Functional Cryogel Microstructures Prepared by Light-Induced Cross

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Functional Cryogel Microstructures Prepared by Light-Induced Cross-Linking of a Photoreactive Copolymer Marc Zinggeler,† Jan-Niklas Schönberg,† Patrick L. Fosso, Thomas Brandstetter, and Jürgen Rühe* Laboratory for Chemistry and Physics of Interfaces, Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany S Supporting Information *

ABSTRACT: A novel, highly efficient method for the preparation of functional, microstructured and surface-attached cryogels is described. Photoinduced C,H-insertion reactions are used to generate cryogels in a single, rapid photo-crosslinking process. To this end, solutions containing both a photoreactive copolymer and the (bio)molecules to be immobilized are placed on a polymeric substrate followed by freezing and a short UV exposure. This strategy combines photolithography and cryogel formation allowing for a simultaneous generation and (bio)functionalization of cryogels in a single reaction step. To demonstrate the potential of the generated materials for bioanalytical applications, we successfully prepared DNA and protein cryogel microarrays. KEYWORDS: functional cryogel(s), cryogel microstructure(s), surface-attached cryogel(s), porous hydrogel(s), photo-cross-linking

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route to obtain cryogels is offered by radiation induced crosslinking of preformed polymers. Frequently used radiation sources are γ-rays,9 electron beams,10 and UV irradiation; the latter is clearly the most convenient means of obtaining crosslinked cryogels (recently reviewed in ref 11). Work published so far on UV irradiation uses low-molecular-weight photoinitiators as a source for radicals, which can transfer to the polymer and cause cross-linking through subsequent recombination of macroradicals. However, such two-component systems suffer from intrinsically limited cross-linking efficiency and the low-molecular-weight compounds might phase-separate upon cooling and/or become entrapped in the gel, could leach out at a later stage and thus contaminate the system.11,12 Furthermore, the generation of porous structures itself is in most cases only the first step in the generation of functional cryogels. To bind biomolecules (mostly proteins) to the gel, the gels are typically functionalized by introducing appropriate reactive groups such as epoxy8 or aldehyde13 groups through copolymerization in the gel-forming polymers. This is followed by a coupling reaction after gel formation.14 Such a two-step (or in some cases even multistep) process increases the time and effort required for the preparation of the final, functional gels substantially. In this work we present a novel, highly efficient method for the preparation of functional, surfaceattached cryogels with tunable pore morphology and spatially defined microstructure within a single, rapid photoreaction

olymeric cryogels have gained much attention in recent years because of their unique porous structure and good mechanical stability. A large number of attractive biotechnological and biomedical applications have been developed, especially in the areas of chromatography, drug release, and tissue engineering.1−3 Cryogels are (macro-)porous polymer networks prepared through polymerization reactions or by cross-linking polymeric precursors in moderately frozen solutions. The term “moderately frozen” in this context means that microphase separation occurs and systems are obtained that consist of crystallized solvent (typically water) and the ’liquid microphase’ (LMP), an unfrozen fraction of concentrated monomer respective polymer solution.4 In such systems, reactions leading to the formation of the polymer networks occur only within the liquid phase. With ongoing reaction progression, this phase becomes more and more viscous and eventually completely solidified. Thus, after thawing the solvent crystals, a network of interconnected pores remains.5 A variety of strategies for cryogel formation have been described (e.g., reviewed in ref 6). In most cases, vinyl monomers are polymerized and cross-linked in situ by free-radical cross-linking polymerization.6 In other cases, natural or synthetic macromolecules are transformed into networks using suitable bifunctional cross-linking agents such as glutaraldehyde or diamines.6 A common problem with both polymerization-based approaches and chemically induced crosslinking reactions is that the reactions have to be slow enough to prevent gelation of the precursors before the freezing process is complete; hence the preparation of cryogels via these routes typically takes several hours.7,8 A much more time-efficient © 2017 American Chemical Society

Received: January 24, 2017 Accepted: March 30, 2017 Published: March 30, 2017 12165

DOI: 10.1021/acsami.7b01232 ACS Appl. Mater. Interfaces 2017, 9, 12165−12170

Letter

ACS Applied Materials & Interfaces

Figure 1. Schematic depiction of the developed process for the preparation of functional cryogel microstructures. The starting solution consists of an aqueous mixture of a photoreactive copolymer (left inset: x = 92.5 mol %, y = 5 mol %, and z = 2.5 mol %) and biomolecules. After freezing a heterophase system consisting of ice crystals and an unfrozen fraction of concentrated copolymer/biomolecule solution, i.e., the liquid microphase (LMP) is obtained. UV-illumination through a mask induces C,H-insertion reactions in the LMP (right inset) which cause copolymer cross-linking, surface-attachment and biomolecule immobilization all at once. Finally, the illuminated sample is thawed and washed to yield functional cryogel microstructures.

Figure 2. Gel content and swelling ratios of the prepared gels as a function of the illumination time. Solid lines serve as a guide to the eye. (a) Gel content of cryogels prepared from different polymer concentrations at −20 °C. (b) The gel content of frozen (cryogel prepared at −20 °C) and unfrozen (hydrogel prepared at room temperature) polymer solution with a polymer concentration of 80 mg/mL. (c) Influence of the UVillumination spectrum on the gelation kinetics of cryogels prepared at −20 °C from a polymer solution with concentration 80 mg/mL. In one case, the frozen system was illuminated with the full Hg spectrum and in the other case a bandpass filter at 365 nm was inserted in the light path. (d) Linear swelling ratio of cryogels prepared from 80 mg/mL polymer solutions at −20 °C as a function of illumination time, measured in water and in PBS buffer.

step. Hereto, we generated a tailor-made copolymer containing a photochemical cross-linker and placed an aqueous solution of

the copolymer mixed with desired biomolecules on a polymeric substrate followed by freezing and a short UV illumination step 12166


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proximity to the surface of the substrate have the possibility for surface attachment. All others are removed during washing, which explains the observed lag phase at the beginning (Phase 1). As the growth of the cluster progresses, at the percolation point, a cluster of interconnected chains spans throughout the complete LMP-volume of the sample. During this phase, many chains become attached to the forming surface-attached network, and a strong increase of the gel content is observed (Phase 2). Further UV exposure only forms cross-links between polymer chains already part of the network, but does not add new chains to the cluster. This results in the steady state seen in Phase 3. A second parameter that is very important for cryogel formation is the polymer concentration. At higher polymer concentrations, the lag phase is shorter and the percolation threshold is reached earlier (Figure 2a). The effect of the cryoconcentration can also be seen when the kinetics of gel formation of two identical copolymer solutions are compared, when one is illuminated while frozen, the other in the unfrozen state (Figure 2b). In these experiments copolymer solutions with a concentration of 80 mg/mL were illuminated either at room temperature to yield hydrogels or at −20 °C to obtain cryogels. In the latter case the cryo-concentration effect leads to a higher copolymer concentration in the liquid phase compared to the unfrozen system, again resulting in a shorter lag phase and a shorter time in which the percolation threshold is reached. Further, it should be noted that irradiation with and without a bandpass filter at 365 nm resulted in practically identical cross-linking kinetics (Figure 2c). This is most likely due to the fact that the absorption coefficient of the polymer solutions at 365 nm is rather low, which allows the radiation to penetrate deep into the sample volume, whereas UV light with a shorter wavelength is rapidly absorbed in the outer skin of the forming gels. Thus, only the long wavelength UV light determines the gel formation kinetics. Even though there is considerable light scattering in the frozen system, the cryogel thicknesses that can be achieved using the presented method are in the range of several millimeters. The cross-link density and thus the swelling properties of cryogels can be precisely tuned by choosing appropriate illumination conditions. Since many cross-linker units are contained in the polymer chains (in the polymers shown here approximately 140 per chain), the percolation point is reached when the conversion of the cross-linker units is still rather small. If the irradiation is continued after adding all chains to the network, the cross-link density further increases. The degree of cross-linking determines the mesh size and thus the swelling of the polymer network when exposed to water.20,21 To study the influence of irradiation time on the swelling behavior, cryogels prepared at −20 °C from copolymer solutions with a concentration of 80 mg/mL were illuminated for varying amount of times. To measure the swelling properties of the different cryogels, fluorescently labeled streptavidin (streptavidin-Cy5) was bound to the gels simply by adding it to the initial copolymer solution at a concentration of 10 μg/mL. The dye-labeled streptavidin contains many C−H groups and is therefore easily susceptible to be attacked by the activated benzophenone units, leading to a covalent attachment of the protein to the network during its formation. Since the label is covalently attached to the network, imaging of the gels in the swollen state is straightforward. To this end, the frozen solutions were illuminated through a mask to yield cylinders with a diameter of 1 mm. The cylinders were then immersed in

using a photomask. The photo-cross-linking process enables the generation of functional cryogel elements and their covalent attachment to numerous substrates, allowing for an effective integration into microfluidic systems. The developed process employs a statistical copolymer, which consists of three molecular building-blocks (Figure 1).15 The first and main component of the copolymer is N,Ndimethyl acrylamide (DMAA, 92.5 mol %), which serves as hydrophilic matrix to prevent nonspecific interactions with the biological environment (e.g., protein adsorption).16 4-methacryloyloxybenzophenone (MABP, 5 mol %) serves as the second building-block and provides the polymer with photoreactive groups. To increase the water solubility of the final copolymer, the sodium salt of 4-styrenesulfonate (SSNa, 2.5 mol %) is used as the third component so that the final copolymer has a solubility of greater than 300 g/L. For the preparation of surface-attached cryogels, an aqueous solution of the copolymer is first placed onto a polymeric substrate and then moderately frozen by lowering the temperature a few degrees below the freezing point of the solution. After freezing, the system is illuminated with UV light through a photomask to induce the chemical reactions in the LMP. Upon exposure the benzophenone groups contained in the copolymer undergo an n−π* transition into a biradicaloid triplet state, which abstracts almost any hydrogen atom from neighboring C−H groups present in close proximity. Consequently, two radicals are formed which can recombine to establish a covalent bond connecting the two chains leading to a (formal) C,H-insertion cross-linking reaction (CHic).17 The presence of water does not significantly hamper the process.18 Additionally, groups close to the surface bind to C− H groups present on the surface of the substrate, so that crosslinking of the polymer chains and surface attachment are accomplished in one step. Simultaneously, any biofunctional molecule mixed into this solution prior to freezing is attached to the polymer matrix. After thawing and washing functional, surface-attached cryogel-microstructures are obtained (Figure 1). To gain more insight into the photo-cross-linking process of the copolymer in the frozen state and to explore which exposure dose is required to form a stable network, the gel content was monitored as a function of illumination time. Seal frames were fixed on standard PMMA microscope slides to obtain wells with dimensions of 1.5 mm × 1.6 mm and a height of 0.25 mm. The wells were filled with aqueous copolymer solutions of varying concentrations and sealed with a coverslip. Subsequently, the samples were frozen at −20 °C, similar to values reported in the literature.19 The frozen samples were illuminated with UV-light from the bottom, i.e., through the PMMA slides, for varying times. After completion of the UV treatment, the nonbound copolymer was removed in a subsequent washing step with distilled water. Finally, the samples were dried and the gel content was determined gravimetrically (Figure 2a). Stable cryogels with high gel contents can be prepared within less than 1 min of illumination. The observed kinetics of the gelation process can be divided into three phases. First, the gel content increases only marginally over time. This is followed by a sudden, strong increase that plateaus so that a steady state is rapidly reached. This behavior is due to the fact that the cross-linking reactions in the LMPs are percolation processes:17 Upon photochemical activation first small isolated clusters of connected polymer chains are formed. Only those that are coincidentally in close 12167


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lower concentrations (Figure 3), in agreement with the cryogel literature.10,28,29 Because the cryogels are generated in a photoreaction, spatially defined gel formation can be achieved simply by restricting the illuminated area. To demonstrate this, a polymer solution (80 mg/mL) containing streptavidin-Cy5 (10 μg/mL) was frozen at −20 °C and subsequently illuminated through a photomask for 400 s. The mask consisted of circular holes with varying diameters (d) ranging from 100 to 500 μm, which were separated by different spacing (d-3*d) resembling a microarray. After thawing, the structures were developed using distilled water. Fluorescence and bright field images of the obtained structures at different magnifications are shown in Figure 4a. Well-separated, cylindrical cryogel elements were obtained for all diameters. The structures were firmly attached to the PMMA substrate and resisted prolonged washing with water on a shaker at 150 rpm for several days. This seems to be a very

water or phosphate-buffered saline (PBS) and allowed to swell for 24 h. The linear swelling ratios of the gels were then determined by fluorescence microscopy and are plotted against the respective illumination times (Figure 2d). In agreement with the expectations,22,23 the swelling ratio decreases with increasing illumination time. It was also found that the structures swell more strongly when immersed in water compared to PBS. This responsive behavior can be explained by the screening of charges on the polymer chains by the salt molecules, which reduces the swelling capacity of the material. The pore structure of cryogels is governed by the size and structure of the ice crystals during gelation. During ice crystals growth, the polymer molecules partition into the concentrated unfrozen phases (the LMP).4 The water in the LMP remains unfrozen due to the high concentration of the cryogel precursors. In the case of macromolecular precursors this effect is most probably caused by the agglomeration of water molecules to the polymer chains (hydration effect).24,25 As shown by Jellinek and Fok, with increasing formation of ice crystals, the viscosity in the LMP is strongly increased and the freezing process is slowed down significantly so that a true equilibrium is not reached on experimental time-scales.24 Such a kinetic effect could well-explain the influence of the polymer concentration on the cross-linking behavior (Figure 2a). Generally, it can be stated that the faster the crystallization, the smaller the ice cells.26,27 Accordingly, the freezing rate is the most widely used parameter to influence cryogel morphologies. In this work the freezing rate was controlled by the Peltier device on which the samples were placed. Figure 3 shows

Figure 3. Fluorescence micrographs of swollen cryogels in PBS prepared from solutions with different polymer concentrations (25, 50, and 80 mg/mL) and frozen at different temperatures (−15 and −30 °C). The parameters are indicated in each image. The pore walls of the cryogels were fluorescently labeled by covalent attachment of streptavidin-Cy5. All images were taken at the same magnification and the shared scale bar is in the bottom right image.

Figure 4. (a) Micrographs of a photo lithographically structured cryogel array consisting of cylinders with varying diameters and spacing. The bright field micrograph on top shows the array slightly tilted, to give an impression of the 3D structure (the height of the structures is around 340 μm). The fluorescence micrograph on the lower left provides an overview of the whole array, whereas the magnified images in the middle and on the right give an indication of the process resolution and the achieved pore structure. (b) Schematics and fluorescence micrographs of cryogel microstructures functionalized with ssDNA or protein (EpCAM). The functionality of the immobilized compounds was confirmed by incubation with fluorescent counterparts (i.e., complementary ssDNA-Cy5 or anti-EpCAM-Cy5). As a control blank (i.e., nonfunctionalized) cryogel microstructures incubated under identical conditions are shown.

micrographs of two swollen cryogels that were prepared from the same precursor solution (80 mg/mL copolymer and 10 μg/ mL streptavidin-Cy5 in water) but frozen at different temperatures (−15 °C and −30 °C, respectively). The measured freezing rates were 2.8 and 4.0 mm/s and yielded quite strongly differing pore sizes. Additionally, cryogels prepared from solutions with higher concentration have smaller pores and thicker walls compared to the ones prepared with 12168


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attractive feature, because it prevents delamination, loss of cryogel elements even under shear flow conditions and enables a stable anchorage of cryogel elements in fluidic systems. All cylinders had a height of around 340 μm and were found to stand upright, except for the smallest structures, which showed some tendency to ground collapse. Because of the universal reactivity of the groups inducing the C,H-insertion reactions, any biofunctional molecule can be easily incorporated in the cryogel matrix. This was demonstrated successfully for the case of DNA molecules as well as for proteins. Therefore, in the first case, a DNA probe (ssDNA, 36 bp) and in the second case a protein (EpCAM) were added to polymer solutions (both 80 mg/mL) prepared in suitable buffers. Both solutions were frozen and illuminated for 120 s through a photomask containing 250 μm holes to yield functional cryogel cylinders. The functionality of the immobilized compounds was confirmed by incubation with its fluorescently labeled counterpart, i.e., complementary ssDNACy5 or an anti-EpCAM-Cy5 antibody as shown in Figure 4b. In both cases, a strong fluorescence caused by the specific binding of the analyte was observed. The buffer composition has a profound influence on the cryogel morphology, which can be explained by the strong influence of the salt on the LMP caused by a more or less strong freezing point depression. In the experiments described in Figure 4b, this salt effect is responsible for the significantly stronger swelling of the protein-functionalized cryogel (prepared in a buffer with an osmotic concentration 300 mOsm/L), compared to the sample containing the DNA (30 mOsm/L). In conclusion, photoinduced C,H-insertion reaction represents a simple and versatile process for the preparation of surface-attached, microstructured, and functional cryogels. A key feature of the process is that it allows simultaneous cryogel formation, biomolecule immobilization and surface attachment all in one single and rapid photoreaction step. The combination of photolithography and cryogel formation opens new horizons for cryogel-based systems. The simplicity of the cryogel formation process allows for the simple integration of functional cryogel elements into various analytical platforms. In following communications, we will report on the use of these gels in a variety of bioanalytical applications.

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Zentrales Innovationsprogramm Mittelstand - ZIM (KF2162028AJ3) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Dr. Richard Kneusel for proofreading the manuscript. This research was supported by the Zentrales Innovationsprogramm Mittelstand - ZIM (KF2162028AJ3).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01232. Experimental details on polymer synthesis and characterization, universal preparation of cryogels, gel content measurements, swelling measurements, pore size measurements, preparation of cryogel microarray and preparation of DNA and protein functionalized cryogel structures (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Marc Zinggeler: 0000-0003-4926-9791 Author Contributions †

M.Z. and J.N-S. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 12169


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Functional Cryogel Microstructures Prepared by Light-Induced Cross

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