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Simple and non-toxic fabrication of poly(vinyl alcohol)-patterned polymer surface for the formation of cell patterns
Applied Surface Science 316 (2014) 179–186
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Simple and non-toxic fabrication of poly(vinyl alcohol)-patterned polymer surface for the formation of cell patterns In-Tae Hwang a , Yu-Ran Jin a , Min-Suk Oh b , Chan-Hee Jung a,∗ , Jae-Hak Choi c,∗ a Radiation Research Division for Industry and Environment, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si, Jeollabuk-do 580-185, Republic of Korea b POSCO Technical Research Laboratories, 699 Gumho-dong, Gwangyang, Jeonnam 545-090, Republic of Korea c Department of Polymer Science and Engineering, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea
a r t i c l e
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Article history: Received 5 March 2014 Received in revised form 22 July 2014 Accepted 28 July 2014 Available online 3 August 2014 Keywords: Poly(vinyl alcohol) Ion irradiation Cell pattern
a b s t r a c t In this study, a facile and non-toxic method for the formation of cell-adhesive poly(vinyl alcohol) (PVA) patterns on the surface of a non-biological polystyrene substrate (NPS) is developed to control cellular micro-organization. PVA thin films spin-coated onto the NPS are selectively irradiated with 150 keV H+ ions through a pattern mask and developed with deionized water to form negative-type PVA patterns. Well-defined stripe patterns of PVA with a width of 100 m are created on the NPS at a higher fluence than 5 × 1015 ions/cm2 , and their surface chemical compositions are changed by ion irradiation without any significant morphological change. Based on the results of the protein adsorption test and in vitro cell culture, cancer cells are preferentially adhered and proliferated onto the more hydrophilic PVA regions of the PVA-patterned NPS, resulting in well-defined cell patterns. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cell patterning on an artificial substrate has received a great amount of attention as an essential prerequisite for a variety of biological applications such as the fundamental study of cell biology, tissue engineering, cell-based bioassays, and cell-based drug screening [1–7]. To facilitate the formation of cell patterns, a variety of surface patterning techniques, including inkjet printing, photolithography, and micro-contact printing, has been extensively explored [8–10]. However, although they provide resolved patterns on the surface of a substrate, they have drawbacks, such as multiple steps and non-biocompatible processes with a necessity of toxic chemicals to form the patterned surfaces [11–13]. Therefore, a simpler and more biocompatible surface patterning method is required to prepare patterned surfaces for cell patterning. An ion beam-based patterning technique is a powerful surface patterning method for the formation of cell patterns. It offers several advantages including convenient and precise controllability, reliability, temperature-independent processing, and non-toxic processing without the use of harsh chemicals owing to the greater liner energy transfer (LET) and straighter penetration trajectory of
∗ Corresponding authors. Tel.: +82 42 821 6664; fax: +82 42 821 8910. E-mail addresses: [email protected] (C.-H. Jung), [email protected] (J.-H. Choi). http://dx.doi.org/10.1016/j.apsusc.2014.07.163 0169-4332/© 2014 Elsevier B.V. All rights reserved.
the ion beams in comparison to other techniques based on electron beams, UV light, ␥-rays, and X-rays [14–18]. Thus, microstructures formed by ion irradiation have been widely used to spatially control the adhesion and proliferation of cells [19–21]. Poly(vinyl alcohol) (PVA) has been used in the biomedical field because of its water solubility, biocompatibility, optical transparency, and good capability of thin film formation [22–29]. Despite these benefits, it has not been extensively used as a cell guiding material for patterned cell culture because the patterns of PVA are difficult to form by conventional photolithography without biologically-undesirable chemicals, such as photoacid generators and a developer [30–32]. Thus, the fabrication of PVA-patterned platforms for the formation of cell patterns by an eco-friendly and biocompatible ion beam-based technique without any toxic chemicals has not been previously studied. To the best of our knowledge, this is the first report on the formation of PVA patterns on a polymer substrate using a simple and biocompatible ion beam-based technique. In this study, ion beam-based patterning of cell-adhesive PVA on a non-biological surface was carried out to control cellular behaviors. This technique offers several advantages including easy and precise controllability, temperature-independence, reliability, and non-toxicity without the need of any harsh chemicals. The ion beam-based patterning of PVA on a non-biological surface was investigated under various conditions to form negative-type PVA
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patterns. The surface properties of the resulting PVA patterns were investigated in terms of the thickness, wettability, chemical composition and structure. Furthermore, selective cell adhesion on a PVA-patterned non-biological surface was investigated by means of an in-vitro cell culture test.
ThermoElectron Co., England) employing MgK␣ radiation. The applied power was 14.5 keV and 20 mA, and the base pressure in the analysis chamber was less than 10−9 mbar.
2. Experimental
The stability test of the PVA-patterned NPS substrates was performed by measuring the thickness of PVA patterns before and after incubation in phosphate buffered saline (PBS, pH 7.4, Life Technologies) solutions. The PVA-patterned NPS substrates were immersed in the PBS solutions, and subsequently incubated at 37 ◦ C and 5% CO2 in a humidified incubator. After incubation for 15 days, the thicknesses of the PVA patterns were measured by a 3D optical surface profiler.
2.1. Materials PVA (weight average molecular weight: 89,000–98,000, degree of hydrolysis: >99%) was purchased from Aldrich Chemical Company. Non-biological polystyrene (NPS) petri dishes supplied from SPL Life Science Company were used as a substrate without any further purification. To form the PVA patterns, a customized metal mask (100 m spaces and 300 m pitches) was obtained from Youngjin Astech Co., Ltd. 2.2. Patterning of PVA on NPS substrates Thin PVA films on NPS substrates were formed by spin-coating a 7 wt% PVA solution in distilled water and drying in a vacuum oven for 24 h. The PVA films formed on the NPS were selectively irradiated with 200 keV H+ ions at fluences ranging from 3 × 1015 to 9 × 1015 ions/cm2 through a pattern mask at room temperature. Ion irradiation was carried out by using a 300-keV ion implanter at the Advanced Radiation Technology Institute (ARTI, Republic of Korea) [33]. The ion current density was kept at approximately 1.0 A/cm2 to prevent the thermal effect. The working pressure of the implanter’s target was kept under 10−5 –10−6 Torr. Afterwards, to generate the PVA patterns, the resulting substrates were developed with a deionized hot water (70 ◦ C) and then dried in an N2 stream. 2.3. Surface characterization of PVA-patterned NPS substrates The surface morphology and profiles of PVA-patterned NPS substrates were analyzed using an optical microscope (Type 020-519, Leica, Germany) and a 3D optical surface profiler (NanoSystem, Korea), and an atomic force microscope (AFM, XE-100, Park system, Korea), respectively. The contact angles of the non-irradiated and irradiated PVA were measured using a contact angle analyzer (Phoenix 300, Surface Electro Optical Company, Korea). A deionized water droplet (4 l) was dropped carefully onto the surface at room temperature. The average contact angle was obtained by five measurements. The chemical structure of the non-irradiated and irradiated PVA was investigated using an attenuated total reflectance Fourier transform infrared spectrometer (ATR-FTIR, Tensor 37, Bruker Co., USA). The change in the chemical composition of PVA before and after ion irradiation was analyzed using an X-ray photoelectron spectrometer (XPS, MultiLab 2000,
2.4. Pattern stability test
2.5. Protein adsorption test The protein adsorption test was performed with FITC-labeled bovine serum albumin proteins (BSA-FITC, Sigma Aldrich) reported in the literature [34]. A 200 l of BSA-FITC in a PBS solution with a concentration of 1 mg/ml was fully covered on the PVA-patterned NPS substrates, and successively incubated at 37 ◦ C and 5% CO2 in a humidified incubator for 1 h. After washing with distilled water several times, the adsorption of BSA-FITC on the PVA-patterned NPS substrates was observed with a fluorescence microscope (DMI4000 B, LEICA). The representative plot profiles of the adsorbed BSAFITC were drawn with the ImageJ software from their fluorescence images. 2.6. In vitro cell culture Pre-confluent H1299 (human lung carcinoma cell), HeLa (human cervical cancer cell), and NIH3T3 (mouse fibroblast cell) cells were detached by trypsin-EDTA and then pipetted several times to disperse them into single cells. Prior to the cell culture, PVA-patterned NPS dishes were sterilized with 70% ethanol. Cells with a density of 2.5 × 104 cells/ml were seeded on PVA-patterned NPS substrates and kept in a RPMI 1640 medium (Gibco) for H1299 and in a Dulbecco’s modified eagle medium (DMEM, Gibco) for HeLa and NIH3T3 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ◦ C and 5% CO2 in a humidified incubator. After 72 h, the adhesion and growth behavior of the cells were observed with an optical microscope (Type 020-519, Leica, Germany). 2.7. Cell proliferation assay and viability test Cell proliferation was measured with a CCK-8 assay kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol [35]. Briefly, H1299 cells with a density of 1 × 104 cells/ml were seeded onto the normal polystyrene (NPS) and thin
Fig. 1. Schematic illustration of ion beam-based patterning of PVA on a non-biological substrate to control cellular micro-organization.
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Fig. 2. Optical microscopic images of PVA patterns on NPS substrates formed at fluences of 5 × 1015 (a), 7 × 1015 (b), and 9 × 1015 ions/cm2 (c): The insets in (b-c) magnify the dotted rectangle in each respective figure.
PVA-coated NPS dishes irradiated at various fluences ranging from 5 × 1015 to 9 × 1015 ions/cm2 . After incubation for various durations of 1, 2, 3, and 4 days, the media were removed and 1 ml of culture medium containing a 10% CCK-8 solution was then added to each sample. After incubation for 1 h, the optical density (OD) of the resulting solution was measured at 450 nm using a UV-Vis spectrophotometer (MQX 200 model, Bio-Tek Instruments, USA).
The OD value of each sample represents the relative proliferation rate of H1299 cells. All experiments were conducted in triplicate. In addition, to evaluate the cell viability on the PVA-patterned NPS substrates, H1299 cells with a density of 1 × 104 cells/ml were seeded onto the tissue culture polystyrene (TCPS) and PVApatterned NPS dishes irradiated at various fluences ranging from 5 × 1015 to 9 × 1015 ions/cm2 . After cultivation for 24 h, a CCK-8
Fig. 3. 3D optical surface profiles of PVA-patterned NPS substrates formed at fluences of 5 × 1015 (a), 7 × 1015 (b), and 9 × 1015 ions/cm2 (c).
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Fig. 4. The water contact angles of the NPS (a), non-irradiated PVA (b), and irradiated PVA films at fluences of 5 × 1015 , 7 × 1015 , and 9 × 1015 ions/cm2 .
assay was performed under the same conditions as the proliferation assay. To estimate the cell viability, the relative percentage of each substrate compared to TCPS was calculated from their OD values. 3. Results and discussion 3.1. Formation of well-defined PVA patterns on NPS substrates The ion beam-based pattering of cell-adhesive PVA thin films on NPS substrates to manipulate cell adhesion and proliferation is illustrated in Fig. 1. PVA thin films on NPS substrates formed by spin-coating of an aqueous PVA solution without any additives such as crosslinkers and initiators were selectively irradiated by accelerated H+ ions through a pattern mask and then developed with deionized water to form negative-type PVA patterns. Finally, the resulting PVA-patterned NPS substrates were further utilized for the spatial control of cellular micro-organization. The optical microscopic images of the formed PVA patterns on the NPS substrates by selective ion irradiation through a pattern mask (100 m space and 300 m pitch) followed by development with deionized water are shown in Fig. 2. As shown in Fig. 2a–c,
Fig. 5. ATR-FTIR spectra of the NPS (a), non-irradiated PVA (b), and irradiated PVA films at fluences of 5 × 1015 (c), 7 × 1015 (d), and 9 × 1015 ions/cm2 (e).
well-defined 100 m negative-type PVA patterns were formed at the given fluences. However, PVA patterns were not generated at less than 5 × 1015 ions/cm2 (data not shown). Thus, ion irradiation above a fluence of 5 × 1015 ions/cm2 is necessary to form negative-type PVA patterns. This ion irradiation-induced formation of negative-type PVA patterns without any additives can be explained as follows. Unlike techniques based on electron beams, UV light, ␥-rays, or X-rays, the larger amount of radicals in solidstate PVA films can be generated by ion irradiation with high LET, thus leading to an increase in the probability of interpolymer radical coupling reactions. Thus, ion irradiation above a certain fluence can result in a crosslinked PVA in a solid state [36,37]. The surface profiles of PVA-patterned NPS substrates prepared at various fluences are presented in Fig. 3. The remaining thickness of the PVA patterns slightly decreased from 385 nm to 351 nm with an increase in the fluence. This reduction in the thickness of the PVA patterns can be attributed to the fact that the formation of volatile species by ion irradiation-induced breaking of covalent bonds in PVA is accelerated with an increase in the fluence, resulting in a reduced thickness [38,39]. Furthermore, as shown in the AFM images related to elucidating the ion beam-induced morphological
Fig. 6. The C1s spectra for non-irradiated (a) and irradiated PVA films at fluences of 5 × 1015 (b), 7 × 1015 (c), and 9 × 1015 ions/cm2 (d).
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change in the surface of PVA (see Fig. S1 of the supplementary information), the non-irradiated PVA films were smoother than the NPS surface. On the other hand, all the patterns PVA formed at the given fluences exhibited almost similar morphologies to that of the non-irradiated PVA and the RMS values of their surface roughness ranging from 0.80 to 1.10 was not much different from that of the non-irradiated one. Therefore, ion irradiation seemed to cause no significant topographical change in the PVA surfaces. To investigate the ion irradiation-induced changes in the wettability of PVA, water contact angle measurements were performed and the results are shown in Fig. 4. The averaged contact angle of the untreated PVA surface was around 28◦ , indicating a highly hydrophilic surface in comparison to the NPS with an average contact angle of about 83◦ . On the other hand, in the cases of irradiated PVA, the average contact angles increased up to 64◦ with an increase in the fluence, which is much lower than that of the NPS. This result indicates that, although its surface was more hydrophobic than that of the non-irradiated PVA, the irradiated surfaces have a more hydrophilic surface, which is more favorable for cell adhesion and proliferation than that of the NPS. This change in wettability
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Fig. 7. [O]/[C] atomic ratio of non-irradiated and irradiated PVA films at various fluences.
can be attributed to the change in the surface chemical structure and composition of the PVA caused by ion irradiation [40]. The changes in the chemical structure of PVA films brought about by ion irradiation were analyzed by ATR-FTIR spectroscopy,
Fig. 8. Fluorescence micrographs of the serum proteins adsorbed on the PVA-patterned NPS substrates prepared at fluences of 5 × 1015 (a), 7 × 1015 (b), and 9 × 1015 ions/cm2 (c).
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Fig. 9. Optical microscopic images of HeLa (a-c), H1299 (d-f), and NIH3T3 (g-i) cells on the PVA-patterned NPS substrates prepared at various fluences.
and the results are shown in Fig. 5. In the non-irradiated PVA spectrum in Fig. 5b, the characteristic bands corresponding to the chemical structure of the PVA backbone and unhydrolyzed pendant acetate group were identified at 3331 (O H ), 2935 (C H ), 1736 (C O ), 1442 (ıC H ), and 1091 cm−1 (ıO H ) [41]. On the other hand, in the irradiated PVA spectra in Fig. 5c–e, new bands assigned to C C and C O C appeared at 1630 (C C ) and 1166 cm−1 (C O C ), and their intensities gradually increased with an increase in the fluence. This change in chemical structure may result from the ion irradiation-induced crosslinking and degradation of the PVA [42]. To further investigate the changes in the chemical structure and composition on the PVA surface after ion irradiation, an XPS analysis was carried out and the results are presented in Fig. 6. The spectrum of the non-irradiated PVA presented in Fig. 6a exhibited characteristic peaks at 285.0 (C C), 286.5 (C OH), and 289.1 eV (C O) O of the unhydrolyzed pendant acetate group. In the irradiated PVA, new peaks corresponding to the C O C and C O groups were generated at 285.8 and 289.2 eV, respectively. Moreover, as shown in Fig. 7, the [O]/[C] elemental ratio of the irradiated PVA gradually decreased to 0.25 with an increase in the fluence in comparison to that of the non-irradiated PVA. This change in the surface chemical structure and composition can be attributed to the crosslinking and degradation of PVA caused by ion irradiation [43]. 3.2. Pattern stability The stability of the PVA-patterned NPS substrates affecting the cell behavior was estimated by measuring the change in the thickness of PVA patterns before and after incubation in PBS solutions for 15 days under the same conditions as the cell culture. As shown in Fig. S2 (see the supplementary information), all the PVA patterns formed at the given fluences exhibited almost identical thickness before and after incubation for 15 days. This result can be ascribed to the fact that PVA was densely crosslinked by ion irradiation at the
given fluences, thereby resulting in the formation of robust patterns without swelling. 3.3. Protein adsorption on PVA-patterned NPS substrates To investigate the feasibility in the application of the PVApatterned NPS substrate to the control of cell adhesion, the protein adsorption on the PVA-patterned surfaces prerequisite for cell adhesion was estimated by a fluorescent microscopic analysis with FITC-labeled BSA. As shown in Fig. 8, the BSA was selectively adsorbed only onto the PVA regions of all the PVA-patterned NPS surfaces formed at the given fluences, resulting in well-organized BSA patterns. However, their corresponding fluorescence intensities were gradually reduced with an increase in the fluence. The phenomenon could be explained by the fact that BSA preferentially bound to the oxygen-containing functionalities on the surfaces of the PVA patterns, and thus the amount of the BSA adsorption was dependent on the numbers of the functionalities that were affected by the fluence [44]. 3.4. In-vitro cell culture on PVA-patterned NPS substrates To demonstrate the possibility for the control of cellular microorganization, three types of cell lines including H1299, HeLa, and NIH3T3 were in-vitro cultured on the PVA-patterned NPS substrates. As shown in Fig. 9, at all given fluences, all types of cells were preferentially adhered and proliferated onto the PVA regions, resulting in well-organized 100 m patterns of the cells on the PVApatterned NPS substrates. This cellular micro-organization on the PVA-patterned NPS surface seemed to result from the preferential protein adsorption on the regions of the PVA patterns rather than those of the NPS. To further examine cell response to the wholly-irradiated PVA surfaces, a cell proliferation assay using CCK-8 was performed on bare NPS and PVA-coated NPS substrates irradiated at various fluences. Fig. 10a shows the proliferation of the H1299 cells cultured
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Fig. 10. Cell proliferation of the H1299 cells cultured on the PVA film irradiated at various fluences (a), and cell viability on the TCPS and PVA-patterned NPS substrates prepared at various fluences (b).
on the irradiated PVA film at various fluences. The proliferation rates of the cells on the irradiated PVA were much higher than that on bare NPS although those was slightly lower in comparison to that on the non-irradiated PVA. This result implies that the PVA surfaces irradiated at various fluences provide a more cell-friendly environment than the NPS surface, which is in good agreement with the above-mentioned protein adsorption result. Moreover, as shown in Fig. 10b, the result of the cell viability test revealed that all PVApatterned NPS substrates at the given fluences with a viability of more than 80% had cytocompatibility, although their viability was slightly lower in comparison to that of the control TCPS [45]. These results indicate that the preferential adhesion of the cells on the surface of the PVA patterns was originated from their recognition of the pre-adsorbed serum proteins on the patterns and that the PVA-patterned substrates were cytocompatible. 4. Conclusion The formation of PVA patterns on the surface of NPS substrates was successfully performed by selective ion irradiation in the absence of any toxic chemicals to control cellular microorganization. The results of the surface profile and AFM analyses revealed that 100 m negative-type PVA patterns were successfully generated on the NPS substrate at a fluence above 5 × 1015 ions/cm2 and their surface morphologies was not significantly changed. Based on the contact angle measurement, ATR-FTIR, and XPS results, the irradiated PVA surfaces were more hydrophilic than the bare NPS surface even though its surface became more hydrophobic than the non-irradiated PVA surface owing to the ion irradiation-induced change in the surface chemical environment. It is clearly shown from the stability test that all the PVA patterns on the NPS at the given fluences exhibited the robustness without dimensional change in the environment of cell culture. Furthermore, the results of the protein adsorption and in vitro cell culture test, clearly showed that two types of human cancer cells were preferentially adhered and proliferated only onto the more hydrophilic PVA regions of the PVA-patterned NPS substrates prepared at all given fluences and therefore resulting in the formation of welldefined 100 m cell patterns due to the preferential adsorption of serum proteins on the more hydrophilic PVA regions of the PVApatterned NPS. These resulting PVA-patterned substrates using a convenient and non-toxic ion beam-based technique are applicable to the study of cell biology and the development of cell-based biosensors and drug discovery. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2013M2A26023510).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsusc. 2014.07.163.
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Simple and non-toxic fabrication of poly(vinyl alcohol)-patterned polymer surface for the formation of cell patterns In-Tae Hwang a , Yu-Ran Jin a , Min-Suk Oh b , Chan-Hee Jung a,∗ , Jae-Hak Choi c,∗ a Radiation Research Division for Industry and Environment, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup-si, Jeollabuk-do 580-185, Republic of Korea b POSCO Technical Research Laboratories, 699 Gumho-dong, Gwangyang, Jeonnam 545-090, Republic of Korea c Department of Polymer Science and Engineering, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea
a r t i c l e
i n f o
Article history: Received 5 March 2014 Received in revised form 22 July 2014 Accepted 28 July 2014 Available online 3 August 2014 Keywords: Poly(vinyl alcohol) Ion irradiation Cell pattern
a b s t r a c t In this study, a facile and non-toxic method for the formation of cell-adhesive poly(vinyl alcohol) (PVA) patterns on the surface of a non-biological polystyrene substrate (NPS) is developed to control cellular micro-organization. PVA thin films spin-coated onto the NPS are selectively irradiated with 150 keV H+ ions through a pattern mask and developed with deionized water to form negative-type PVA patterns. Well-defined stripe patterns of PVA with a width of 100 m are created on the NPS at a higher fluence than 5 × 1015 ions/cm2 , and their surface chemical compositions are changed by ion irradiation without any significant morphological change. Based on the results of the protein adsorption test and in vitro cell culture, cancer cells are preferentially adhered and proliferated onto the more hydrophilic PVA regions of the PVA-patterned NPS, resulting in well-defined cell patterns. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cell patterning on an artificial substrate has received a great amount of attention as an essential prerequisite for a variety of biological applications such as the fundamental study of cell biology, tissue engineering, cell-based bioassays, and cell-based drug screening [1–7]. To facilitate the formation of cell patterns, a variety of surface patterning techniques, including inkjet printing, photolithography, and micro-contact printing, has been extensively explored [8–10]. However, although they provide resolved patterns on the surface of a substrate, they have drawbacks, such as multiple steps and non-biocompatible processes with a necessity of toxic chemicals to form the patterned surfaces [11–13]. Therefore, a simpler and more biocompatible surface patterning method is required to prepare patterned surfaces for cell patterning. An ion beam-based patterning technique is a powerful surface patterning method for the formation of cell patterns. It offers several advantages including convenient and precise controllability, reliability, temperature-independent processing, and non-toxic processing without the use of harsh chemicals owing to the greater liner energy transfer (LET) and straighter penetration trajectory of
∗ Corresponding authors. Tel.: +82 42 821 6664; fax: +82 42 821 8910. E-mail addresses: [email protected] (C.-H. Jung), [email protected] (J.-H. Choi). http://dx.doi.org/10.1016/j.apsusc.2014.07.163 0169-4332/© 2014 Elsevier B.V. All rights reserved.
the ion beams in comparison to other techniques based on electron beams, UV light, ␥-rays, and X-rays [14–18]. Thus, microstructures formed by ion irradiation have been widely used to spatially control the adhesion and proliferation of cells [19–21]. Poly(vinyl alcohol) (PVA) has been used in the biomedical field because of its water solubility, biocompatibility, optical transparency, and good capability of thin film formation [22–29]. Despite these benefits, it has not been extensively used as a cell guiding material for patterned cell culture because the patterns of PVA are difficult to form by conventional photolithography without biologically-undesirable chemicals, such as photoacid generators and a developer [30–32]. Thus, the fabrication of PVA-patterned platforms for the formation of cell patterns by an eco-friendly and biocompatible ion beam-based technique without any toxic chemicals has not been previously studied. To the best of our knowledge, this is the first report on the formation of PVA patterns on a polymer substrate using a simple and biocompatible ion beam-based technique. In this study, ion beam-based patterning of cell-adhesive PVA on a non-biological surface was carried out to control cellular behaviors. This technique offers several advantages including easy and precise controllability, temperature-independence, reliability, and non-toxicity without the need of any harsh chemicals. The ion beam-based patterning of PVA on a non-biological surface was investigated under various conditions to form negative-type PVA
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patterns. The surface properties of the resulting PVA patterns were investigated in terms of the thickness, wettability, chemical composition and structure. Furthermore, selective cell adhesion on a PVA-patterned non-biological surface was investigated by means of an in-vitro cell culture test.
ThermoElectron Co., England) employing MgK␣ radiation. The applied power was 14.5 keV and 20 mA, and the base pressure in the analysis chamber was less than 10−9 mbar.
2. Experimental
The stability test of the PVA-patterned NPS substrates was performed by measuring the thickness of PVA patterns before and after incubation in phosphate buffered saline (PBS, pH 7.4, Life Technologies) solutions. The PVA-patterned NPS substrates were immersed in the PBS solutions, and subsequently incubated at 37 ◦ C and 5% CO2 in a humidified incubator. After incubation for 15 days, the thicknesses of the PVA patterns were measured by a 3D optical surface profiler.
2.1. Materials PVA (weight average molecular weight: 89,000–98,000, degree of hydrolysis: >99%) was purchased from Aldrich Chemical Company. Non-biological polystyrene (NPS) petri dishes supplied from SPL Life Science Company were used as a substrate without any further purification. To form the PVA patterns, a customized metal mask (100 m spaces and 300 m pitches) was obtained from Youngjin Astech Co., Ltd. 2.2. Patterning of PVA on NPS substrates Thin PVA films on NPS substrates were formed by spin-coating a 7 wt% PVA solution in distilled water and drying in a vacuum oven for 24 h. The PVA films formed on the NPS were selectively irradiated with 200 keV H+ ions at fluences ranging from 3 × 1015 to 9 × 1015 ions/cm2 through a pattern mask at room temperature. Ion irradiation was carried out by using a 300-keV ion implanter at the Advanced Radiation Technology Institute (ARTI, Republic of Korea) [33]. The ion current density was kept at approximately 1.0 A/cm2 to prevent the thermal effect. The working pressure of the implanter’s target was kept under 10−5 –10−6 Torr. Afterwards, to generate the PVA patterns, the resulting substrates were developed with a deionized hot water (70 ◦ C) and then dried in an N2 stream. 2.3. Surface characterization of PVA-patterned NPS substrates The surface morphology and profiles of PVA-patterned NPS substrates were analyzed using an optical microscope (Type 020-519, Leica, Germany) and a 3D optical surface profiler (NanoSystem, Korea), and an atomic force microscope (AFM, XE-100, Park system, Korea), respectively. The contact angles of the non-irradiated and irradiated PVA were measured using a contact angle analyzer (Phoenix 300, Surface Electro Optical Company, Korea). A deionized water droplet (4 l) was dropped carefully onto the surface at room temperature. The average contact angle was obtained by five measurements. The chemical structure of the non-irradiated and irradiated PVA was investigated using an attenuated total reflectance Fourier transform infrared spectrometer (ATR-FTIR, Tensor 37, Bruker Co., USA). The change in the chemical composition of PVA before and after ion irradiation was analyzed using an X-ray photoelectron spectrometer (XPS, MultiLab 2000,
2.4. Pattern stability test
2.5. Protein adsorption test The protein adsorption test was performed with FITC-labeled bovine serum albumin proteins (BSA-FITC, Sigma Aldrich) reported in the literature [34]. A 200 l of BSA-FITC in a PBS solution with a concentration of 1 mg/ml was fully covered on the PVA-patterned NPS substrates, and successively incubated at 37 ◦ C and 5% CO2 in a humidified incubator for 1 h. After washing with distilled water several times, the adsorption of BSA-FITC on the PVA-patterned NPS substrates was observed with a fluorescence microscope (DMI4000 B, LEICA). The representative plot profiles of the adsorbed BSAFITC were drawn with the ImageJ software from their fluorescence images. 2.6. In vitro cell culture Pre-confluent H1299 (human lung carcinoma cell), HeLa (human cervical cancer cell), and NIH3T3 (mouse fibroblast cell) cells were detached by trypsin-EDTA and then pipetted several times to disperse them into single cells. Prior to the cell culture, PVA-patterned NPS dishes were sterilized with 70% ethanol. Cells with a density of 2.5 × 104 cells/ml were seeded on PVA-patterned NPS substrates and kept in a RPMI 1640 medium (Gibco) for H1299 and in a Dulbecco’s modified eagle medium (DMEM, Gibco) for HeLa and NIH3T3 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ◦ C and 5% CO2 in a humidified incubator. After 72 h, the adhesion and growth behavior of the cells were observed with an optical microscope (Type 020-519, Leica, Germany). 2.7. Cell proliferation assay and viability test Cell proliferation was measured with a CCK-8 assay kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol [35]. Briefly, H1299 cells with a density of 1 × 104 cells/ml were seeded onto the normal polystyrene (NPS) and thin
Fig. 1. Schematic illustration of ion beam-based patterning of PVA on a non-biological substrate to control cellular micro-organization.
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Fig. 2. Optical microscopic images of PVA patterns on NPS substrates formed at fluences of 5 × 1015 (a), 7 × 1015 (b), and 9 × 1015 ions/cm2 (c): The insets in (b-c) magnify the dotted rectangle in each respective figure.
PVA-coated NPS dishes irradiated at various fluences ranging from 5 × 1015 to 9 × 1015 ions/cm2 . After incubation for various durations of 1, 2, 3, and 4 days, the media were removed and 1 ml of culture medium containing a 10% CCK-8 solution was then added to each sample. After incubation for 1 h, the optical density (OD) of the resulting solution was measured at 450 nm using a UV-Vis spectrophotometer (MQX 200 model, Bio-Tek Instruments, USA).
The OD value of each sample represents the relative proliferation rate of H1299 cells. All experiments were conducted in triplicate. In addition, to evaluate the cell viability on the PVA-patterned NPS substrates, H1299 cells with a density of 1 × 104 cells/ml were seeded onto the tissue culture polystyrene (TCPS) and PVApatterned NPS dishes irradiated at various fluences ranging from 5 × 1015 to 9 × 1015 ions/cm2 . After cultivation for 24 h, a CCK-8
Fig. 3. 3D optical surface profiles of PVA-patterned NPS substrates formed at fluences of 5 × 1015 (a), 7 × 1015 (b), and 9 × 1015 ions/cm2 (c).
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Fig. 4. The water contact angles of the NPS (a), non-irradiated PVA (b), and irradiated PVA films at fluences of 5 × 1015 , 7 × 1015 , and 9 × 1015 ions/cm2 .
assay was performed under the same conditions as the proliferation assay. To estimate the cell viability, the relative percentage of each substrate compared to TCPS was calculated from their OD values. 3. Results and discussion 3.1. Formation of well-defined PVA patterns on NPS substrates The ion beam-based pattering of cell-adhesive PVA thin films on NPS substrates to manipulate cell adhesion and proliferation is illustrated in Fig. 1. PVA thin films on NPS substrates formed by spin-coating of an aqueous PVA solution without any additives such as crosslinkers and initiators were selectively irradiated by accelerated H+ ions through a pattern mask and then developed with deionized water to form negative-type PVA patterns. Finally, the resulting PVA-patterned NPS substrates were further utilized for the spatial control of cellular micro-organization. The optical microscopic images of the formed PVA patterns on the NPS substrates by selective ion irradiation through a pattern mask (100 m space and 300 m pitch) followed by development with deionized water are shown in Fig. 2. As shown in Fig. 2a–c,
Fig. 5. ATR-FTIR spectra of the NPS (a), non-irradiated PVA (b), and irradiated PVA films at fluences of 5 × 1015 (c), 7 × 1015 (d), and 9 × 1015 ions/cm2 (e).
well-defined 100 m negative-type PVA patterns were formed at the given fluences. However, PVA patterns were not generated at less than 5 × 1015 ions/cm2 (data not shown). Thus, ion irradiation above a fluence of 5 × 1015 ions/cm2 is necessary to form negative-type PVA patterns. This ion irradiation-induced formation of negative-type PVA patterns without any additives can be explained as follows. Unlike techniques based on electron beams, UV light, ␥-rays, or X-rays, the larger amount of radicals in solidstate PVA films can be generated by ion irradiation with high LET, thus leading to an increase in the probability of interpolymer radical coupling reactions. Thus, ion irradiation above a certain fluence can result in a crosslinked PVA in a solid state [36,37]. The surface profiles of PVA-patterned NPS substrates prepared at various fluences are presented in Fig. 3. The remaining thickness of the PVA patterns slightly decreased from 385 nm to 351 nm with an increase in the fluence. This reduction in the thickness of the PVA patterns can be attributed to the fact that the formation of volatile species by ion irradiation-induced breaking of covalent bonds in PVA is accelerated with an increase in the fluence, resulting in a reduced thickness [38,39]. Furthermore, as shown in the AFM images related to elucidating the ion beam-induced morphological
Fig. 6. The C1s spectra for non-irradiated (a) and irradiated PVA films at fluences of 5 × 1015 (b), 7 × 1015 (c), and 9 × 1015 ions/cm2 (d).
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change in the surface of PVA (see Fig. S1 of the supplementary information), the non-irradiated PVA films were smoother than the NPS surface. On the other hand, all the patterns PVA formed at the given fluences exhibited almost similar morphologies to that of the non-irradiated PVA and the RMS values of their surface roughness ranging from 0.80 to 1.10 was not much different from that of the non-irradiated one. Therefore, ion irradiation seemed to cause no significant topographical change in the PVA surfaces. To investigate the ion irradiation-induced changes in the wettability of PVA, water contact angle measurements were performed and the results are shown in Fig. 4. The averaged contact angle of the untreated PVA surface was around 28◦ , indicating a highly hydrophilic surface in comparison to the NPS with an average contact angle of about 83◦ . On the other hand, in the cases of irradiated PVA, the average contact angles increased up to 64◦ with an increase in the fluence, which is much lower than that of the NPS. This result indicates that, although its surface was more hydrophobic than that of the non-irradiated PVA, the irradiated surfaces have a more hydrophilic surface, which is more favorable for cell adhesion and proliferation than that of the NPS. This change in wettability
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Fig. 7. [O]/[C] atomic ratio of non-irradiated and irradiated PVA films at various fluences.
can be attributed to the change in the surface chemical structure and composition of the PVA caused by ion irradiation [40]. The changes in the chemical structure of PVA films brought about by ion irradiation were analyzed by ATR-FTIR spectroscopy,
Fig. 8. Fluorescence micrographs of the serum proteins adsorbed on the PVA-patterned NPS substrates prepared at fluences of 5 × 1015 (a), 7 × 1015 (b), and 9 × 1015 ions/cm2 (c).
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Fig. 9. Optical microscopic images of HeLa (a-c), H1299 (d-f), and NIH3T3 (g-i) cells on the PVA-patterned NPS substrates prepared at various fluences.
and the results are shown in Fig. 5. In the non-irradiated PVA spectrum in Fig. 5b, the characteristic bands corresponding to the chemical structure of the PVA backbone and unhydrolyzed pendant acetate group were identified at 3331 (O H ), 2935 (C H ), 1736 (C O ), 1442 (ıC H ), and 1091 cm−1 (ıO H ) [41]. On the other hand, in the irradiated PVA spectra in Fig. 5c–e, new bands assigned to C C and C O C appeared at 1630 (C C ) and 1166 cm−1 (C O C ), and their intensities gradually increased with an increase in the fluence. This change in chemical structure may result from the ion irradiation-induced crosslinking and degradation of the PVA [42]. To further investigate the changes in the chemical structure and composition on the PVA surface after ion irradiation, an XPS analysis was carried out and the results are presented in Fig. 6. The spectrum of the non-irradiated PVA presented in Fig. 6a exhibited characteristic peaks at 285.0 (C C), 286.5 (C OH), and 289.1 eV (C O) O of the unhydrolyzed pendant acetate group. In the irradiated PVA, new peaks corresponding to the C O C and C O groups were generated at 285.8 and 289.2 eV, respectively. Moreover, as shown in Fig. 7, the [O]/[C] elemental ratio of the irradiated PVA gradually decreased to 0.25 with an increase in the fluence in comparison to that of the non-irradiated PVA. This change in the surface chemical structure and composition can be attributed to the crosslinking and degradation of PVA caused by ion irradiation [43]. 3.2. Pattern stability The stability of the PVA-patterned NPS substrates affecting the cell behavior was estimated by measuring the change in the thickness of PVA patterns before and after incubation in PBS solutions for 15 days under the same conditions as the cell culture. As shown in Fig. S2 (see the supplementary information), all the PVA patterns formed at the given fluences exhibited almost identical thickness before and after incubation for 15 days. This result can be ascribed to the fact that PVA was densely crosslinked by ion irradiation at the
given fluences, thereby resulting in the formation of robust patterns without swelling. 3.3. Protein adsorption on PVA-patterned NPS substrates To investigate the feasibility in the application of the PVApatterned NPS substrate to the control of cell adhesion, the protein adsorption on the PVA-patterned surfaces prerequisite for cell adhesion was estimated by a fluorescent microscopic analysis with FITC-labeled BSA. As shown in Fig. 8, the BSA was selectively adsorbed only onto the PVA regions of all the PVA-patterned NPS surfaces formed at the given fluences, resulting in well-organized BSA patterns. However, their corresponding fluorescence intensities were gradually reduced with an increase in the fluence. The phenomenon could be explained by the fact that BSA preferentially bound to the oxygen-containing functionalities on the surfaces of the PVA patterns, and thus the amount of the BSA adsorption was dependent on the numbers of the functionalities that were affected by the fluence [44]. 3.4. In-vitro cell culture on PVA-patterned NPS substrates To demonstrate the possibility for the control of cellular microorganization, three types of cell lines including H1299, HeLa, and NIH3T3 were in-vitro cultured on the PVA-patterned NPS substrates. As shown in Fig. 9, at all given fluences, all types of cells were preferentially adhered and proliferated onto the PVA regions, resulting in well-organized 100 m patterns of the cells on the PVApatterned NPS substrates. This cellular micro-organization on the PVA-patterned NPS surface seemed to result from the preferential protein adsorption on the regions of the PVA patterns rather than those of the NPS. To further examine cell response to the wholly-irradiated PVA surfaces, a cell proliferation assay using CCK-8 was performed on bare NPS and PVA-coated NPS substrates irradiated at various fluences. Fig. 10a shows the proliferation of the H1299 cells cultured
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Fig. 10. Cell proliferation of the H1299 cells cultured on the PVA film irradiated at various fluences (a), and cell viability on the TCPS and PVA-patterned NPS substrates prepared at various fluences (b).
on the irradiated PVA film at various fluences. The proliferation rates of the cells on the irradiated PVA were much higher than that on bare NPS although those was slightly lower in comparison to that on the non-irradiated PVA. This result implies that the PVA surfaces irradiated at various fluences provide a more cell-friendly environment than the NPS surface, which is in good agreement with the above-mentioned protein adsorption result. Moreover, as shown in Fig. 10b, the result of the cell viability test revealed that all PVApatterned NPS substrates at the given fluences with a viability of more than 80% had cytocompatibility, although their viability was slightly lower in comparison to that of the control TCPS [45]. These results indicate that the preferential adhesion of the cells on the surface of the PVA patterns was originated from their recognition of the pre-adsorbed serum proteins on the patterns and that the PVA-patterned substrates were cytocompatible. 4. Conclusion The formation of PVA patterns on the surface of NPS substrates was successfully performed by selective ion irradiation in the absence of any toxic chemicals to control cellular microorganization. The results of the surface profile and AFM analyses revealed that 100 m negative-type PVA patterns were successfully generated on the NPS substrate at a fluence above 5 × 1015 ions/cm2 and their surface morphologies was not significantly changed. Based on the contact angle measurement, ATR-FTIR, and XPS results, the irradiated PVA surfaces were more hydrophilic than the bare NPS surface even though its surface became more hydrophobic than the non-irradiated PVA surface owing to the ion irradiation-induced change in the surface chemical environment. It is clearly shown from the stability test that all the PVA patterns on the NPS at the given fluences exhibited the robustness without dimensional change in the environment of cell culture. Furthermore, the results of the protein adsorption and in vitro cell culture test, clearly showed that two types of human cancer cells were preferentially adhered and proliferated only onto the more hydrophilic PVA regions of the PVA-patterned NPS substrates prepared at all given fluences and therefore resulting in the formation of welldefined 100 m cell patterns due to the preferential adsorption of serum proteins on the more hydrophilic PVA regions of the PVApatterned NPS. These resulting PVA-patterned substrates using a convenient and non-toxic ion beam-based technique are applicable to the study of cell biology and the development of cell-based biosensors and drug discovery. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2013M2A26023510).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsusc. 2014.07.163.
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