Creating “Living” Polymer Surfaces to Pattern Biomolecules and

Creating patterns of biomolecules and cells has been applied widely in many fields associated with the life sciences, including diagnostics. In these ...
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Creating “Living” Polymer Surfaces to Pattern Biomolecules and Cells on Common Plastics Chunyan Li,†,‡ Andrew Glidle,‡ Xiaofei Yuan,‡ Zhixiong Hu,‡,§ Ellie Pulleine,‡ Jon Cooper,‡ Wantai Yang,*,† and Huabing Yin*,‡ †

State Key Laboratory of Chemical Resource Engineering, Key Laboratory of Carbon Fiber and Functional Polymers, Ministry of Education, College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing, China 100029 ‡ College of Science and Engineering, Division of Biomedical Engineering, School of Engineering, University of Glasgow, Glasgow, United Kingdom § Division of Medical and Biological Measurements, National Institute of Metrology, Beijing, China 100013 S Supporting Information *

ABSTRACT: Creating patterns of biomolecules and cells has been applied widely in many fields associated with the life sciences, including diagnostics. In these applications it has become increasingly apparent that the spatiotemporal arrangement of biological molecules in vitro is important for the investigation of the cellular functions found in vivo. However, the cell patterning techniques often used are limited to creating 2D functional surfaces on glass and silicon. In addition, in general, these procedures are not easy to implement in conventional biological laboratories. Here, we show the formation of a living poly(ethylene glycol) (PEG) layer that can be patterned with visible light on plastic surfaces. This new and simple method can be expanded to pattern multiple types of biomolecule on either a previously formed PEG layer or a plastic substrate. Using common plastic wares (i.e., polyethylene films and polystyrene cell culture Petri-dishes), we demonstrate that these PEG-modified surfaces have a high resistance to protein adsorption and cell adhesion, while at the same time, being capable of undergoing further molecular grafting with bioactive motifs. With a photomask and a fluid delivery system, we illustrate a flexible way to immobilize biological functions with a high degree of 2D and 3D spatial control. We anticipate that our method can be easily implemented in a typical life science laboratory (without the need for specialized lithography equipment) offering the prospect of imparting desirable properties to plastic products, for example, the creation of functional microenvironments in biological studies or reducing biological adhesion to surfaces.

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INTRODUCTION Cell patterning is of great importance in many applications, including biosensors,1 tissue engineering,2 and fundamental research into cellular development, such as growth, migration, and differentiation.3−7 Various methods of cell patterning have been developed using techniques such as photolithography,8−12 photochemical activation/degradation,13,14 and soft lithography.15 Use of these technologies has enabled biological studies that have relied on making patterns of single cell types on twodimensional surfaces.16−18 As our understanding of cell interactions with their environment advances, the importance of the spatial and dynamic arrangement of biological functions is becoming increasingly apparent (including, for example, patterns of proteins, growth factors, enzymes, multiple cell types). One implication of this understanding has manifested itself in the design and production of in vitro systems that mimic physiological conditions, so allowing in-depth investigation of the cellular activities found in live tissue.3,4,19 In this context, spatiotemporal patterning of multiple biomolecules without compromising their bioactivity is of importance but remains technically challenging. One serious issue stems from the © XXXX American Chemical Society

conformational instability of large proteins, making them susceptible to degradation. Here, we report a simple method to perform multicomponent patterning of bioactive ligands to create cell microenvironments on common plastics. The method involves visible-light patterning, which has the advantage of being both widely available and having high spatiotemporal resolution.20 The use of visible light not only has negligible effects on proteins and cells, but also facilitates its ready implementation as a low-cost technique in the laboratory. In general, one key element when making patterned surfaces for cell studies is the creation of an inert background with a high resistance to protein adsorption. To date, the most effective and widely used methods are those based on poly(ethylene glycol) (PEG),21 with the majority of reports using photochemical methods to pattern photolabile PEG selfassembled monolayers on a surface (e.g., via photoactivation or photodegradation).22−26 Upon irradiation with light these Received: September 27, 2012 Revised: March 7, 2013

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Visible Light Induced Graft Polymerization. A medium pressure mercury lamp fitted with a band gap filter (between 320 and 480 nm) was used as a visible light source. With this setting, the majority of light comes from the emission lines at 365, 405, and 436 nm of the mercury lamp. Except where noted, the light intensity incident on the assembly was 1.8 mW/cm2 (using a Thorlabs S210 power meter fitted with a thermal head and set to 380 nm). Exposure times ranging from 2 to 20 min were examined. Non-patterned Graft Polymerization over a Whole Surface. A similar film assembly to that described above was used for graft polymerization onto ITX-immobilized polyethylene films. Here, the top film was a piece of the previously prepared ITX modified PE film and the bottom film, a piece of plain, unmodified PE. The solution (50 μL) placed between these two PE films was the monomer of interest, either neat, or in an aqueous or organic carrier solvent. Before the monomer solution was introduced into the film assembly, it was sparged with nitrogen gas for 10 min to remove dissolved oxygen. The monomer solutions used in the study reported here include aqueous EGMA solutions (33% by volume), neat GMA monomer, a mixture of GMA and GMA−polylysine, or a mixture of GMA and GMA-MP11 at the ratio of 2:1. After visible light induced polymerization reaction, the modified polyethylene films were immersed in acetone for 24 h and dried at room temperature before subsequent characterization. This procedure was slightly modified for ITX-immobilized Petri-dishes. Here, for example, 40 μL of aqueous EGMA (33% by volume) was placed inside the Petri-dish and covered with a quartz coverslip. After irradiation, the modified Petri-dish was rinsed with RO water. Patterned Grafting. For the formation of a single component patterned surface, the blank quartz plate on the top of the PE film assemblies or Petri dishes was simply replaced by a photomask prior to irradiation, as above. This approach is suitable for single component patterning. However, to create multiple component patterns spatiotemporally, we developed a flow chamber system (see illustration in Results and Discussions). A chamber (1.5 cm long, 1 cm wide, and 20 μm deep) was etched into a glass substrate. A modified plastic substrate, for example, an ITX-modified PE film, was placed with the ITX face inward and clamped in place using a 125 μm thick quartz plate and a photomask. A monomer solution was then delivered into the chamber using a syringe pump and PTFE tubing. Once the solution filled the chamber, the flow was halted prior to subsequent irradiation. For multiple component patterning, after irradiation of the first monomer solution was complete, the chamber was flushed with a suitable carrier solvent and replaced with the new monomer solution. As a result of the easy exchange of monomer solutions and the registration of different masks, precisely controlled multiple component patterns can be readily realized. Grafting Yield. Grafting yield (GY) is defined as GY = (Wb − Wa)/A (μg/cm2), where Wa and Wb represent the weight of the dried films before and after grafting, respectively; A is the film area (∼15 cm2). Data are presented as mean ± one standard deviation (n = 3). Films were weighed using an analytical balance (Sartorius BP211D, Germany) with a resolution of 0.01 mg. Absorption Spectra. UV−vis absorption spectra of the films were measured using a Hitachi U2000 spectrophotometer with wavelength range from 190 to 600 nm. Attenuated total reflectance (ATR) FTIR spectra of the polymer films were measured with a Bomem MB-120 infrared spectrometer by placing a film onto the face of a 25 reflection KRS-5 crystal, which was then mounted in a Grasby Specac variable angle ATR accessory (set to 45°) in the sample compartment of the spectrometer. Contact Angle. Stationary contact angles were measured on an Easy Drop system (Kruss GmbH, Germany) using a 3 μL droplet of deionized water. DSM software based on the Young−Laplace method was used to analyze results. Each sample was measured three times at different sites. Contact angle of one type of film is given as a mean ± one standard deviation from three replicate samples. Atomic Force Microscopy (AFM). The topography of the patterned films was characterized on a NanoWizard II Atomic force microscope (JPK instruments AG, Germany) using MCLT (Veeco)

become active, allowing the subsequent physisorption or covalent immobilization of biomolecules. However, because both reactive and protein repelling moieties are present on the same (PEG-based) structure, it remains a challenge to maintain a surface with high resistance to nonspecific protein adsorption, while at the same time generating specific bioactivities that target cells. In this work, we demonstrate the clean formation of a living poly(ethylene glycol) (PEG) brush that has high resistance to protein adsorption, while at the same time, being capable of further conjugation with functional biomolecules. Our method requires mild irradiation with visible light in the presence of olefin monomers (neat or in aqueous solutions), making the procedure highly compatible with biomolecules and cells. Using a photomask and a simple microfluidic delivery system enables the attachment to be dynamically controlled and so modulate cell microenvironments.

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EXPERIMENTAL SECTION

Materials. Low density polyethylene film (PE; Beijing No. 7 Plastic Factory, Beijing, China) of 85 μm thickness was cut into squares of 3 × 5 cm2 for use. All film discs were extracted with acetone for 12 h to remove any impurities and then dried under reduced pressure at room temperature. Cell culture Petri-dishes (Nunc, Thermoscientific, made from polystyrene) were used as received. Isopropyl thioxanthone (ITX, purity ≥ 98%), poly(ethylene glycol methacrylate) (EGMA, n = 30), glycidyl methacrylate (GMA), poly-L-lysine hydrobromide (Mw = 500−2000 g/mol), microperoxidase-11 (MP11), and common solvents were purchased from Aldrich-Sigma Chemical Co. EGMA and GMA were stripped of inhibitors using disposable inhibitorremoval columns (Sigma Chemical Co.) and then stored under an argon atmosphere at −4 °C. Coupling Poly-L-lysine to GMA. GMA (40 μL, ≈0.28 mmol) and poly-L-lysine hydrobromide (40 mg, ≈0.03 mmol) were added to a 400 μL buffer solution (0.1 M NaHCO3, pH 9) in a glass tube. They were mixed rapidly on a shaker overnight at room temperature. Due to the low water solubility of GMA, the reaction with the amine groups in poly-L-lysine occurred at the interface between the aqueous poly-Llysine phase and the oil GMA phase. After reaction, to facilitate separation of the two phases, 400 μL of ethyl acetate was added, and the mixture was centrifuged for 5 min at 800 rpm. This led to separation of the two phases and the ethyl acetate organic phase was retained. Following evaporation of the ethyl acetate the colorless GMA-polylysine conjugate was obtained. The same procedure was employed to synthesize a GMA-MP11 conjugate. ITX Immobilization on Plastic Surfaces. Two pieces of thin polyethylene film (3 × 5 cm2) were overlaid on each other face to face, on top of which a 1 mm blank quartz plate was placed in order to flatten the assembly.27 Prior to this, ITX (20 μL, 0.5 M in acetone) was added between the two PE films. The use of the quartz plate ensured the ITX solution spread evenly between the films to give a thin liquid layer ∼50 μm thick and prevented rapid evaporation during the subsequent irradiation step. Irradiation using a UV light (low pressure mercury lamp) was performed through the quartz plate for predetermined periods of time. The irradiation intensity measured at the surface of the quartz plate was 9 mW/cm2 at a wavelength of 254 nm. After irradiation, the films were separated by immersing the assembly in deionized water. Only the top-film of the assembly was retained and this was washed by immersing in acetone for at least 4 h to remove any residual ITX. The ITX-modified films were blown dry with nitrogen gas and stored in the dark. In the case of cell culture Petri-dishes, ITX (40 μL, 0.1 M in dimethyl sulfoxide) was applied to the inside of a Petri-dish and covered with a 2.5 cm diameter, 150 μm thick quartz coverslip. Here, UV irradiation was performed through the quartz coverslip due to the strong optical absorption of polystyrene at wavelengths below 300 nm. After irradiation, the Petri-dishes were rinsed with ethanol to remove residual ITX. B

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Scheme 1. (A) Proposed Chemical Pathway; (B) Illustration of the Procedure Involved and the Assembly Developed when Using Polyethylene Films (PE) and EGMA Monomers As an Examplea

a

(A) In stage (1), ITX abstracts hydrogen atoms from C−H bonds on a polymer surface via deep UV irradiation, giving rise to ITX semipinacol radicals and free radicals on the polymer surface. These two radicals preferentially recombine with each other, forming “dormant” ITX semipinacol groups on the surface. In stage (2), visible light (320−480 nm) photolyses the ITX semipinacol groups back into ITX semipinacol radicals and free radicals on the polymer surface (i.e. Pm•). The surface radicals (Pm•) initiate grafting of monomers (i.e. M) which then recombine with ITX semipinacol radicals, reforming ITX semipinacol groups. These reversible dissociation/combination equilibria are established at the termini of the propagating graft chain radicals, leading to the formation of polymer grafts. (B) EGMA and GMA structures. (C) Orange dots, ITX semipinacol groups; black-green brushes: poly(EGMA) graft which have a polyethylene backbone structure (i.e. black lines) and branched chains of PEG30 (i.e. green lines). ITX semipinacol groups initially on the PE film in (1) are transferred to the terminus of poly(EGMA)’s grafted backbone in (2). In the right hand panel of (2), it is tentatively suggested that the solution interface of the “living” polymer formed comprises a mobile mixture of protein repelling hydrated PEG groups and small ITX motifs, either at the immediate interface, or buried slightly below it.

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probe having a spring constant of 0.5 N/m. The scanning was carried out in air using tapping mode at a scan rate of 1 Hz. Optical Microscopy. Phase and fluorescence images were collected using an Olympus IX71 microscope (Olympus, Japan). Phase images of cells on substrates were taken after culture for the indicated periods. The samples were washed with fresh medium prior to imaging. Fluorescence images of FITC-BSA and calcein-AM stained cells (for viability assay) were collected using a 485 ± 10 nm filter for excitation and a 520 ± 10 nm filter for emission. Cell Viability Assay. MG63 cells were maintained in cell culture flasks at 37 °C and 5% CO2 in Dulbecco’s modified Eagle medium containing nutrient mixture F-12 (DMEM/F12, GIBCO) supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL of penicillin and streptomycin (denoted as full culture medium). Prior to cell culture, the modified PE and polystyrene substrates were sterilized under UV illumination for 15 min and rinsed with PBS buffer. In the case of PE films, they were placed in a six-well culture plates and seeded with the cell suspension at a density of 1 × 104 cells/well. Modified 3.5 cm diameter polystyrene Petri-dishes were seeded using 1.2 × 106 cells/dish. After incubation for predetermined times (e.g., 2 h, 24 h, and 2 days), cell suspensions were aspirated to remove unattached cells and the surface washed twice with fresh full culture medium. Viability of cells adhered onto the films was measured using a calcein-AM live cell assay. Cells were incubated with 5 μM calceinAM/PBS at 37 °C for 45 min followed by fluorescence imaging. Live cells appear fluoresent by converting nonfluorescent calcein-AM into green fluoresent calcein (excitation at 495 nm and emission at 515 nm).

RESULTS AND DISCUSSION Formation of “Living” and Cell-Repellent PEG Brush. Our strategy utilizes a simple photoassisted surface living radical polymerization system developed in our laboratory.27,28 It consists of two-step reactions, as shown in Scheme 1. In step (1), we introduce “living” terminal groups on a polymer surface by immobilizing ITX on it. Since ITX has adsorption peaks around 260 nm (strong) and around 380 nm (weak), UV irradiation with a low pressure mercury lamp (∼254 nm) may promote ITX to abstract hydrogen atoms from C−H bonds on an organic surface. This results in free radicals on the polymer surface and ITX semipinacol radicals. These then preferentially recombine with each other, leading to the immobilization of ITX on the surface. In step (2), visible light photolyzed the immobilized ITX groups back into ITX semipinacol radicals and free radicals on the polymer surface. The surface radicals initiated graft polymerization of solution based olefin monomers, following which ITX semipinacol radicals combine with radicals at the terminus of the extended polymer chain.27 Here, we illustrate the method by using thin sheets of flexible low-density polyethylene film as a model substrate due to its lack of strong UV-vis absorption band and ease of handling. We first optimized the ITX immobilization on polyethylene film in terms of irradiation time, which varied from 2 min to 5, 10, 15, and 18 min. After thorough Soxhlet extraction and washing to remove noncovalently bound ITX,29,30 the UV spectrum of the C

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Figure 1. (A) ATR-FTIR spectra of polyethylene film (i.e., PE) and poly(EGMA) grafted polyethylene film (i.e., poly(EGMA)−PE), (B) UV− visible spectra, and (C) resistance to cell adhesion of PE and poly(EGMA)−PE films. MG63 cells were cultured for 2 h and 2 days, respectively.

resulting films was measured. All modified films showed a broad absorption band from 380 to 420 nm (Supporting Information, Figure S1), similar to that of ITX. Increasing the irradiation time led to higher absorption intensity, indicating an increased amount of immobilized ITX. The modified ITX films so prepared were stable when stored in the dark for several months prior to further use (Note, films irradiated for 18 min were used for the rest study). We then evaluated grafting of PEG brushes on the surface of ITX-immobilized films by visible light irradiation for different periods, using EGMA monomers. An aqueous EGMA solution (33.3% by volume) was used, allowing the potential for it to be applied in the presence of biomolecules (e.g., proteins or cells). A plain polyethylene film was used as a control and exposed to EGMA solutions under the same irradiation conditions. As shown in Figure 1A, the ATR-FTIR spectra of the plain polyethylene films before and after irradiation treatment is identical, indicating that no EGMA was grafted. In contrast, characteristic peaks corresponding to vibration bands of the ester groups in EGMA at 1257 and 1726 cm−1 appeared in the ATR-FTIR spectra of the ITX-immobilized films, suggesting that the ITX groups immobilized on the films have initiated the graft polymerization of EGMA. Furthermore, the contact angles of poly(EGMA) surfaces decreased with increased polymerization time, for example, from 105 ± 3° for a plain polyethylene film to 38 ± 3° after 18 min grafting with EGMA (Table 1). This enhanced hydrophilicity suggests the surface coverage of poly(EGMA) on ITX-immobilized films is increased with irradiation time. It is notable that the ITX groups remain on the poly(EGMA) brushes, as shown by the characteristic UV adsorption bands of the poly(EGMA) grafted films (Figure 1B). Since it is known that PEG formation and composition greatly influences its resistance to protein adsorption and hence cell adhesion,31,32

Table 1. Average Water Contact Angle of the Surface of Functionalized Film sample plain polyethylene film (PE) ITX-immobilized film (ITX−PE)a poly(EGMA)−PE 1b poly(EGMA)−PE 2b poly(EGMA)−PE 3b poly(GMA/GMA-polylysine)− poly(EGMA) 1c poly(GMA/GMA-polylysine)− poly(EGMA) 2c poly(GMA/GMA-polylysine)− poly(EGMA) 3c

reaction time (min)

contact angled (±3°)

5 2 10 18 2

110 105 77 54 38 42

10

58

18

76

a

Irradiation under deep UV (∼254 nm, see section 2.3 in Experimental Section). bIrradiation under visible light range (320− 480 nm). ITX-immobilized films (ITX−PE) and aqueous EGMA solutions (33% by volume) were used. cIrradiation under visible light range (see above). Poly(EGMA) grafted films (i.e. poly(EGMA)−PE 3) and a mixture of GMA and GMA-polylysine at ratio of 2:1 were used. dAll measurements had one standard deviation of ±3° or less.

the suitability of poly(EGMA) as an inert background to cell adhesion was evaluated by MG63 cell culture in full medium. As shown in Figure 1C, very few, if any attached to poly(EGMA) grafted films after 2 days of cell culture. In contrast, MG63 cells spread well on plain polyethylene films (Figure 1C) and on ITX-immobilized polyethylene films (Supporting Information, Figure S2) after only a 2 h incubation. Thus, the poly(EGMA) grafted films have excellent resistance to cell adhesion regardless of the terminal “dormant” ITX semipinacol groups. An explanation for the negligible effect on cell adhesion of the remaining ITX semipinacol groups is that these D

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Figure 2. (A) ATR-FTIR spectra of a series of films after successive reactions. Poly(EGMA)−PE and poly(GMA)−PE films were formed by visible light grafting of EGMA and GMA on ITX-immobilized polyethylene films, respectively. Poly(EGMA)−PE films were employed further for subsequent grafting of GMA or a mixture of GMA/GMA-polylysine comonomers, giving rise to poly(GMA)−poly(EGMA) or poly(GMA/GMApolylysine)−poly(EGMA) films. (B) Dependence of graft yield (GY) of the poly(EGMA)−PE and poly(GMA/GMA-polylysine)−poly(EGMA) films on the reaction time. Error bars are ± one standard deviation.

In addition, Figure 2B shows an approximately linear increase in their graft yield with copolymerization time, which is similar to that of EGMA grafted from the ITX-immobilized films. This implies a well-defined chain growth mechanism in both cases,27 which provided an effective way of tuning the thickness of each functional layer. Importantly, the capability of “passing on” light activated reactivity to successive layers opens up a powerful avenue to create three-dimensional, multifunctional structures. Dynamic Formation of Biomolecule Patterns. The dependence of the reaction on visible light irradiation provides a flexible way to form active biomolecule patterns with spatiotemporal control. To realize this, we developed a simple flow chamber for easy exchange of solutions and coupled it with a photomask (Figure 3A). By repeating the exposure with different photomasks and monomers, multiple component patterns in both 2D and 3D can be readily generated. Figure 3B shows the GMA/GMA-polylysine copolymer patterns formed on a poly(EGMA) grafted film using a photomask. An array of 50 μm squares (i.e., one example of a range of designs) is clearly visible in the optical phase image. However, the edges of the features do not appear sharp. This is perhaps a consequence of the 2-D and 3-D nature of the polymer growth leading to polymer chains growing along the surface, thereby reducing the lateral resolution of the patterns. The average height of the pattern after 15 min irradiation is approximately 1.8 μm, as measured by AFM. As shown above in Figure 2B, the thickness of the pattern can be tuned via the irradiation period. Although an extensive optimization to determine the limit of patterning resolution was not undertaken, dot and line patterns using masks with features varying from 100 μm down to 10 μm, suggested that, with the conditions used, the practical resolution limit was ∼16 μm (Supporting Information, Figure S3). Following the above GMA/GMA-polylysine copolymer patterning, patterned films can be further modified to create surfaces consisting of multiple functionality by reaction of other proteins with the pendent epoxide groups of the co-deposited GMA polymer. To demonstrate this, a patterned film was incubated with fluorescein-labeled BSA solution (pH 7.4) for 24 h. Fluorescence images were collected after the film was thoroughly washed with 0.1% Tween 80 in PBS solution to remove nonspecifically bound BSA. As shown in Figure 3C,

functionalities are masked by hydration effects of the PEG30 side chains on the poly(EGMA) brushes (Scheme 1B). However, this raises the question of whether the ITX semipinacol groups are still accessible to initiate further grafting of active biomolecules. To evaluate this, glycidyl methacrylate (GMA) was applied to a poly(EGMA) grafted film and exposed to visible light irradiation for 18 min. As shown in Figure 2A, new peaks at 906 and 849 cm−1, belonging to the stretching of the epoxy group in GMA, appear on the ATR-FTIR spectrum of the poly(EGMA) grafted film after GMA polymerization. In parallel, the ITX-immobilized films also gave the same peaks after direct GMA polymerization on it (Figure 2A). All of these results are indicative of the “living” ITX terminals of the poly(EGMA) brushes toward further photoassisted polymerization. Immobilization of Multiple Bioactivities. A distinct feature of this grafting methodology when patterning a succession of layers, is that there is no need for additional initiators, multistep coupling reactions or purification. Such clean and mild reaction conditions offer a promising route for the immobilization of biomolecules. Only alkene moieties are required. This general functionality can be conjugated to biomolecules (including proteins) via established methods,33,34 thus enhancing the capability of simultaneous immobilization of molecules with different bioactivities. To illustrate this potential, we created thin films of biomolecules directly (covalently) bound to a poly(EGMA) grafted film as follows: poly-L-lysine, a motif well-known to promote cell adhesion, was used as a model biomolecule. It was coupled to GMA monomer through a nucleophilic ringopening reaction between its amine groups and the epoxide groups of GMA35 (denoted as GMA-polylysine). Then the mix monomer of GMA/GMA-polylysine (weight ratio of 2:1) were directly copolymerized onto the poly(EGMA) grafted film. This copolymerization method allows flexible modulation of poly-L-lysine density immobilized on the films. As a result, characteristic peaks for the epoxide groups of GMA and amide I groups of poly-L-lysine (1639 cm−1) were both present in the ATR-FTIR spectrum of the films after the polymerization procedure (Figure 2A), indicating the linkage of both GMA and poly-L-lysine onto the surface. E

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For proteins and enzymes, it is critical to maintain their biological function after being immobilized on a surface. We therefore evaluated the enzymatic reactivity of microperoxidase11 (MP11) patterned on poly(EGMA)-ITX modified PE films. MP11 was linked to GMA using the same procedure as for the GMA-polylysine conjugates. A mixture of GMA and GMAMP11 monomers (at ratio of 2:1) were patterned on poly(EGMA) grafted films or ITX-immobilized films under different exposure times (5−18 min). A TMB (3,3′,5,5′tetramethylbenzidine)/H2O2 based assay was used to evaluate the MP11 activity of the resultant films. A blue color was developed on all the patterned films, whereas no color was seen on the controls. The deepness of the blue color that developed on the patterned region (Figure 4) indicated both the successful preservation of MP11 activity and that the amount immobilized increased with increasing light exposure (grafting) time.

Figure 4. Enzymatic activities of grafted MP11. (A) On poly(GMA/ GMA-MP11)−poly(EGMA) films. The poly(GMA/GMA-MP11) patterns were formed by visible light grafting of a mixture of GMA and GMA-MP11 monomers on poly(EGMA) grafted films. Therefore, a poly(EGMA)−PE film was used as a control, denoted as A-control. The patterns on films A1−A4 were formed at different exposure times, namely, A1 (18 min), A2 (15 min), A3 (10 min), and A4 (5 min). (B) On poly(GMA/GMA-MP11)−PE films. The patterns were formed on ITX immobilized films, and thus an ITX−PE film was used as a control, denoted as B-control. Patterns on films B1 and B2 were formed at exposure times of 15 and 18 min, respectively. A total of 500 μL of TMB liquid substrate were placed on top of the films.

Finally, the bioactivity of the GMA/GMA-polylysine copolymer patterns on a poly(EGMA) grafted film was evaluated using MG63 cell culture. As shown in Figure 5, cells spread well on the patterns after overnight culture with all of them remaining alive. This illustrates the immobilized poly-Llysine promoted cell adhesion and growth. In contrast, no cells were found on the surrounding poly(EGMA) background, further confirming the excellent resistance of poly(EGMA) to nonspecific protein adsorption and, hence, cell adhesion. Implementation of the “Living” Grafting Using Common Plastic Products. According to the mechanism of Scheme 1, the method described above is applicable to any of the C−H containing polymers, such as polystyrene, polypropylene, polyethylene, poly(ethylene terephthalate), and poly(methyl methacrylate), that are widely used in plastic products. To evaluate this potential, we employed this approach to modify commercial cell culture Petri-dishes that are normally made from polystyrene. We first investigated the immobilization of ITX on Petri-dishes via UV irradiation (low

Figure 3. (A) Schematic illustration of the dynamic configuration consisting of a photomask integrated onto a flow chamber for visible light patterning. (B) Representative phase image of the formed poly(GMA/GMA-polylysine) patterns on a poly(EGMA) grafted polyethylene film. (C) Fluorescence image of the poly(GMA/GMApolylysine) patterns after reaction with fluorescein-labeled BSA, showing covalent immobilization of the protein.

fluorescence was restricted to the square patterns, indicating the immobilization of BSA on the pattern (note: immobilization occurs through reaction of residual amino groups from lysine in BSA with the epoxide groups). Minimal fluorescence was found on the poly(EGMA) background, in good agreement with the previous results (Figure 1C). F

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Figure 7. (A) DIC and (B) fluorescence image of MG63 cells cultured on poly(EGMA) patterned Petri-dish after a live cell assay. Note, the background line texture is from the Petri-dish. All of the cells are alive, as indicated by the presence of fluorescence. Cells were cultured on the Petri-dish for 24 h before performing the live assay.

Figure 5. Fluorescence image of MG63 cells cultured on poly(GMA/ GMA-polylysine) patterns (indicated by the white circles) after a live cell assay. All of the cells are alive, as indicated by the presence of fluorescence. Cells were cultured on the film for 24 h before performing the live assay.

to UV and visible light sources, our approach can be readily carried out in common biological and medical laboratories, providing researchers with greater capability toward challenging investigations that cannot be met by traditional methods.

pressure mercury lamp). As shown in Figure 6A, with the increase in irradiation time, the absorbance band between 380 to 420 nm appeared in the UV spectra of the treated Petridishes, suggesting the surface immobilization of ITX. Cell culture experiments showed that there were no significant differences in cell adhesion between ITX modified Petri dishes, and the as received, untreated Petri-dishes (Supporting Information, Figure S4). Subsequent visible light grafting (20 min) of EGMA onto the ITX-immobilized Petri-dishes resulted in poly(EGMA) grafted Petri-dishes, which showed an excellent resistance to cell adhesion (Figure 6B). Furthermore, via illumination through a photomask, poly(EGMA) patterns were formed on the Petri-dish that restricted cell attachment to the non-poly(EGMA) region and, subsequently, the formation of cell patterns (Figure 7 and Supporting Information, Figure 5). These results agree well with those observed for polyethylene films, illustrating a simple way for modification of commercial Petri-dishes. Further immobilization of biomolecules and patterning of different biomolecules on the poly(EGMA) grafted Petri-dishes can be readily performed via the same approach as described above. Considering the wide accessibility

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CONCLUSION

We have established a versatile technique for patterning multiple biomolecules with 2D and 3D spatial control. We have demonstrated for the first time that live PEG layers are capable of ongoing molecular grafting, leading to surfaces that can have a patterned combination of motifs, each of which retain their own specific properties. Most importantly, this method can be used with many common plastics, whether they are in the solid, rigid, or flexible thin film/sheet form, and the substrate topography can be either 2D or 3D. Within the field of biology, we anticipate that this method offers great promise for the controlled fabrication of structured protein assemblies on surfaces and subsequently for the dynamic modulation of cell microenvironments in situ.

Figure 6. (A) UV−visible spectra of ITX immobilized Petri-dishes that were obtained at different irradiation periods. The untreated Petri-dish and ITX in DMSO were given as references. (B) Cell adhesion to a modified Petri-dish after 24 h culture. Cells adhered and spread well in the ITXimmobilized area (i.e., above the white line). However, no cell ahered to the poly(EGMA) grafted area (i.e., below the white line). G

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1, UV−visible spectra of plain polyethylene film, and ITX-immobilized polyethylene films; Figure S2, MG63 cells cultured on ITX-immobilized polyethylene films; Figure S3, poly(EGMA) patterns with features ranging from 100 to 16 μm; Figure S4, cell growth in (A) unmodified and (B) ITXmodified Petri dishes; Figure S5, cell pattern formation on a Petri-dish. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for financial assistance from the “Scotland-China Higher Education Research Partnership for Ph.D.” project supported by the Scottish government and the Chinese Ministry of Education. E.P. thanks LGC for a case studentship award. Support was also given by EPSRC (EP/ H04986X/1), The Leverhulme Trust, and NSFC (Nos. 51033001, 51221002).

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dx.doi.org/10.1021/bm4000597 | Biomacromolecules XXXX, XXX, XXX−XXX