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Communications Technique of Surface Modification of a Cell-Adhesion-Resistant Hydrogel by a Cell-Adhesion-Available Inorganic Microarray Jianguo Sun,† Stefan V. Graeter,†,‡ Lin Yu,† Shifeng Duan,† Joachim P. Spatz,‡ and Jiandong Ding*,† Key Laboratory of Molecular Engineering of Polymers of Ministry of Education, Department of Macromolecular Science, Advanced Materials Laboratory, Fudan University, Shanghai 200433, China, Department of New Materials and Biosystems, Max-Planck-Institute for Metals Research, and Department of Biophysical Chemistry, University of Heidelberg, Heisenbergstrasse 3, D-70569 Stuttgart, Germany Received April 30, 2008; Revised Manuscript Received June 25, 2008
A microtransfer technique for micropattern fabrication using a dithiol macromolecular linker is suggested by transferring a conventionally photolithography-prepared gold microarray on a hard inorganic substrate to a polymeric substrate. The linker was synthesized by end-capping a poly(ethylene glycol) (PEG) chain by the thiol groups. The efficiency of this technique is demonstrated by the transfer of gold microdots from glass to a cell-adhesionresistant PEG hydrogel, which was formed by polymerizing PEG diacrylate macromers. The stability and biocompatibility of the resulting polymeric-inorganic hybrid material and cell-adhesion contrast of the patterned surface is confirmed by preliminary cell experiments. Recently, microarrays have emerged as a topic of great interest in many fields,1–5 especially with regard to gene and protein chips6 and cell arrays.7,8 Patterned surfaces have also been found to be powerful tools to reveal cell-biomaterial interactions,9–13 and the associated fundamental studies might be quite meaningful for the development of tissue engineering and reconstruction.14–17 On the other hand, much progress has been made in studies of biomaterials in the latest decade,18–20 and especially hydrogels, as a wet and mechanically biomimetic material, constitute a unique topic of research.21–31 While a micropattern on a viscoelastic biomimetic substrate is highly desirable, the conventional techniques of micropattern fabrication, based mainly upon hard inorganic substrates, especially silicon and glass,32 are difficult to be used to fabricate micropatterns on elastic hydrogel substrates. Although some techniques have been extended to silicone rubber substrates,10 it is hard to decorate the cross-linked poly(ethylene glycol) (PEG) hydrogels with a microarray because of the chemically inactive property of PEG. On the other hand, hydrogels are more suitable than silicone rubbers for mimicking the extracellular matrix (ECM).33 Hence, a new technique to fabricate microarrays on a hydrogel surface is desired. With established methods it is almost impossible to straightforwardly generate a gold array on the surface of a hydrogel. This paper suggests a transfer strategy to prepare micropatterns: the microarrays are first fabricated on a conventional hard substrate and then transferred onto a hydrogel surface. As a demonstration of the feasibility of this technique, we present the successful fabrication of a gold microarray on the surface of a PEG hydrogel. PEG and oligo(ethylene glycol) are well* Fax: +86 21 65640293; Tel: +86 21 65643506, E-mail: jdding1@ fudan.edu.cn (J. DING). † Fudan University. ‡ Max-Planck-Institute for Metals Research and University of Heidelberg.
known biomaterials preventing nonspecific adsorption of proteins and, thus, of cells.34,35 They exhibit excellent properties: biocompatible, nonfouling in complex environments, and biologically inert due to hydrophilicity and charge neutrality. On the other hand, gold is a popular “bridge chemical” suitable for further functionalization, based upon which variety of scientific topics could be investigated.11,36,37 The basic transfer procedure is schematically presented in Figure 1a. At first, a gold microarray on a hard substrate (glass in this paper) was prepared by conventional lift-off photolithography technique.38 The gold surface was then chemically modified via immobilization of a linker. Subsequently, a hydrogel substrate was formed due to cross-linking PEG diacrylate macromers. After removing the hard substrate, a gold microarray was formed on the PEG hydrogel surface. Photolithography has been used to prepare chemically structured PEG microarrays39–41 or merely gold microarrays42 on an inorganic hard substrate such as silicon. So far, however, this technique does not allow the preparation of gold micropatterns on the surface of a PEG hydrogel. As a significant improvement of photolithography, soft lithography techniques, like microcontact printing, have been successfully employed to fabricate microstructures on soft substrates. Nevertheless, neither the photolithography nor its combination with softprinting techniques can be applied to prepare “stable-in-water” metallic microstructures on hydrogels. PEG is chemically inactive and the metallic microarray on PEG could either fail to be generated or lose its order under water unless strong interaction between the array and the substrate could be introduced. In this paper, we present a technique that allows the successful transfer of photolithographically prepared metallic microstructures from glass substrates to soft hydrogel surfaces. To this end, a PEGdithiol linker is introduced, which binds to the micropattern by forming a gold-sulfur bond. The synthesis route of the dithiol-
10.1021/bm800477s CCC: $40.75 2008 American Chemical Society Published on Web 07/23/2008
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Figure 1. (a) Schematic presentation of the photolithography transfer technique for decorating hydrogel substrate surface with a gold microarray; (b) schematic presentation of the function of the linker in the transfer technique; and (c) the synthesis route of the linker, HSPEG-SH.
Figure 2. Optical images of micropatterns in a reflection optical microscope: (a) a photoresist micropattern on glass; (b) the associated gold micropattern on PEG hydrogel after transferring; and (c) the gold micropattern after the PEG hydrogel was swollen in water. The bars refer to 50 µm.
terminated PEG is shown in Figure 1c. In our experiments, p-toluenesulfonic acid was used as catalyst, and the molecular weight (MW) of the initial PEG was 4000. A medium-MW PEG was used in linker synthesis because an overly large MW might lead to significant stereohindrance in reaction of the linkers with the gold surface and an overly small MW might not ensure sufficient entanglement of the linkers with the cross-linked macromers. The characterization results of the synthesized linker are shown in the Supporting Information. A permanent topological entanglement of the bifunctional linkers with the hydrogel network might be formed during polymerization of PEG diacrylate macromers, as schematically presented in Figure 1b. The resultant gold micropatterns on the surfaces of PEG hydrogels were found to be stable in water, as seen in Figure 2c. The original micropattern on glass was generated by photolithography: a positive photoresist was exposed to ultraviolet light through a mask with the desired opaque pattern, and the exposed part of the photoresist was then solubilized in a developer solution, resulting in a photoresist pattern as shown
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in Figure 2a. Subsequently, gold was evaporated onto the photoresist micropattern, and the photoresist was “lifted off” by sequential sonication in acetone resulting in the gold microarray on the glass support. The thickness of the resulting gold dots is about 23 nm according to our atomic force microscopy measurements. To finally transfer the gold structures from the glass to the hydrogels, the glass substrates were immersed in aqueous solutions of the PEG dithiol linker with concentrations of 10 mM for 12 h. After the samples were rinsed with water and dried by pure nitrogen, PEG diacrylates (MW 700) with 0.05% (w/w) of photoinitiator D2959 were dropped onto the sample surface and exposed to ultraviolet light under nitrogen. The photopolymerization of the bifunctional macromers led to gel formation. The glass substrates were then removed and, eventually, the gold micropatterns on the hydrogels were obtained. The gold structures could be transferred with high precision to the gels (Figure 2b). After immersing the samples in water, gels were swelled, but no significant detachment of the gold from the polymer substrate was found (Figure 2c). By swelling the hydrogels in water, the distances within the gold microdots were increased to a certain extent (about 9% in Figure 2c compared to Figure 2b). Therefore, the resulting micropatterns on the gels were determined by the predesigned photolithography mask and also influenced by the swelling ratio of the gels. Because it is well-known that the swelling ratio of a cross-linked hydrogel could be controlled by many factors such as MW and concentration of the original macromers, the dot distance could be adjusted readily. The experiments demonstrated the feasibility of the transfer lithography for the preparation of microstructured hydrogels. Thus, the presented transfer technique represents a unique tool for the decoration of biomaterials with metallic microstructures. The role of the linker was tested by immersing metallicmicrostructured glass substrates in solutions with different linker concentrations followed by the cross-linking of PEG diacrylates. The linker solutions with concentration ranging from 0 to 10 mM have been tested. The reaction of gold and the thiol groups lasted for a sufficiently long time (12 h in our experiments). Without any linker added, just a small residue of gold remained on the surface of the hydrogels after glass being removed, and the gold microarrays could not be transferred to PEG hydrogels, as seen in Figure 3a. The amount of gold coverage on the microdot positions on the hydrogel surface depends on the concentration of the linker, HS-PEG-SH (Figures 3a-d), under a given volume of the linker solution. Only when the amount of linker is sufficiently large, most of the gold microdots can be transferred to the PEG hydrogel (Figure 3d). The transfer efficiency of the gold microarrays was also characterized by measuring the gold transfer percent, as shown in Figure 3e. It is rather interesting to note that a ring-like array was observed under a moderate concentration of linker due to an incomplete transfer (Figure 3b). But why does an incomplete transfer prefer the peripheral region of the gold dots? Because the gold dots on a glass substrate prepared by a lift-off lithography technique must have a small height, parts of the S-Au bonds might be formed on the side faces of the gold microdiscs, as schematically indicated in Figure 1b. Therefore, the edge of a gold dot has a higher probability to be transferred to the PEG hydrogel. Further, due to the wrapping by the hydrogel, the edge is more easily transferred, as can be seen in the case of no linker (Figure 3a), where still a bit of gold was transferred and the gold on the PEG was just located around the edges of the original gold dots on the glass. As a result, our experiments have not only confirmed the feasibility of our
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the DAPI fluorescence micrograph (Figure 4b) illustrate that the gold microarrays are cell-adhesion available, while the PEG hydrogel itself is adhesion-resistant (Figure 4c). In summary, the present communication has suggested and confirmed a transfer technique to fabricate gold microarrays on the surface of viscoelastic, biologically inert and chemically inactive PEG hydrogels. The transfer is for matter instead of just topological shapes. The pattern is first generated on a conventional hard substrate suitable for photolithography, and then transferred to another substrate. The transfer resulted in an inorganic metal array on a polymeric substrate. We demonstrate that a linking agent is necessary to ensure a successful transfer and the stability of the micropattern on a hydrogel under a wet environment. The linker chosen in this paper is thought to covalently bond gold and topologically entangle the PEG network. Apart from confirming the feasibility of this transfer technique, our experimental results have offered an interesting technique to prepare edge patterns via an incomplete transfer. The material biocompatibility and cell-adhesion contrast of the patterned surface have been confirmed by the preliminary cell experiments. It is not difficult to adjust surface pattern forms and perform further chemical modifications based upon gold dots. This technique platform reveals much potential for further investigations of cell-biomaterial interactions in various wellcontrolled microgeometries.
Figure 3. SEM images of the transferred gold micropatterns assisted by the linker with concentrations of 0 (a), 1 (b), 5 (c), and 10 mM (d) and data of the gold transfer efficiency (e). The transfer percentage was calculated by the gold area in the microdots in the SEM images over the gold dot area in the mask.
Acknowledgment. This research was supported in part by NSF of China (Nos. 50533010, 20574013, and 20774020), Chinese Ministry of Science and Technology (a 973 Project No. 2005CB522700), Chinese Ministry of Education (a Key Grant No. 305004), the Science and Technology Developing Foundation of Shanghai (No. 07JC14005), Shanghai Education Committee (Project No. B112), Visiting Ph. D Student Scholarship (in China) from DAAD (S.V.G.), Senior Visiting Scholarship jointly from DFG and Chinese Ministry of Education (J.D.D.), and Senior Visiting Scholarship of Key Laboratory of Fudan University (J.P.S.). Supporting Information Available. Experimental details of sample preparation and characterization of the synthesized linker. This material is available free of charge via the Internet at http://pubs.acs.org.
References and Notes Figure 4. Optical micrographs of adhesion of 3T3 fibroblasts on gold microarrays on a PEG hydrogel: (a) a phase contrast micrograph; (b) the associated DAPI fluorescence micrograph; and (c) a PEG surface as the control region without significant cell adhesion.
transfer technique and the necessity of the linker, but also offered an approach to prepare “edge” patterns via an incomplete transfer. Preliminary cell experiments have also been done to confirm the material biocompatibility and the cell-adhesion contrast of the resulting micropatterned surfaces, as shown in Figure 4. Prior to cell culture, the micropatterned hydrogels were sterilized by immersing in 70% ethanol for 30 min and then exchanged with a phosphate buffer saline (PBS) solution three times. The 3T3 fibroblasts cells were seeded onto the hydrogel surfaces at 6000 cells per cm2. After 18 h, the media was removed and cells were rinsed with PBS solution twice. Cells were then fixed by 1.5% glutaraldehyde solution, and cell nucleus were stained by a fluorescence dye, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), followed by washing with the PBS solution twice. The phase contrast micrograph (Figure 4a) and
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BM800477S
