Japan). A droplet of distilled water (2 μL) was deposited on an albumin film-coated substrate before and after UV irradiation. The measurement was conducted at 10 s after the water drop was placed on the surface of a sample. X-ray Photoelectron Spectroscopy (XPS) Analysis. The surface of albumin films before and after UV irradiation was characterized by XPS equipped with a Mg source (Mg Kα energy of 1253.6 eV) using the PHI ESCA 5700 with the MultiPak data treatment system. An analysis spot size was 800 μm. The binding energies of spectra were corrected on the basis of the lowest bonding energy of the C 1s peak set at 285.0 eV. The peak positions and areas were optimized by a weighted least-squares fitting method using Gaussian line shapes. Three independent experiments were performed, and the average values are shown in the results. Protein Adsorption. Bovine plasma fibronectin (Invitrogen, Carlsbad, CA) was labeled using a peroxidase labeling kit (Dojindo Laboratories, Kumamoto, Japan). The albumin films before and after UV irradiation were immersed in 20 μg/mL labeled fibronectin in PBS at room temperature for 5 h. After washing with PBS, the amount of
adsorbed fibronectin was determined using Amplex UltraRed reagent (Invitrogen) according to the instructions of the manufacturer. Nonfluorescent Amplex UltraRed reagent is converted into a fluorescent product by reacting with hydrogen peroxide in the presence of peroxidase. The fluorescent intensity of the samples was measured using a fluorescence spectrophotometer (Fluoroskan Ascent FL, Thermo Fisher Scientific, Inc., Waltham, MA). As a control, the adsorption of labeled fibronectin on a gold surface was similarly measured. The substrate with a gold surface was prepared by coating the glass plates (22 × 26 mm; Matsunami, Osaka, Japan) with a 1 nm layer of chromium followed by 19 nm of gold using vacuum deposition equipment (SGC-22SA, Showa Shinku Co., Ltd., Kanagawa, Japan). The surface was cleaned with piranha solution [3:1 (vol/vol) mixture of sulfuric acid and 30% hydrogen peroxide solution] just before the adsorption measurement. Statistical Analysis. All quantitative results were expressed as means ± standard deviation (SD). Statistical analyses were performed using Student’s t tests. The differences were considered significant for p values of less than 0.05.
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RESULTS Cell Adhesion Behavior on UV-Irradiated Albumin Films. Cell adhesiveness of albumin films was evaluated for various UV irradiation times. At 5 h after the seeding of fibroblast L929 cells on each dish, cells were observed under a microscope (Figure 1). The albumin films before UV irradiation were resistant to cell adhesion, similar to the albumin coating (Figure 1A). When films were exposed to UV light for a short time, the surface changed to allow for cell adhesion and the cell adhesiveness tended to increase with UV irradiation time for up to 2 h (panels B and C of Figure 1). Interestingly, more prolonged UV irradiation decreased cell C
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Langmuir adhesiveness in a time-dependent manner (panels D−H of Figure 1). Cell adhesion on the film was minimal after a 14 h UV irradiation period, and no cell attachment was observed on the film at 20 and 24 h of UV irradiation, similar to before UV irradiation. Human epithelial carcinoma HeLa cells also showed similar cell adhesive behaviors on UV-irradiated albumin films (see Figure S1 of the Supporting Information). Additional cell adhesion did not occur on the films even if cells were cultured for more than 5 h; thus, the evaluation of cell adhesiveness at 5 h after cell seeding was valid. To eliminate the possibility that the albumin film was removed by UV irradiation, its presence was confirmed by protein staining with Coomassie Brilliant Blue (see Figure S2 of the Supporting Information). Further detailed investigations of UV-irradiated albumin films were performed using four samples that represented various degrees of cell adhesiveness, including non-adhesiveness (before UV irradiation), maximum cell adhesiveness (2 h), decreasing adhesiveness (14 h), and a sample that returned to the initial state of non-adhesiveness (24 h). Figure 1I shows the ratio of the number of adhered cells to inoculated cells at 5 h after inoculation on the films irradiated with UV light for 0, 2, 14, and 24 h. Cell adhesion took place on the albumin film for 2 h of UV irradiation and was comparable to that of culture dishes. Meanwhile, cell adhesion decreased to 12% after 14 h of UV irradiation. Figure 2 shows the micrographs of L929 cells on the culture dish and the albumin films at 1 day after cell seeding. Cells elongated to show spindle-like morphology on the albumin film at 2 h of UV irradiation in a manner similar to that observed on the culture dish, while cells retained a round shape on the film at 14 h of UV irradiation. Of note, we previously employed 5 h of UV irradiation to convert a cell non-adhesive surface to an adhesive surface.19 This inconsistency may be due to the differences in UV irradiation conditions (i.e., the UV irradiation equipment and distance between UV lamps and albumin films). Spectroscopic Investigation of UV-Irradiated Albumin Films. To examine conformational changes in albumin molecules for UV-irradiated albumin films, CD measurements were performed in the range of 190−250 nm (Figure 3A). The CD spectrum of the albumin films before UV irradiation showed the characteristic minima at 208 and 222 nm, which corresponded to an α-helical structure of albumin.23,24 UV irradiation induced a marked decrease in the CD signals around 208 and 222 nm, which decreased more as the irradiation time increased. In addition, the minimum at 208 nm tended to shift to a lower wavelength. These results suggested that the conformational changes in albumin, that is, the loss of α-helixes and increased random coil conformation, are caused by UV irradiation. The percentages of residual α-helix content after UV irradiation are summarized in Figure 3B. The α-helical structure was slightly destroyed by 2 h of UV irradiation, and only 20% of the α-helical structure was retained after 24 h of UV irradiation. We also investigated changes in the primary structure of albumin upon UV irradiation using a spectrophotometer. Figure 4 shows the UV absorption spectra of albumin films before and after UV irradiation. Commonly, proteins show an absorption peak at 280 nm, which is mainly due to the absorbance of cysteine (i.e., the disulfide bonds) and the aromatic amino acids, such as tryptophan and tyrosine. Additionally, the peptide bonds, the basic structural units of proteins, give strong signals in the region of 200−220 nm.25,26 There was no significant change in the absorbance peak derived from peptide bonds after 2 h of UV irradiation. However,
Figure 4. UV absorption spectra of albumin films before and after UV irradiation. UV irradiation was conducted for 2, 14, and 24 h. The inset shows magnified spectra for a wavelength of 240−320 nm.
prolonged UV irradiation (14−24 h) caused a decrease in absorbance, indicating the cleavage of peptide bonds. As shown in the inset of Figure 4, UV irradiation also caused an increase in the absorption intensity in the 260−300 nm regions as a function of the irradiation time. Surface Characterization of UV-Irradiated Albumin Films. The effects of UV irradiation on the surface characteristics of albumin films were analyzed on the basis of the contact angle and XPS measurements. The water contact angle of albumin films before UV irradiation was 87°, which is more hydrophobic than that of the intact cell culture dish (approximately 56°) (Figure 5). The film surface gradually
Figure 5. Water contact angles of albumin films before and after UV irradiation or control culture dish. UV irradiation was conducted for 2, 14, and 24 h. Data are represented by the means ± SD (n = 6). (∗) p > 0.05. (∗∗) p < 0.03.
became hydrophilic as the UV irradiation time increased. Thus, the wettability of the film surface changed dramatically from hydrophobic to hydrophilic upon UV irradiation. XPS measurements were performed to investigate the atomic composition of the albumin films (Table 1). The oxygen content increased as the UV irradiation time increased. We also performed a curve-fitting analysis of the C 1s spectra in accordance with the method by Bomben et al. 27 A representative C 1s spectrum and curve fitting are shown in D
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Langmuir Table 1. Surface Atomic Composition (%) UV irradiation time (h)
C (1s)
O (1s)
N (1s)
0 2 14 24
66 64 60 58
20 22 26 27
14 14 14 15
Figure S3 of the Supporting Information. The C 1s spectrum can be deconvoluted into three components with peak maxima at 288.1, 286.4, and 285.0 eV, which mainly originated from carbonyl carbons of peptide bonds at the main chain (oxygenbonded carbons, CO), α carbons at the main chain (nitrogen-bonded carbons, C−N), and carbon bonding with hydrogen and carbon at the side chain (C−H and C−C), respectively.27 Notably, each peak also contained contributions from other types of carbons, for example, C−O, CN, and CC, respectively. As shown in Figure 6, UV irradiation
Figure 7. Adsorption of fibronectin onto albumin films before and after UV irradiation or control gold surface. UV irradiation was conducted for 2, 14, and 24 h. Data are represented by the means ± SD (n = 7). Experiments were performed 3 times, with two or three samples examined in each experiment. (∗) p < 0.05. (∗∗) p < 0.005.
adhesion on a substrate is thought to initially involve the adsorption of cell adhesion proteins on the substrate and the subsequent adherence of cells on the substrate via the adsorbed proteins. In fact, fibronectin adsorption and cell adhesion on albumin films exhibited similar responses to UV irradiation times (Figures 1I and 7). No cells adhered to the films without UV or with 24 h of UV irradiation, despite the adsorption of fibronectin. This discordance may be explained by the fact that a certain amount of cell adhesion proteins is required for cell adhesion. Roberts et al. investigated cell adhesion by changing the density of the cell adhesion sequence RGD on the surface and found that there is a threshold RGD density that enables cell adhesion.28 It is also worth noting that cell adhesion is correlated with not only the amount of adsorbed fibronectin but also the orientation of fibronectin on the substrate.29 Cells can attach to adsorbed fibronectin only when fibronectin molecules are placed in the proper orientation such that the cell-adhesion sequence RGD is exposed (i.e., toward the cells). Here, we used gold surfaces as a control to determine the amount (in ng/cm2) of fibronectin adsorbed on an albumin film. Dolatshahi-Pirouz et al. examined fibronectin adsorption on a gold surface at a bulk concentration of 20 μg/mL, identical to our experimental conditions, using ellipsometry and a quartz crystal microbalance with dissipation (QCM-D).30 They estimated 300−400 ng/cm2 fibronectin was adsorbed on a gold surface. On the basis of this value and Figure 7, 240−320 ng/cm2 fibronectin was expected to be adsorbed on an albumin film with 2 h of UV irradiation. Approximately 350−400 ng/ cm2 fibronectin is necessary to form a monolayer on a surface based on theoretical calculations.31 Therefore, the surface of an albumin film after 2 h of UV irradiation would be almost covered by a monolayer of adsorbed fibronectin. The adsorption of cell adhesion proteins, such as fibronectin, on albumin films is highly dependent upon the surface properties of the films. Here, the surface chemical composition, wettability, and conformations of albumin molecules each changed upon UV irradiation. As indicated by the CD measurements, UV irradiation caused large conformational changes in albumin molecules (Figure 3). Such conformational changes are thought to promote protein adsorption via hydrophobic interactions and subsequent cell adhesion on the
Figure 6. Percentage of the peak area ratio of the carbon-related bond in albumin films before and after UV irradiation. UV irradiation was conducted for 2, 14, and 24 h.
increased the ratio of carbon−oxygen bonds and reduced the ratio of carbon−nitrogen bonds to total bonds, indicating the cleavage of carbon−nitrogen bonds and the generation of oxygen-containing groups. Adsorption of Fibronectin on the UV-Irradiated Albumin Films. The adsorption of cell adhesion proteins on albumin films was evaluated using peroxidase-labeled fibronectin (Figure 7). The fibronectin adsorption was suppressed on the albumin films before UV irradiation when compared to the control gold surface. UV irradiation for 2 h resulted in increased fibronectin adsorption onto the film to a degree that was comparable to that of the gold surface. However, prolonged UV irradiation (14 and 24 h) decreased the amount of adsorbed fibronectin as a function of the irradiation time. Thus, the adsorption behavior of fibronectin onto the films depended upon UV irradiation time, similar to the observations for cell adhesive behavior (Figure 1).
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DISCUSSION In the present study, we discovered the unique properties of albumin films. Specifically, cell adhesiveness after UV irradiation varied depending upon the irradiation time according to the following progression: non-adhesiveness (before UV irradiation), increase in adhesiveness (0−2 h), maximal cell adhesiveness similar to that of culture dishes (2 h), decrease in adhesiveness (2−20 h), and return to the initial state of non-adhesiveness (20−24 h). The process of cell E
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Langmuir film as a result of the exposure of the hydrophobic core embedded inside the albumin molecule. We previously reported that preferable cell adhesion occurred on a denatured albumin film prepared by cleaving intramolecular disulfide bonds in albumin followed by reforming the inter- and intramolecular disulfide bonds, after which the native albumin structures were completely disrupted.32 Furthermore, the promotion of protein adsorption on denatured albumin has been observed using radioactive-labeled albumin or fibrinogen.33,34 Several reports have also shown a strong correlation between the conformational change of albumin molecules adsorbed on a substrate and platelet adhesion.23,35 Latour et al. have proposed a new, interesting molecular mechanism, although it remains controversial; they suggested that platelets adhere directly to denatured albumin via interactions between the specific receptor on platelets and the unidentified platelet binding site on albumin, which are exposed and/or formed upon conformational changes of albumin.36 Therefore, it remains possible that cells directly attach to denatured albumin without the aid of cell adhesion proteins. UV irradiation caused prominent changes in not only the conformation but also the primary structure of albumin molecules. As seen in Figure 4, UV irradiation caused the partial cleavage of peptide bonds in albumin, which might result in conformational changes. Increased absorption intensity in the range of 260−300 nm was also observed upon UV irradiation (inset of Figure 4), suggesting that aromatic amino acids, such as tryptophan and tyrosine, are influenced by UV irradiation. Kivatinitz et al. reported that, when proteins are exposed to UV light, dimerization of tyrosine and carbonylation of tryptophan occur.37 Although they used a milk protein solution as a sample, it is possible that similar changes occurred in UV-irradiated albumin films. The hydrophilicity of albumin film surfaces increased according to UV irradiation time (Figure 5). UV irradiation, which is widely used to modify the surfaces of materials by oxygenation, is thought to cause polymer chain cleavage followed by the production of a variety of oxygen-containing groups, such as hydroxyl, carboxylic, or carbonyl groups, on the surfaces of materials exposed to air.38−41 Consistent with this proposed mechanism, an increased oxygen content and an increased ratio of carbon−oxygen bonds were observed on the surfaces of UV-irradiated albumin films (Table 1 and Figure 6). Therefore, increased hydrophilicity of albumin films after UV irradiation would be partly, if not completely, explained by the production of oxygen-containing hydrophilic functional groups at film surfaces. The change in the surface chemical composition is thought to highly affect protein adsorption on the films. The effects of surface functional groups on protein adsorption have been investigated using self-assembled monolayers of alkanethiols carrying various functional groups as a model surface.43,44 The results showed that protein adsorption tended to be suppressed on surfaces presenting hydroxyl groups but promoted on surfaces presenting carboxylic groups. It should be noted that, before UV irradiation, the albumin film had a higher water contact angle (87°) than expected (Figure 5). We previously measured the water contact angle of an albumin-adsorbed surface, and its value was found to be 75°,32 which was consistent with the result (70°) reported by Hickman et al.42 Although albumin is a water-soluble protein, the surfaces with adsorbed albumin and the albumin film did not show clear hydrophilic characteristics. Because air is hydrophobic, the hydrophobic core embedded
inside the albumin molecule might be partly exposed to the surface in the outermost layer of the adsorbed albumin and the albumin film. Also, the surface composition of nitrogen did not change with UV irradiation (Table 1), although a reduced ratio of carbon−nitrogen bonds was found in Figure 6, suggesting that nitrogen remained on the film surface after cleavage of the carbon−nitrogen bonds. Thus, UV irradiation simultaneously induced multiple changes in albumin films. Denaturation of the albumin structure and carboxylic group production promote protein adsorption and subsequent cell adhesion, while hydroxyl group production has the opposite effects. It is possible that the positive UV effects for protein adsorption and cell adhesion are stronger than the negative effects within 2 h of UV irradiation and vice versa for greater than 2 h of UV irradiation. To fully elucidate the mechanism of UV-induced alteration of the albumin film with respect to cell adhesive properties, further investigations regarding UV-induced changes in the stiffness and mobility of peptide chains, which are closely related to cell adhesive behavior,45,46 are required. Furthermore, it is important to examine the cell adhesive behaviors on UVirradiated albumin films using various types of cells to gain insight into the interaction between cells and albumin films, although we selected the widely used cell lines L9292 fibroblasts and HeLa cancer cells in this study.
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CONCLUSION
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ASSOCIATED CONTENT
We demonstrated that the cell adhesive properties of albumin films were altered in diverse ways upon UV irradiation in a time-dependent manner. UV irradiation induced various changes in albumin films, such as a decreased α-helical structure, cleavage of peptide bonds, increased hydrophilicity of film surfaces, and increased oxygen content, which are expected to greatly influence the adsorption behavior of cell adhesion proteins on films. In addition, a positive correlation was observed between the degree of cell adhesion and amount of fibronectin adsorbed on the films, indicating that cell adhesion occurs via the adsorbed cell adhesion proteins. Taken together, we expect that UV-induced changes in films affect the amount of cell adhesion proteins adsorbed on the films depending upon irradiation time. Cells then adhere to the films via adsorbed proteins according to their amount. Although further investigations are required to fully understand the molecular mechanism for UV-induced alterations of albumin films toward cell adhesiveness, the unique features of albumin films could be useful for developing novel biomaterials, such as materials with desired cell adhesive properties and intricately patterned substrates that contain multiple regions with different degrees of cell adhesiveness.
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b03958. Phase-contrast microscopic observations of HeLa cells 5 h after inoculation on UV-irradiated albumin films (Figure S1), Coomassie Brilliant Blue staining of albumin films before and after UV irradiation (Figure S2), and representative C 1s spectrum and curve fit for albumin films (Figure S3) (PDF) F
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(18) Yamazoe, H.; Tanabe, T. Preparation of Water-insoluble Albumin Film Possessing Nonadherent Surface for Cells and Ligand Binding Ability. J. Biomed. Mater. Res., Part A 2008, 86A, 228−234. (19) Yamazoe, H.; Uemura, T.; Tanabe, T. Facile Cell Patterning on an Albumin-coated Surface. Langmuir 2008, 24, 8402−8404. (20) Nakanishi, J.; Kikuchi, Y.; Takarada, T.; Nakayama, H.; Yamaguchi, K.; Maeda, M. Photoactivation of a Substrate for Cell Adhesion under Standard Fluorescence Microscopes. J. Am. Chem. Soc. 2004, 126, 16314−16315. (21) Edahiro, J.; Sumaru, K.; Tada, Y.; Ohi, K.; Takagi, T.; Kameda, M.; Shinbo, T.; Kanamori, T.; Yoshimi, Y. In situ Control of Cell Adhesion Using Photoresponsive Culture Surface. Biomacromolecules 2005, 6, 970−974. (22) Goubko, C. A.; Basak, A.; Majumdar, S.; Cao, X. Dynamic Cell Patterning of Photoresponsive Hyaluronic Acid Hydrogels. J. Biomed. Mater. Res., Part A 2014, 102, 381−391. (23) Tanaka, M.; Motomura, T.; Kawada, M.; Anzai, T.; Kasori, Y.; Shiroya, T.; Shimura, K.; Onishi, M.; Mochizuki, A. Blood Compatible Aspects of Poly(2-methoxyethylacrylate) (PMEA)-relationship between Protein Adsorption and Platelet Adhesion on PMEA Surface. Biomaterials 2000, 21, 1471−1481. (24) Barreca, D.; Laganà, G.; Ficarra, S.; Tellone, E.; Leuzzi, U.; Magazù, S.; Galtieri, A.; Bellocco, E. Anti-aggregation Properties of Trehalose on Heat-induced Secondary Structure and Conformation Changes of Bovine Serum Albumin. Biophys. Chem. 2010, 147, 146− 152. (25) Beaven, G. H.; Holiday, E. R. Ultraviolet Absorption Spectra of Proteins and Amino Acids. Adv. Protein Chem. 1952, 7, 319−386. (26) Wetlaufer, D. B. Ultraviolet Spectra of Proteins and Amino Acids. Adv. Protein Chem. 1963, 17, 303−390. (27) Bomben, K. D.; Dev, S. B. Investigation of Poly(L-amino acids) by X-ray Photoelectron Spectroscopy. Anal. Chem. 1988, 60, 1393− 1397. (28) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber, D. E.; Whitesides, G. M. Using Mixed Self-assembled Monolayers Presenting RGD and (EG)3OH Groups to Characterize Long-term Attachment of Bovine Capillary Endothelial Cells to Surfaces. J. Am. Chem. Soc. 1998, 120, 6548−6555. (29) Iuliano, D. J.; Saavedra, S. S.; Truskey, G. A. Effect of the Conformation and Orientation of Adsorbed Fibronectin on Endothelial Cell Spreading and the Strength of Adhesion. J. Biomed. Mater. Res. 1993, 27, 1103−1113. (30) Dolatshahi-Pirouz, A.; Jensen, T.; Foss, M.; Chevallier, J.; Besenbacher, F. Enhanced Surface Activation of Fibronectin upon Adsorption on Hydroxyapatite. Langmuir 2009, 25, 2971−2978. (31) García, A. J.; Vega, M. D.; Boettiger, D. Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation. Mol. Biol. Cell 1999, 10, 785−798. (32) Yamazoe, H.; Yamauchi, K.; Tanabe, T. Preparation of S-sulfo Albumin Film and Its Cell Adhesive Property. Mater. Sci. Eng., C 2009, 29, 1105−1108. (33) Holmberg, M.; Hou, X. Fibrinogen Adsorption on Blocked Surface of Albumin. Colloids Surf., B 2011, 84, 71−75. (34) Holmberg, M.; Hou, X. Competitive Protein Adsorptionmultilayer Adsorption and Surface Induced Protein Aggregation. Langmuir 2009, 25, 2081−2089. (35) Hylton, D. M.; Shalaby, S. W.; Latour, R. A., Jr. Direct Correlation between Adsorption-induced Changes in Protein Structure and Platelet Adhesion. J. Biomed. Mater. Res., Part A 2005, 73A, 349−358. (36) Sivaraman, B.; Latour, R. A. The Adherence of Platelets to Adsorbed Albumin by Receptor-mediated Recognition of Binding Sites Exposed by Adsorption-induced Unfolding. Biomaterials 2010, 31, 1036−1044. (37) Scheidegger, D.; Pecora, R. P.; Radici, P. M.; Kivatinitz, S. C. Protein Oxidative Changes in Whole and Skim Milk after Ultraviolet or Fluorescent Light Exposure. J. Dairy Sci. 2010, 93, 5101−5109.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +81-29-861-6264. Fax: +81-72-751-9517. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Junko Neki of the National Institute of Advanced Industrial Science and Technology (AIST) for her assistance. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant-in-Aid for Young Scientists (B), 15K16334].
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