Fabrication of Flexible Hydrogel Sheets Featuring Periodically Spaced

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Fabrication of flexible hydrogel sheets featuring periodically spaced circular holes with continuously adjustable size in real time Joachim Jelken, Pandiyarajan Chinnayan Kannan, Jan Genzer, Nino Lomadze, and Svetlana A. Santer ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09580 • Publication Date (Web): 16 Aug 2018 Downloaded from http://pubs.acs.org on August 18, 2018

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ACS Applied Materials & Interfaces

Fabrication of flexible hydrogel sheets featuring periodically spaced circular holes with continuously adjustable size in real time Joachim Jelken,1 C. K. Pandiyarajan,2 Jan Genzer,2 Nino Lomadze,1 Svetlana Santer1*

1

2

Institute of Physics and Astronomy, University of Potsdam, 14476 Potsdam, Germany

Department of Chemical & Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA

AUTHOR EMAIL ADDRESS: [email protected] Keywords: Photosensitive polymers, PNIPAm, hydrogels, UV cross-linking, stimuli responsive structured polymer films, azobenzene containing molecules

ABSTRACT We report on the formation of stimuli responsive structured hydrogel thin-films whose pattern geometry can be adjusted on demand and tuned reversibly by varying solvent quality or by changing temperature. The hydrogel films, ~100 nm in thickness, were prepared by depositing layers of random copolymers comprising N-isopropylacrylamide (NIPAm) and UV-active methacryloyoxybenzophenone (MABP) units onto solid substrates. A two-beam interference pattern technique was used to cross-link the selected areas of the film; any unreacted material was extracted using ethanol after ultraviolet (UV) light assisted crosslinking. In this way we produced nano-holes, perfectly ordered structures with a narrow size *

Corresponding author: [email protected] 1

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distribution, negligible tortuosity, adjustable periodicity, and a high density. The diameter of the circular holes ranged from a few micrometers down to several tens of nanometers; the holes periodicity could be adjusted readily by changing the optical period of the UV interference pattern. The holes were reversibly closed and opened by swelling/shrinking the polymer networks in the presence of ethanol and water, respectively, at various temperatures. The reversible regulation of the hole diameter can be repeated many times within a few seconds. The hydrogel sheet with circular holes periodically arranged may also be transferred onto different substrates and be employed as tunable templates for deposition of desired substances.

Introduction During the past few years stimuli responsive structured polymer films have gained increased attention due to a wide range of applications in tissue engineering, cell seeding, bionics, or microfluidics.1,2,3,4,5,6,7,8 The unique feature of stimuli responsive structured polymer sheets is the ability to change the pattern size upon altering an external stimulus. Depending on the functionality integrated within the polymer films thermal,9,10,11,12,13,14 chemical, 15,16,17,18,19,20,21 magnetic, or electro-magnetic stimuli 22 , 23 , 24 , 25 can be employed to control the pattern geometry and size, and thus control the function of the structured polymer film on demand. Even though different geometries such as pillars, stripes, holes and etc. can be produced within the polymer film to obtain distinct patterns, in this paper we will discuss exclusively the formation of circular holes, leading to a pattern reminiscent of a membrane. Polymer films containing circular holes can be produced by different fabrication methods such as templating with microstructures and colloidal particles of polymer thin films,2,26,27 microphase separation of diblock or triblock copolymers,28,29,30,31,32,33,34,35,36 and in gel thin-films, 37,38,39 or using protein-polymer conjugates. 40 In all these methods, the size 2


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distribution and ordering of pattern are governed either by the template geometry (i.e., colloids or proteins) limiting also the polymer film thickness, or be restricted by the size of the formed nanophases (e. g. Self-assembly and Non-solvent Induced Phase-Separation (SNIPS) method). To obtain a uniform well-ordered distribution of holes within the polymer sheet of desirable thicknesses, one can choose photo-lithography methods based on selective cross-linking of the polymer material followed by appropriate development. The challenge to this approach is to employ suitable polymers that fulfill the requirements, such as photoresponsivity for cross-linking, chemical and mechanical stability for a reliable long-term performance of the structured polymer film, controllability of the film thickness, pore size and distribution, low tortuosity and small pore thickness for shorter diffusion time through the patterned polymer film, or biocompatibility in case of biological applications. Herein, we disclose a versatile system employing a photo cross-linking process, which meets all of the above requirements. Specifically, we synthesized a precursor random copolymer that incorporates N-isopropylacrylamide (NIPAm) and ultraviolet (UV)-active methacryloyoxybenzophenone (MABP) units. PNIPAm is a thermo-responsive polymer, which exhibits a lower critical solution temperature (LCST) of 32°C in water. Above LCST, the PNIPAm chains undergo a coil-to-globule transition due to breaking intermolecular Hbonding between PNIPAm and water, while promoting the formation of intramolecular Hbonding among the polymer. The presence of MABP facilitates cross-linking of the NIPAm chains upon UV-irradiation at 254/365 nm; it also decreases the LCST of the PNIPAm copolymer (poly(NIPAm-co-y%MABP)), depending on the content of MABP (i.e., y).41,42 The responsivity of the hydrogel film can thus be achieved by varying solvent type and/or temperature. Applying UV irradiation by varying the UV dose allows for selective crosslinking of the polymer layers. Subsequent exposure of the film to a good solvent removes any uncross-linked material from the polymer layer. In this study we utilize irradiation with UV

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interference pattern to produce well-order checkerboard patterns within the polymer film. The size and distribution of the produced circular holes (termed also hereinafter pores) are controlled by the periodicity of the UV interference pattern and the UV irradiation dose. Since the UV-cross-linking reaction is very fast (few seconds under the given UV dosage, see below), we add UV absorbing agent (azobenzene containing molecules by simply mixing it within the polymer) to control the spatial distribution of polymer cross-linking density in the film. UV-irradiated azobenzene molecules convert optical energy into mechanical work through a photo-isomerization process in which the molecule conformation changes from trans- to cis-state.43 The photo-isomerization is completely reversible and free of any sideproducts. The energy excess of the incoming UV light is transferred to multiple photoisomerization processes of the incorporated azobenzene molecules. Since the employed azobenzene (DR1) is not covalently attached to the polymer, the azobenzene molecules are completely removed from the hydrogel sheet by rinsing the irradiated film with ethanol. This method of producing a flexible hydrogel films with periodically spaced circular holes is fast and can be readily adjusted to generate any type of desired holes size, order and periodicity. The solvent- and temperature-driven swelling of the polymer network causes a reversible change in the size of the holes. This allows using the fabricated polymer films as porous membranes for selective deposition of different substances.

Experimental Part Materials Synthesis of MABP. 5.25 g of methacryloylchloride in 20 ml of dichlormethane was added to the solution of 5.06 g of 4-hydroxybenzophenone in 100 ml of dichloromethane at 0°C. The mixture was allowed to reach room temperature and stirred for ~16 h. After completion of the reaction, monitored via thin layer chromatography (TLC), the reaction mixture was 4


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filtered, and the organic phase was washed with the solution of 5 % HCl, 5% NaHCO3 and water. The resulting organic phase was dried over anhydrous MgSO4 and added to ~200 ml of cold n-hexane and allowed to precipitate at -20 °C overnight. The precipitated monomer was filtered and subsequently recrystallized using ethyl acetate-hexane mixture. Synthesis of poly(NIPAm-co-y% MABP). The copolymer was synthesized through freeradical polymerization using azobisisobutyronitrile (AIBN) as a free radical initiator. Calculated amounts of PNIPAm, MABP and AIBN were dissolved in 10 ml of 1,4-dioxane, degassed under nitrogen air for three freeze-thaw cycles. The mixture was stirred in a preheated oil bath at 60°C for ~16-18 hours (see Table S1 in Supporting Information). The polymer was precipitated in petroleum ether and washed thoroughly with petroleum ether using 100 ml (repeated 4 times). The molecular weight of polymers was determined by static light scattering (see Table S1, Supporting Information ). 39,40 Dispersed red one (DR1) was purchased from Sigma Aldrich and used without further purification. Poly(3-hexylthiophen-2,5-diyl) (P3HT) was purchased from Merck and dissolved in chlorobenzene to prepare a solution with the concentration of 20 mg/ml. Polyallylamine-hydrochloride was purchased from Sigma Aldrich and dissolved in water with the concentration of 0.1 mg/ml. Polydimethylsiloxane (PDMS) was purchased from Sigma Aldrich. The PDMS stamps were prepared by cast molding using Sylgard 184. This was done by mixing elastomer and curing agent in a 10:1 ratio, pouring the mixture onto the polymer film and curing it for 24h at RT. The stamps were then detached from polymer films and deposited on glass slides. Polydimethylsiloxane (PDMS) was purchased from Sigma Aldrich. The PDMS stamps were prepared by cast molding using Sylgard 184. This was done by mixing elastomer and curing agent in a 10:1 ratio, pouring the mixture onto the polymer film and curing it for 24h at RT. The stamps were then detached from polymer films and deposited on glass slides.

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Preparation of surface-attached PNIPAm hydrogel. The precursor solution of poly(NIPAmco-y% MABP) with a concentration of 60 mg/ml was prepared in ethanol. The solution was spin casted on silicon or glass substrates, which was pre-deposited with a monolayer of 3Aminopropyl)triethoxysilane or 3-(trimethoxysilyl)propylmethacrylate) at 800 rpm for 1 minute to obtain a film thickness of 800 nm. Afterwards the film was irradiated with UV light (UV dosage noted below) with orthogonally aligned interference patter (IP): after the first UV irradiation the sample was rotated by 90° to perform the second UV illumination. Rinsing the sample with ethanol after UV exposure removes any un-reacted material from the film resulting in a checkerboard pattern within the hydrogel film.

Afterwards the sample was dried in a stream of dry nitrogen gas. For

controlling the kinetic of the cross-linking of PNIPAM the azobenzene-dye DR1 (60 mg/ml) was mixed with the precursor copolymer solution. The solution was stirred for 24 h and filtered afterwards. The polymer film was deposited in the same way as described earlier. The irradiation time varied from 3 s to 10 min. The organic semi-conductor P3HT was spin casted at 1200 rpm for 1 min on the structured poly(NIPAm-co-y%MABP) + DR1 hydrogel film to perform the fluorescence microscopy measurements. For the patterned hydrogel film transfer the water soluble polyallylaminehydrochloride (PAH) was spin casted on a glass substrate. Afterwards the poly(NIPAm-coy%MABP) was deposited onto the PAH layer and the structured hydrogel sheet was created as described above.

Methods The height and the morphology of the hydrogel sheet were measured by Atomic Force Microscopy (AFM). The measurements were carried out using NTEGRA (NT-MDT) operating in intermittent contact mode. We used commercial tips (Nanoworld-Point probe)

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with a resonance frequency of 320 kHz, and a spring constant of 42 N/m. The experiments were carried out under ambient conditions, i.e., at room temperature with a relative humidity of 55%. The UV interference pattern was generated using a homemade Lloyd’s mirror interferometer with an UV-Laser (KIMMON He-Cd Laser, λ = 325 nm, I = 13 mW/cm2). The period of the interference pattern was controlled by adjusting the angle between the two interfering beams. To record the diffraction pattern generated during irradiation (λ = 491 nm, 50 mW, Cobolt Calypso) of the structured polymer film, we used a homemade setup equipped with a digital camera. Fluorescence measurements were carried out using an optical microscope (Olympus BX 51, 100x Objective UMPlanFl) with a mercury lamp for the excitation. The bandpass filter (510-550 nm) provides a green light irradiation of the sample; an additional edge filter (590 nm) in front of the camera was used to record the fluorescence. The scanning electron microscope (SEM) measurements were performed with a Zeiss Ultra plus field- emission-scanning-electron-microscope. The samples were prepared on a silica waver and coated with an additional 20 nm thick gold layer. The gold layer was applied by thermal evaporation.

Results and Discussion To produce patterned hydrogel films with reversible tunable holes we synthesized a poly (NIPAm-co-y% MABP) copolymer (Figure 1a), where the MABP mole percentage in the copolymer (i.e., y%) is systematically varied to control the cross-link density of the polymer network. We specifically chose NIPAm as our model system due to its hydrophilicity and LCST behavior in water. The presence of MABP in the precursor copolymer allows us to cross-link the polymer film photo-chemically using UV light at 254 or 365 nm. Briefly, the 7



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UV-active benzophenone units present in the polymer absorbs UV light instantaneously and form triplet bi-radicals on the carbonyl group (─C•─O•). These radicals then abstract a proton from any neighboring CH2 group (backbone or side chain) in the vicinity and create two carbon-based radicals (─C•─C•─). Upon recombination of these two radicals form a covalent (─C─C─) linkage among the polymer chains and with the substrate surface due to the presence of alkoxysilanes, thus forming the surface-attached networks.41,42,44

Figure 1. (a) Chemical structure of the poly(NIPAm-co-y%MABP), where y = 5.0, 7.5, or 10%. (b) Chemical structure of azobenzene molecule (Disperse Red 1). (c) Sketch of the cross-linking process. The thin film (300 – 800nm in height) consisting of the polymer (blue lines) and the azobenzene molecules (schematically represented as red rod) is irradiated with interference pattern (λ = 325 nm, I = 13 mW/cm2) of periodicity D resulting in cross-linking of the polymer (indicated by the yellow dots).

To engineer pores/holes within the polymer layer, we apply cross-irradiation with a two-beam interference pattern. In this method, the sample is initially irradiated with an 8


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interference pattern (λ = 325 nm) forming periodic lines of non- and cross-linked polymer material. The periodicity of cross-linking is equal to the optical periodicity of the interference pattern. Afterwards the sample is rotated by 90° and a second irradiation is conducted with similar irradiation parameters. Rinsing the sample with ethanol removes any uncross-linked polymer (at the areas of minimal UV light exposure) and the patterned hydrogel sheet is formed (Figure 1). A series of AFM micrographs showing step by step the formation of the structure within the hydrogel film (sample as prepared, after irradiation and after treatment with ethanol) is presented in the Supporting Information (see Figure S1). Figure 2 shows AFM micrograph of the structured hydrogel film produced using the methodology described above. The periodicity of the interference pattern is 5 µm, and the diameter of the holes is 1.6 µm. The depth of the produced holes is equal to the initial thickness of the polymer film, i.e., ~700 nm.

Figure 2. AFM micrograph of structured poly(NIPAm-co-5%MABP) hydrogel film irradiated with interference pattern (λ=325 nm, I =13 mW/cm2, tirr = 5 sec) of 5 µm 9



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periodicity. Below the cross-section of the patterned hydrogel sheet acquired along the white line is depicted. The pores are 700 nm deep and span across the film. The pore diameter at the substrate is ~1.6 µm.

The cross-linking of the MABP-containing polymer thin films with UV light is very fast; it takes only a few seconds with the current irradiation dosage (I =13 mW/cm2). Further reduction of the UV irradiation dosage and time results in inhomogeneous cross-linking of the film throughout the film depth. To solve this problem, we integrated within the polymer film a “UV absorber”, an azobenzene-containing molecule, which absorbs excess energy of the incoming light during a photo-isomerization process involving a trans- to cis-state transition of the UV absorber. Azobenzene-containing molecule DR1 (Disperse red 1) (Figure 1b) was blended into the poly(NIPAm-co-y%MABP) polymer film (see Materials and Methods). The content of the DR1 molecules was chosen in such a way that per one monomer unit of the polymer two DR1 molecules were added. Since the azobenzenes do not cross-link during the UV exposure, simple rinsing with a good solvent (i.e., ethanol) for DR1 of the UV-irradiated sample removed the azobenzenes completely. This has been proved by the UV/vis absorption spectra of the polymer film recorded before and after rinsing with ethanol (see Supporting Information, Figure S2). The DR1 has a characteristic absorption band at 461 nm, which is visible in the film before and disappears completely after the exposure to ethanol. Moreover, it is also visible by naked eye, since DR1 has a pronounced red color, which disappears after ethanol rinsing. Using this “excess energy absorber”, we were able to control the size of the pores within the structured hydrogel film by adjusting the irradiation time at fixed UV light dosage (I =13 mW/cm2) and periodicity of the IP. Figure 3 shows the structured polymer film fabricated as shown in Figure 1c. In this experiment, the azobenzene-containing polymer film, comprising poly(NIPAm-co-10%MABP) and DR1, of initial thickness 200 nm was cross-irradiated with UV interference pattern of periodicity 2

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µm for 120 seconds followed by treatment with ethanol to remove any uncross-linked material. The 1.2 µm wide holes with the depth of 200 nm penetrate through the whole thickness of the polymer film (Figure 3c). The perfectly aligned checkerboard patterns are produced over the whole irradiation area as large as ~ 1 cm2.

Figure 3. (a-b) AFM micrographs of the structured poly(NIPAm-co-10%MABP) + DR1 hydrogel film cross-irradiated during 120 s with UV interference pattern of D = 2 µm. The depth of the generated pores is h = 200 nm (equal to the total thickness of the polymer film), and the diameter d =1.2 µm. (c) Schematic representation of the patterned hydrogel sheet. The yellow dots indicate schematically cross-links in the polymer layer.

The size of the pores within the patterned hydrogel sheet can be turned by varying the irradiation time. Figure 4 shows the dependence of the hole diameter as a function of irradiation time for different periodicities of the interference pattern. The pore diameter of the structured poly(NIPAm-co-10%MABP) hydrogel sheet decreases with increasing the irradiation time. The shortest exposure time to cross-link the polymer and maintain the film was ~60 seconds (Figure 4a), given the employed UV dosage quoted earlier.

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Figure 4. (a) AFM micrographs of the structured poly(NIPAm-co-10%MABP) + DR1 hydrogel film with a 2 µm period for different irradiation times. The pore diameter decreases and the pore spacing increases with increasing irradiation time. The depth of the pores is about 280 nm. (b) The dependence of the pore size on irradiation time at different periodicities of the interference pattern.

As evidenced in Figure 4b, the periodicity of the interference pattern alters the pore size significantly as well. Specifically, for a 5 µm period the pore diameter can be adjusted from 2.75 µm (90 s UV irradiation time) to 317 nm (600 s UV irradiation time). Increasing the irradiation time is further cross-linking the whole polymer layer and the structure within the hydrogel sheet disappears. Changing the period of the interference pattern to 2 µm and 1 µm, the pore size decreases to 270 nm and 160 nm, respectively (Figure 4b). From this it follows that the pore diameter can be controlled from the micrometer to the nanometer range by simply adjusting the UV irradiation dose. The smallest pore size we produced was 90 nm (sheet thickness of 280 nm) at an irradiation periodicity of 500 nm resulting in the density of 4.108 pores/cm2.

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The structured hydrogel film can be transferred to any substrate using a sacrificial layer placed between the polymer film and the glass substrate (Figure 5). Here we utilize poly(allylamine hydrochloride) (PAH) as a water-soluble layer. The PAH was first spin coated onto a glass substrate onto which a thin (200 nm) layer of poly(NIPAm-co7.5%MABP) + DR1 was deposited (Figure 5a).

Figure 5. (a) Scheme of the structured hydrogel sheet transfer process. A water soluble poly(allylamine hydrochloride) (PAH) layer was added between the polymer film and the glass substrate. After irradiation and development, the patterned hydrogel film was generated followed by the evaporation of the gold layer on top. When the sample was taken into a water bath, the PAH layer was dissolved and the hydrogel film is floating on the water surface. The structured polymer film was then picked out with a silicon substrate. (b) Scanning electron microscopy (SEM) and (c) atomic force microscopy (AFM) images of the patterned hydrogel sheet after the film transfer, which created a fold and is changing the shape of the pores.

The sample was then cross-irradiated with a UV-light (at 325 nm) and extracted in ethanol (to remove any unreacted material), which resulted in a structured polymer film (periodicity D = 5 µm). Subsequently, the sample was immersed in water, which dissolved the sacrificial layer PAH thus leaving the hydrogel film floating on top of the water (Figure 5a). The specimen was then collected on a silicon substrate by submerging it in the water 13



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bath. Figures 5b and 5c represent the SEM and AFM micrographs, respectively, of the patterned hydrogel film transferred onto the silicon substrate. For SEM measurements, the structured hydrogel sheet was covered by 20 nm gold layer. From the AFM measurement it is evident that during the transfer process the polymer film was stretched thus resulting in an elliptical shape of the pores (Figure 5c). Such structured polymer films can be used for the selective deposition of different materials in a controlled spatial manner. For example, we deposited an organic semiconductor poly(3hexylthiophene) (P3HT) selectively inside the pores of the patterned polymer sheet (Figure 6). The P3HT exhibits strong fluorescence and thus can be easily viewed. For the deposition, the P3HT was dissolved in chlorobenzene to obtain a solution with the concentration of 20 mg/ml and spin casted on the structured poly(NIPAm-co-10%MABP) film. The P3HT was excited by green light irradiation (510-550 nm) and the florescence was recorded with an edge filter (590 nm) in front of the camera. The fluorescence microscope images show that the P3HT enters the pores of the structured polymer film and filling them up (Figure 6). The diameter and the periodicity of the deposited material spots are governed by the geometry of the pores inside the polymer sheet. In Figure 6 we changed the periodicity from 5 µm to 1 µm and the diameter of the spots between 3 µm and 250 nm. This gives the possibility to structure the organic semiconductor P3HT in a well-defined manner, which is of high interest for device production, e.g., of OLED devices. In general, one can use this structure in order to selectively deposit any type of materials. For instance, we have prepared in the similar way a PDMS stamp having posts-shaped surface (see Figure S3 in Supporting Information).

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Figure 6. (a) Fluorescence microscope images of structured poly(NIPAm-co-10%MABP) + DR1 hydrogel film with a 5 µm period and a pore size of 3 µm. The organic semiconductor poly(3-hexylthiophene) (P3HT) used as a fluorescence marker was spin cast on the patterned hydrogel sheet filling up the pores. The sample thickness was 280 nm. In (b) the period of the pores was 1 µm and the pore diameter 250 nm. (c) Chemical structure of the P3HT.

Once the patterned polymer film has been produced, the size of the pores can be controlled by external means such as solvent type and temperature. Moreover, it is possible to open and close the pores completely and reversibly. To demonstrate the tunable pore size of the hydrogel film, we construct the following optical set-up. A blue laser (λ = 491 nm) is focused on the backside of the glass surface carrying the structured polymer film and is diffracted at the porous structure. The reflected diffraction pattern is recorded at the white screen (Figure 7a).

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Figure 7. Photos of diffraction pattern produced by irradiation of the structured poly(NIPAm-co-10%MABP) +DR1 hydrogel sheet (D =1 µm, d =160 nm) with a laser of λ = 491 nm. The laser beam was focused on the backside of the structured polymer film and the diffraction pattern was recorded in reflection. (a) Shows dry hydrogel film with open pores. (b) Due to pore closing in the presence of ethanol, diffraction pattern disappeared while the polymer film swelling in water results in partial closing of the pores. The photo of the diffraction pattern in the presence of water is shown as well. (c) After drying (tdry=10 seconds) the pores open and the diffraction pattern appears. (d-e) Swelling of the hydrogel film (h =280 nm) with pores of 5 µm periodicity and 317 nm diameter. The corresponding video is shown in Figure S4, Supporting information.

When ethanol droplet is deposited on the structured hydrogel sheet, the polymer swells and the pores close, as documented by the disappearance of the diffraction pattern (Figure 7b). During drying of the hydrogel film, the pores open and the diffraction pattern appears again (Figure 7c). Using this approach, one can measure the kinetic of the hydrogel swelling (see real-time video of the structured hydrogel film swelling and drying in Figure S4, Supporting information). As can been seen from the video, the process of pore opening

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is completed within 10 seconds of the drying processes. Swelling of the porous hydrogel sheet is fully reversible and can be conducted many times. Partial closing of the pores within the hydrogel film can also be achieved. Since the swelling ratio of the poly(NIPAm-co-5%MABP) polymer network in the presence of water (swelling ratio ~ 2.8) is less than those induced by ethanol (swelling ratio ~ 5.0), the size of the pores decreases, but the pores do not close completely.44 The diffraction pattern does not disappear fully; only the intensity of the diffraction maximum is reduced (Figure 7). These experiments demonstrate that the pores of the patterned hydrogel film can be opened and closed on demand by swelling and shrinking of the polymer network. The pores can also be opened and closed on demand by varying temperature. Decreasing the water temperature from 50°C to 23°C results in partial closing of the pores due to swelling of the poly(NIPAm-co-MABP) in the presence of water. During this process the intensity of the diffraction pattern decreases (Figure 8 and Figure S5). As shown in Figure S6 (Supporting Information) the pores of the patterned hydrogel sheet close during decreasing of the temperature. The intensity of the pattern in warm water (red line in Figure 8) is less than in dry state that we assign to scattering of the diffracted light and partial swelling of the structured polymer film.

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Figure 8. The change in the intensity of diffraction pattern generated under irradiation (λ= 491nm) of the structured polymer film (683 nm pore diameter). The intensity is highest when the hydrogel sheet is in dry state (black line), the pores are partially closed when the hydrogel film is exposed to water of 23°C resulting in the lowest diffraction pattern intensity (blue line). At elevated water temperatures, the PNIPAm collapses and the pore size increases (red line).

Conclusions We have demonstrated that utilizing photo-active poly(NIPAm-co-y%MABP) copolymer one can fabricate patterned hydrogel sheets with a uniform, well-ordered distribution of pores and pore density up to 4.108 pores/cm2. We employed photo-lithography based method via selective cross-linking of the polymer layers in a controlled manner. Poly(NIPAm-co-y% MABP) was chosen since it possesses photo-responsivity (due to the presence of MABP) for UV light induced well-defined local cross-linking, and the thermo- and solvent responsivity for reversible tunable pore size manipulation. The pore distribution and size of the produced

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structured polymer films can be adjusted by UV irradiation time and periodicity of the interference pattern generated during interference of two UV laser beams. Perfectly ordered checkerboard patterns with a narrow size distribution, negligible tortuosity, adjustable periodicity, and a high porosity were produced. The pore diameter ranged from ~3 µm down to ~90 nm, the periodicity can be adjusted readily from 5 µm down to 500 nm, while the thickness of the hydrogel film is in the range between 300 nm and 800 nm. To achieve such a precise control of spatial distribution of the cross-linking density, we added absorbing agent (azobenzene containing molecules) by mixing it within the parent random copolymer. UVirradiated azobenzene molecules absorb energy excess by converting optical energy in mechanical work during multiple photo-isomerization process from trans- to cis-state. Since the used azobenzene (DR1) is not covalently attached to the polymer, the azobenzene molecules are completely removed out of the hydrogel by rinsing the irradiated film with ethanol. The fabricated nano- structured hydrogel films exhibit reversible opening and closing of the pores during swelling/shrinking process of the polymer network in the presence of different solvents such as water and ethanol, and/or by changing temperature. The reversible regulation of the pore diameter was repeated many times and took place within a few seconds. The structured polymer films may be transferred onto different substrates and be employed as tunable templates for deposition of desired substances.

Supporting Information: Table S1 depicts the molecular parameters of poly(NIPAm-co-y% MABP). Figure S1 illustrates step the formation of the structure within the hydrogel sheet as characterized by AFM measurements.

Figure S2 demonstrates UV-vis spectra of the poly(NIPAm-co-

10%MABP)+DR1 film as prepared, after cross-linking and after rinsing with ethanol. Figure S3 shows PDMS stamp prepared using structured hydrogel film as a stamp. Figure S4: real

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time video of the swelling and drying process of the structured polymer film. Figure S5 demonstrates the intensity change of the diffraction pattern between the dry and swollen state (solvent: water) of the structured hydrogel film. Figure S6 shows video of shrinking of the pores with time when the hot water is cooling down to room temperature.

ACKNOWLEDGMENTS: This research is supported by the Helmholtz Graduate School on Macromolecular Bioscience (Teltow, Germany). CKP and JG acknowledge financial support from the National Science Foundation under Grant No. DMR 1809453.

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Table of Contents

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Fabrication of Flexible Hydrogel Sheets Featuring Periodically Spaced

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