Facile Fabrication of a Modular 'Catch and Release' Hydrogel

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Biological and Medical Applications of Materials and Interfaces

Facile Fabrication of a Modular ‘Catch and Release’ Hydrogel Interface: Harnessing Thiol-Disulfide Exchange for Reversible Protein Capture and Cell Attachment Tugce Nihal Gevrek, Merve Cosar, Duygu Aydin, Elif Kaga, Mehmet Arslan, Rana Sanyal, and Amitav Sanyal ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00802 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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ACS Applied Materials & Interfaces

Facile Fabrication of a Modular ‘Catch and Release’ Hydrogel Interface: Harnessing Thiol-Disulfide Exchange for Reversible Protein Capture and Cell Attachment Tugce Nihal Gevrek,a Merve Cosar,a Duygu Aydin,a Elif Kaga,a Mehmet Arslan,a Rana Sanyala,b and Amitav Sanyal*a,b a

b

Department of Chemistry, Bogazici University, Bebek, Istanbul, 34342, Turkey

Center for Life Sciences and Technologies, Bogazici University, Bebek, Istanbul, 34342, Turkey Email: [email protected] Tel: 90-212-359-7613 Fax: 90-212-287-2467

Keywords: Hydrogel interfaces, thiol-disulfide exchange chemistry, bioconjugation, reversible conjugation, protein and cell attachment.

ABSTRACT Surfaces engineered to ‘specifically capture’ and ‘release on demand’ analytes ranging from biomolecules to cells find niche applications in areas such as diagnostics and detection. Utilization of a disulfide-based linker as a building block allows fabrication of a novel hydrogel

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based platform that incorporates a ‘catch and release’ attribute. Hydrogels incorporating pyridyl disulfide group as a thiol-reactive handle were prepared by photopolymerization in presence of a PEG-based crosslinker. A range of bulk and micro-patterned hydrogels with varying amounts of the reactive group were prepared using PEG-based monomers with different chain lengths. Thiolcontaining molecules were conjugated to these hydrogels through the thiol-disulfide exchange reaction under ambient conditions with high efficiencies, as determined by UV-vis spectroscopy. Facile conjugation of a thiol-containing fluorescent dye, namely BODIPY-SH, was demonstrated, followed by its effective cleavage in the presence of dithiothreitol (DTT), a thiol-containing disulfide reducing agent. Conjugation of a biotin-containing ligand onto the hydrogels allowed specific binding of the protein extravidin when exposed to a mixture of extravidin and bovine serum albumin. The bound protein could be released from the hydrogel by simple exposure to a DTT solution. Likewise, hydrogels modified with a cell adhesive peptide unit containing the RGD-sequence acted as favorable substrates for cellular attachment. Incubation of these cell attached hydrogel surfaces in a DTT-containing solution leads to facile detachment of cells from the surfaces, while retaining high level of cell viability. It can be envisioned that the benign nature of these hydrogels, their facile fabrication and modular functionalization will make them attractive platforms for many applications.

INTRODUCTION Selective attachment or capture of analytes ranging from small molecules to large biomolecules on device interfaces plays a crucial role in biomedical applications such as diagnostics.1 After the analyte selectively interacts with the surface e.g. through non-covalent 2 ACS Paragon Plus Environment

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molecular recognition based events such as protein-ligand interactions, detection of the event involves an optical or electrochemical readout. While this approach is effective for simple detections, undertaking the full structural assignment of the bound analyte at the detection interface may not be possible, and their detachment from the surface for a detailed analysis may be desirable. Such applications necessitate the development of new and efficient methodologies that provide facile engineering of the interface to selectively attach and ‘on demand’ release the analytes. Several preferred biochemical protocols involve the utilization of affinity based protein separation platforms that exploit either non-covalent2 or covalent interactions.3 In recent years, this concept has been extended to catch specific cells4 and bacteria.5 While the design of an effective ‘catch and release’ system entails facile immobilization of the ligand that enables the selective attachment of the target, subsequent non-destructive release of the bound entity under mild conditions is also crucial. For instance, it is important to maintain that the post-isolation detachment of the proteins and cells from the surface does not compromise their functional aspects.6 In a recent report, Wang and co-workers engineered an aptamer-coated hydrogel that was able to bind to the target cancer cells.7 In the study, parent hydrogels containing an oligonucleotide-based monomer was designed to allow the attachment of particular aptamers through hybridization. Captured cancer cells could subsequently be released from the hydrogel surface by using restriction endonucleases. In another example, selective extraction of a target protein from a mixture of proteins was reported by employing a chemo-mechanical system based on pH change.8 A thrombin capture platform was designed using pH-dependent thrombin-binding aptamer on a pH-responsive actuable hydrogel layer. Along similar lines, polymeric systems that are responsive toward temperature,9,10 photochemical11 or electroactive12,13 cues have been reported to undertake specific binding and release of biomacromolecules and cells. Although, the reported approaches are quite elegant, many of them involve designs that are not straightforward 3 ACS Paragon Plus Environment


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and easily adaptable, which unfortunately limits their widespread adaptation. The vast potential of such ‘catch and release’ interfaces in various areas of materials and biological sciences necessitates development of easily applicable and simpler approaches. Several bioconjugation strategies employ thiol-based chemical reactions since thiol groups are either naturally occurring or can be engineered into a biomolecule using routine techniques. Among the various thiol-based chemical methodologies, the nucleophilic and radical thiol-ene reactions have been extensively used for conjugation onto hydrogels.14-19 While these thiol-based chemistries are efficient and conveniently implemented, the non-reversible nature of these conjugations limits their usage for designing ‘catch and release’ platforms. Alternatively, another important thiol-based methodology that relies on the thiol-disulfide exchange reaction is reversible in nature. The newly formed disulfide linkage undergoes effective cleavage to release the conjugated thiol-containing molecule from the attached platform under mild conditions. The thiol-disulfide exchange chemistry has been employed to design stimuli responsive materials that undergo cleavage on demand when exposed to thiol-containing molecules such as glutathione (GSH) or dithiothreitol (DTT).20,21 This specific cleavage chemistry has been exploited to engineer redox-responsive polymer-drug conjugates,22 drug loaded micelles,23-25 biomoleculepolymer conjugates,26 and protein and cell-encapsulated hydrogels.27-29 As a versatile thiol conjugation anchor, pyridyl disulfide (PDS) functional group has been incorporated into a variety of polymeric materials to effectively and reversibly conjugate thiol-containing (bio)molecules.3033

While, the thiol-pyridyl disulfide exchange-based conjugation and release strategies have been

reported for a variety of soluble polymers, hydrogel-based functional interfaces harnessing this exchange reaction have found limited applications. In an example, Wang and co-workers reported the fabrication of degradable hydrogels by crosslinking of pyridyl disulfide


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functionalized hyaluronic acid with PEG-dithiols.34 The fabricated hydrogels could be degraded either enzymatically in the presence of hyaluronidase, or chemically upon the introduction of glutathione. Immobilization and release of thiol-containing chemokine, and encapsulation and release of different types of cells were demonstrated. In most of the reported examples, either synthetic or natural polymers were modified with the PDS group and the disulfide linkages were utilized for obtaining reversible crosslinking to trigger hydrogel degradation. However, a nonreversible crosslinking-based hydrogelation strategy would be desirable in certain applications of PDS functional gels. Especially in hydrogel platforms for biomolecular sensing and cellular proliferation, contaminations from hydrogel degradation could cause interference with assay. Hence, a methodology where robust hydrogels containing the PDS functional group can be directly fabricated will provide access to reversibly thiol-reactive hydrogel interfaces amenable to facile functionalization in a modular fashion. Herein, we disclose bulk and micro-patterned hydrogel interfaces that can be used as reversible conjugation platform for biomolecules and cells (Scheme 1). In particular, a pyridyldisulfide containing methacrylate (PDSMA) monomer is photopolymerized with hydrophilic PEG-based methacrylate monomers in the presence of a PEG-based crosslinker to obtain hydrogels. The PDS functional groups in the gel network provides reactive handles for selective and reversible conjugation of thiol-containing molecules, while the hydrophilic PEG-based matrix imparts anti-biofouling characteristics. A series of hydrogels employing varying amounts of PDS monomer and different molecular weight PEG-based monomers were fabricated and characterized. The post-polymerization modification efficiencies of bulk hydrogels was investigated by conjugation of GSH. The functionalization of the patterned hydrogels was performed via reversible conjugation of thiol-containing fluorescent dye molecules.



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Micropatterned hydrogels were also conjugated with a protein binding ligand to demonstrate ligand-directed protein attachment in a selective manner from a mixture of proteins, followed by its release. Finally, efficient conjugation of a cell binding ligand onto the patterned hydrogels and subsequent attachment and release of human umbilical vein endothelial cells (HUVECs) from the surface while maintaining excellent viability was demonstrated.

Scheme 1. Illustration of modular ‘catch and release’ hydrogel platform tailored for reversible protein and cell attachment. EXPERIMENTAL SECTION Materials. The pyridyl disulfide methacrylate (PDSMA) monomer was synthesized according to a literature procedure.35 Di(ethylene glycol) methyl ether methacrylate (DEGMEMA, Mw = 188 g/mol), poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, Mn = 300 and 1100 6 ACS Paragon Plus Environment

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g/mol),

poly(ethylene

glycol)

dimethacrylate

(PEGDMA,

Mn

=

550

g/mol),

3-

(trimethoxysilyl)propyl methacrylate (TMSMA), L-glutathione reduced (GSH), TRITCextravidin, 2,2-dimethoxy-2-phenylacetophenone (DMPA) and sodium dodecyl sulphate (SDS) were purchased from Sigma Aldrich (USA). Commercially available monomers were purified before use by passing through a short plug of aluminum oxide. Albumin–fluorescein isothiocyanate conjugate (FITC-albumin) was obtained from Sigma (USA). L-dithiothreitol (DTT) was obtained from Fluka (USA). Biotinylated hexa(ethylene glycol)undecanethiol (BiotinSH) was obtained from Nanoscience Instruments (Phoenix, USA). 4,4-Difluoro-1,3,5,7tetramethyl-8-[(10-mercapto)]-4-bora-3a,4a-diaza-s-indacene (BODIPY-SH) was prepared as reported in literature.36 Coomassie blue R-250 dye was obtained from Thermo Scientific (USA). Organic solvents were obtained from Merck (Germany) and used as received. Phosphate buffered saline (PBS) was purchased from Pan Biotech (Germany) and RGD-SH (cyclo (Arg-Gly-Asp-DPhe-Cys) was obtained from Peptides International (USA). Human umbilical vein endothelial cells (HUVEC) and endothelial cell growth media-2 kit (EBM-2 supplemented with EGM-2 SingleQuot kit) were obtained from Lonza Inc. (USA). Live/dead cell assay kit containing calcein-AM (acetoxymethyl ester of calcein) and propidium iodide (PI) was obtained from Sigma-Aldrich (USA). Measurements and Characterization. Surface morphology of dry samples of bulk hydrogels was examined without metal sputtering using scanning electron microscopy (SEM) performed with ESEM XL-30 (Philips, Eindhoven, The Netherlands) instrument operating at 10 kV at a working distance between 6.7-8.6 mm. Freeze-dried hydrogel samples were immersed in liquid nitrogen and broken for morphology analysis. Photopolymerization was done using a Blak-Ray, B-100 AP/R High Intensity UV Lamp (100W, 365 nm). UV-vis spectra were collected on a Cary



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Varian UV-vis spectrometer (Santa Clara, CA, USA). Anton PAAR MCR 302 rheometer (Anton Paar Germany GmbH, Ostfildern, Germany) with 8 mm parallel plate geometry was utilized for evolution of rheological behaviors of hydrogels by measuring their loss (G′′) and storage (G′) moduli as a function of angular frequency and time at 25 oC by using 0.5 % strain and 1.5 mm plate gap. Fluorescently labelled materials were observed using a Zeiss Axio Observer inverted microscope. A Zeiss Filter set 38 (Excitation BP 470/40, Emission BP 525/50) was used for visualizing the BODIPY functionalized hydrogel surfaces, presence of FITC-albumin and stained live cells. Zeiss Filter set 43 (Excitation BP 545/25, Emission BP 605/70) was used for imaging TRITC-extravidin immobilized hydrogel patterns and stained dead cells. Fluorescence microscopy images were processed using Zeiss AxioVision software. Synthesis of Bulk Hydrogels. Bulk hydrogels were synthesized by UV-initiated free radical photopolymerization at ambient temperature. A series of bulk hydrogels containing varying amounts of PDSMA monomer was prepared with different hydrophilic monomers: DEGMEMA (Mw: 188 g/mol), PEGMEMA (Mn: 300 g/mol) and PEGMEMA (Mn: 1100 g/mol) (Table1). Representative procedure for synthesis of hydrogel H-PEG300-10: PDSMA (6.61 mg, 0.0259 mmol), PEGMEMA (69.93 mg, 0.233 mmol), PEGDMA (14.24 mg, 0.0259 mmol) and DMPA (4.97 mg, 0.0194 mmol) were dissolved in dimethyl sulfoxide (DMSO) (20 µL) in a transparent glass vial and sonicated for 5 min. The reaction mixture was exposed to UV-light from 10 cm exposure interval for 30 min.

After the gelation reaction, the unreacted monomers and

oligomeric fractions were removed by washing the hydrogels with copious amount of tetrahydrofuran (THF) and distilled water. Dry hydrogel samples were obtained by lyophilization of the gel sample. Gel content was obtained by dividing dry weight of the obtained hydrogels by their expected theoretical value upon full conversion.


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Swelling Studies. A dry hydrogel sample (~10 mg) was placed a flask containing distilled water at room temperature. Hydrogel was removed from the medium at regular time intervals and its weight was recorded until the hydrogel showed constant weight. The percent equilibrium swelling ratio (%ESR) or water uptake percentage (Wup %) was obtained using the following equation (eq 1): Wup (%) = (Wmax – Wdry) / Wdry × 100

eq1

where Wmax represents the gravimetrically determined weight of water adsorbed hydrogel at the known time point and Wdry represents dry weight of the analyzed hydrogel sample. Pyridothione Release Studies. Hydrogel sample (2 mg) was transferred into a GSH solution (3 mL, 10 mM in PBS) in a UV-cuvette and incubated at 37 °C. At regular intervals, UV-Vis spectrum of the solution was collected to determine the absorbance at 343 nm, belonging to the released pyridothione fragment. The total pyridothione release was calculated based on the molar extinction coefficient of pyridothione37 and the percent release was obtained using the equation below (eq 2): nrel. (%) = (nact / ntheo.) × 100

eq 2

Where nrel, nact and ntheo represents released, actual and theoretical molar amounts of pyridothione. Modification of Silicon Oxide Surfaces with TMSMA. Silicon wafers and glass surfaces were soaked in 10% (w/w) solution of TMSMA in dry toluene for 12 h at room temperature. Modified surfaces were washed several times with toluene and methanol, and then dried under vacuum. Fabrication of Hydrogel Micro-Patterns. A micro-patterned polydimethylsiloxane (PDMS) stamp was carefully placed on the surface of the TMSMA-modified silicon wafer. A hydrogel precursors solution of hydrogel H-PEG300-10 in CH2Cl2 was dropped at one of the open end of 9 ACS Paragon Plus Environment


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PDMS stamp. The channel between the stamp and surface was filled with the mixture driven by capillary action. The filled channels were exposed to UV irradiation (100W, 365 nm) for 30 min. After the gelation process, the PDMS stamp was gently peeled off and hydrogel micropatterns on the surface were washed with THF to remove any unreacted reactants. Fabrication of Hydrogel Layers. Hydrogel precursor mixture (H-PEG300-10, 30 µL) in CH2Cl2 was dropped onto a TMSMA modified glass surface (1.5 cm x 2.5 cm), sandwiched with a cover slide and placed under UV light for 30 min. After removing the cover glass, a hydrogel layer covalently bound to the glass surface was obtained. Hydrogel coated surface was washed with THF to remove any unreacted reactants. Conjugation and Release of BODIPY-Thiol. A silicon surface with PDS containing micropatterned hydrogel was incubated in a solution of BODIPY-SH (1 mg/mL) in THF for 12 h. Thereafter, the pattern was washed with THF several times in order to remove any unbound dye. Subsequently, the BODIPY-SH immobilized patterned hydrogel was incubated in GSH solution (20 mM) or DTT solution (10 mM) for 12 h and washed several times with THF. As a control experiment, patterned hydrogel surface devoid of pyridyl disulfide monomer was treated with BODIPY-SH in similar manner. The hydrogel patterns were visualized using fluorescence microscopy after dye attachment and release. Immobilization and Release of TRITC-Extravidin. PDS containing hydrogel patterns were treated with Biotin-SH (1 mg/mL) in MeOH solution for 12 h. Biotinylated patterns were washed several times with MeOH and incubated with a solution of TRITC-extravidin (20 µL, 1 mg/mL 1x PBS) for 30 min. Protein-immobilized patterns were purified by washing several times with 1x PBS and water, followed by analysis of the samples via fluorescence microscopy. As a control,


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hydrogel pattern that was not biotinylated was incubated in TRITC-extravidin solution and washed several times with water and analyzed. To release the extravidin, surfaces were incubated in DTT solution (10 mM, 3 mL) at room temperature for 3 h. Catch and Release of TRITC-Extravidin from Protein Mixture. TRITC-extravidin (1 mg) and FITC-albumin (1 mg) were dissolved in 1x PBS (0.5 mL). 20 µL solution was spread over the biotinylated hydrogel patterns on silicon surface and left for 30 min. Then, excess solution was pipetted off and fluorescence microscopy images were taken using both filter set 38 and 43. After washing the unbound protein away from the surface with PBS and water, fluorescence microscopy images were acquired. Finally, surfaces were incubated in DTT solution (10 mM, 3 mL) for 3 h and visualized by fluorescence microscopy. Control experiments were performed with similar protocol using non-biotinylated hydrogel patterns. Analysis of Captured Protein using Gel Electrophoresis. Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) was used for analysis of proteins released from the hydrogel. Since extravidin and albumin have similar molecular weights, for analysis extravidin was heated to 90 °C in 1% SDS buffer for 40 min to decompose its tetrameric form. Albumin was also treated under the same conditions as a control. SDS-PAGE was performed using 12% polyacrylamide gel in a Mini-PROTEAN electrophoresis cell (Bio-rad, USA). After electrophoresis, gel was stained using Coomassie blue R-250 and image was captured using a ChemiDoc MP imaging system (Bio-rad, USA). Cell Culture and Live/dead Cell Assay. HUVECs were cultured in EBM-2 supplemented at 37 °C and 5% CO2. The cells were seeded in a six-well plate, containing hydrogel surface, at a density of 105 cells/well and incubated at 37 °C with 5% CO2 for 24 h to adhere completely.



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Subsequently, the cells were co-incubated with DTT solution (5 mM) for 20 min at 37 °C. After the incubation, the cells were washed three times with PBS and stained by live/dead cell assay kit (SIGMA) according to the standard protocol supplied by the manufacturer. Brieﬂy, cells were soaked in staining solution containing calcein-AM (5 µmol/L) and propidium iodide (5 µmol/L) and incubated at 37 °C for 15 min. After washing three times with PBS, the cellular live/dead viability was visualized using fluorescence microscopy. RESULTS AND DISCUSSION Fabrication, Characterization and Functionalization of Bulk Hydrogels. A series of bulk hydrogels containing varying amounts of pyridyl disulfide groups were synthesized via free radical photopolymerization. Thiol reactive monomer PDSMA, along with different PEG-based monomers (DEGMEMA, PEGMEMA-300 and PEGMEMA-1100) were utilized in crosslinking process in the presence of PEGDMA crosslinker and DMPA photoinitiator (Figure 1). PEGbased monomers with different molecular weights were chosen to modulate the hydrophilicity of the hydrogel matrix. While higher amounts of the thiol-reactive PDSMA monomer would allow higher amount of functionalization, its hydrophobic nature can be expected to decrease swelling and hence affect efficiency of conjugation. In such cases, utilization of more hydrophilic PEGbased component would compensate the increased hydrophobicity. In order to investigate the role of these parameters on gelation behavior and properties, a library of hydrogels were synthesized at the onset (Figure 1). In all cases, gelation proceeded with good conversions within 30 min to afford clear and transparent hydrogels (Table 1, Entry 1-9). The type of the hydrophilic PEGbased monomer employed was determinant toward the physical properties of the obtained gels. While the hydrogels obtained using DEGMEMA were quite brittle, and the hydrogels containing PEGMEMA-1100 were very soft and fragile, the hydrogels obtained with PEGMEMA-300 were 12 ACS Paragon Plus Environment

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robust and easy to handle. The exact amount of PDS monomers in the PEGMEMA-300 based hydrogels was determined from HNMR analysis of non-gelated residual material. Results showed good agreement with the feed composition, whereby 8%, 18% and 37% PDS monomer incorporation was observed for hydrogels H-PEG300-10, H-PEG300-20 and H-PEG300-40, respectively.

Figure 1. Reaction scheme of pyridyl disulfide group containing thiol-reactive hydrogel fabrication via free radical photo-crosslinking.

The equilibrium swelling of these hydrogels in water was determined gravimetrically and tabulated in Table 1 as equilibrium swelling ratio (ESR). According to the water uptake profiles, all hydrogels exhibited rapid swelling in water but their equilibrium uptakes varied significantly depending on their composition (Figure S1). As expected, an increase in the content of the hydrophobic PDS monomer led to a decrease in the overall swelling of the hydrogels (Table 1 and Figure 2d). Moreover, the hydrophilic PEG chain length played a vital role in determining the overall swelling characteristics. Increasing the number of hydrophilic ethylene glycol repeating units on the side chains drastically increased the water uptake capacity of the hydrogels. 13 ACS Paragon Plus Environment


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Morphological analysis based on scanning electron microscopy revealed low porosity of DEGMEMA-based network, which explains their lowest water uptake capacity (Figure 2a). Increasing the PEG chain length yielded slightly more porous network structure (Figure 2b,c). The effect of the hydrophobic PDSMA content on gel microstructure was apparent since increasing the amount of the PDSMA monomer in the hydrogels led to decrease in their porosity (Figure S2 and Figure S3). Thermogravimetric analysis (TGA) of PEGMEMA-300 based hydrogels showed thermal behavior expected for such polymeric materials with onset of decomposition near 300 oC under nitrogen atmosphere, and no distinct effect of variation of the PDS monomer amount in the hydrogel was observed on the weight loss (Figure S4).

Table 1. Properties of the bulk hydrogels fabricated with varying amounts of PDSMA and PEGbased comonomer.

a

% yield of obtained hydrogel.

b

Equilibrium swelling ratio i.e. water uptake % at equilibrium swelling. 14 ACS Paragon Plus Environment

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Figure 2. Scanning electron micrographs of 10 % PDS containing bulk hydrogels synthesized using (a) DEGMEMA, (b) PEGMEMA-300 and (c) PEGMEMA-1100. (d) Graphical representation of the equilibrium swelling values of hydrogels.

Rheological characteristics of the gelation process and resulting gels were examined. Though, the gelation reaction was continued for 30 min to assure the maximum cross-linking, the mechanical gel point was achieved in 2 to 4 min as determined by the time scan analysis of the modulus values (Figure 3a and Figure S5a,b) where in situ rheological measurement was conducted on 50 µL of hydrogel precursor with UV irradiation from the start. Dynamic frequency scan analysis of the gel samples at equilibrium swelling revealed storage (G′) and loss (G″) modulus values ranging from 101 to 105 Pa, in which the storage modulus was greater than the loss modulus in all gels (Figure 3b and Figure S5c). The storage and loss moduli of the hydrogels 15 ACS Paragon Plus Environment


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exhibited low oscillation frequency dependence indicating the viscoelastic nature of the materials. It was observed that, both molecular weight of the hydrophilic comonomer affected the rheological properties. More enhanced viscoelastic properties were obtained upon increasing the number of ethylene glycol units of PEG-based monomer (Figure S3c).

Figure 3. Representative rheological analysis of hydrogel H-PEG300-10: (a) time-dependent scan during gelation and (b) angular frequency dependence of modulus values in water-swollen state (G′, solid symbols and G″, open symbols).

The PDS-containing thiol-reactive hydrogels were investigated for their ability to undergo effective functionalization under mild conditions. A thiol-containing tripeptide, glutathione (GSH) was employed as a thiol probe and the associated pyridothione release from the hydrogels was probed via UV-vis spectroscopy (Figure 4). Upon exposure to GSH, the pyridothione release increased over time and reached a plateau after 12 h. It was observed that lower functionalization efficiency was attained in case of increasing the amount of PDS groups in network. While hydrogels H-PEG300-10 and H-PEG300-20 showed an overall release of 94 % and 83 % after 24 h, the hydrogel H-PEG300-40 containing 40 % PDS group showed a total release of 16 ACS Paragon Plus Environment

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50 %. One can find this plausible since the hydrogels become less hydrophilic with increasing amount of the hydrophobic PDS monomer and lower amount of PEG units, which impedes the diffusion of GSH and thus lowers the release of pyridothione. The high functionalization efficiency prompted us to select the composition of hydrogel H-PEG300-10 (10 % PDSMA and 90 % PEGMEMA) for subsequent investigations of functionalization and release.

Figure 4. Functionalization of the hydrogels with glutathione and associated pyridothione release profiles from PEGMEMA-300 based hydrogels.



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Fabrication and Functionalization of Micropatterned Hydrogels. Hydrogel patterns on silicon surface were fabricated using micro-molding in capillaries (MIMIC) method (Figure 5a).38,39 Firstly, silicon oxide surfaces were treated with a silyl ether containing methacrylate monomer TMSMA to promote the interfacial bonding between hydrogel and the substrate. Thereafter, photopolymerization of hydrogel precursors (containing 10 % PDS monomer) confined in PDMS capillaries led to gelation within the channels. Peeling the stamp yielded hydrogel microstructures bound to the surface. Survey of the micropatterns with an optical microscope revealed uniform and defect free fabrication of hydrogel microstructures (Figure 5b). Thiol reactive hydrogel micropatterns were functionalized with a thiol-containing fluorescent dye, BODIPY-SH. Treatment of the micropatterned hydrogel with BODIPY-SH resulted in the conjugation of the dye into the gel network and rendered fluorescent patterns, as deduced from fluorescence microscopy analysis (Figure 5c). As a control experiment to ensure that the dye was not physically trapped in the gel network, hydrogel micropatterns devoid of pyridyl disulfide functional groups were fabricated and treated with BODIPY-SH under same conditions. After washing the dye treated hydrogel micropatterns, fluorescence microscopy analysis was undertaken. Lack of fluorescence suggested absence of any dye molecules, which supports that the origin of the fluorescent patterns in PDS-containing hydrogels was due to the thiol-pyridyl disulfide exchange reaction (Figure S6).


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Figure 5. Functionalization of the micropatterned hydrogel via thiol-disulfide exchange reaction: (a) Thiol-containing dye immobilization on patterned hydrogel H-PEG300-10 on Si/SiO2. (b) Optical microscope image of micropatterned hydrogel on glass surface. (c) Fluorescence microscopy image of BODIPY-SH immobilized micropatterned hydrogel.

Since the BODIPY-SH dye is attached to the hydrogel patterns through disulfide linkages, they should be amenable towards release from the surface by treatment with thiol-containing reagents. To demonstrate this, release studies of the dye molecules from the surface were performed by using thiol containing reducing agents GSH and DTT (Figure 6a). Hydrogel micropatterns containing the fluorescent dye were treated with 10 mM DTT and washed with THF to remove unbound species (Figure 6b). Analysis of thus treated hydrogels with fluorescence microscopy revealed significant loss of fluorescence intensity, becoming nearly similar to what was observed for control experiments in dye conjugation (Figure 6c and 6d). Cleavage of disulfide bound dye



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from hydrogels could also be accomplished through treatment with 20 mM GSH, where similar loss of fluorescence was observed (Figure S7).

Figure 6. (a) Schematic illustration of the release of dye molecules from the micropatterned hydrogel in the presence of 10 mM DTT. (b) Fluorescence microscopy image of BODIPY attached hydrogel via disulfide bonding. (c) Fluorescence microscope image of hydrogel patterns after BODIPY release. (d) Normalized fluorescence intensities of hydrogels.

Protein Immobilization and Release. After demonstrating the successful attachment and release of thiol-containing molecules on the micropatterned hydrogels, biomolecular immobilization and subsequent detachment of TRITC-extravidin on biotinylated micropatterns was investigated (Figure 7a). PDS functional micropatterns were first conjugated with thiol-containing biotin (Biotin-SH), a ligand known to bind strongly to the protein avidin. The biotinylated surfaces obtained via thiol-disulfide exchange reaction were exposed to TRITC-extravidin and efficient 20 ACS Paragon Plus Environment

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binding of the protein onto the hydrogel was confirmed with fluorescence microscopy analysis (Figure 7b). Subsequently, the protein-immobilized hydrogel micropatterns were treated with the disulfide reducing agent DTT to release the bound protein. Fluorescence microscopy analysis of DTT treated hydrogel micropatterns revealed significant loss of red fluorescence thus suggesting the detachment of protein from the hydrogel (Figure 7c). Decrease of relative fluorescence intensity showed that about 60 % of the protein was detached within 3 hours using only a 10mM DTT solution (Figure S8).

Figure 7. a) Scheme of Biotin-SH and TRITC-extravidin immobilization on micropatterned hydrogel and subsequent release via disulfide bond cleavage. b) Fluorescence microscopy image of TRITC-extravidin immobilized patterns on hydrogel. c) Fluorescence microscopy image after release of protein upon treatment with DTT.

Encouraged by the facile immobilization and detachment of proteins on these hydrogels, we further explored the selective ‘catch and release’ of a biomolecule of interest in the presence of other biomolecules. To test this approach, biotinylated hydrogel pattern was incubated with a mixture of fluorescently labeled proteins TRITC-extravidin and FITC-albumin. The treatment of 21 ACS Paragon Plus Environment


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hydrogels and the successive washing steps were thoroughly investigated by fluorescence microscopy analysis (Figure 8). While the incubation of surfaces with the mixture of extravidin and albumin exhibited the expected concurrent red and green fluorescence (Figure 8b and 8c), PBS washing removed the physically surface adhered FITC-albumin, thus eliminating the green fluorescence (Figure 8e), while retaining the red-fluorescent target protein (Figure 8d). The strong binding of biotin with extravidin resulted in the selective attachment of TRITC-extravidin on micropatterned hydrogel, while the anti-biofouling nature of the hydrogel inhibited nonspecific absorption of FITC-albumin. Thereafter, the TRITC-extravidin immobilized hydrogel was treated with DTT to demonstrate the ‘on demand’ release of the biomolecule. As expected, significant loss in red fluorescence was observed by fluorescent microscopy (Figure 8f), thus suggesting the cleavage of TRITC-extravidin from hydrogel. Additionally, the surface-cleaved protein was analyzed using gel electrophoresis to gather further information about its composition (Figure S9). Importantly, only the monomeric avidin containing residue was observed, thus indicating specific binding and release of only the target protein. This observation demonstrates the high efficiency of these PDS-containing hydrogels decorated with appropriate ligands to selectively catch and release a target protein. However, one should note that all proteins may not be amenable to this protocol since some proteins may possess disulfide units that may not re-oxide to yield the native conformation upon reconstitution after removal of the reducing agent.


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Figure 8. (a) Schematic illustration of selective immobilization of TRITC-extravidin on biotinylated surface in the presence of FITC-albumin and release of captured protein. Fluorescence microscopy images of TRITC-extravidin (b) and FITC-albumin (c) on biotinylated hydrogel pattern (H-PEG300-10) after incubation, and after washing the excess proteins with 1x PBS (d, e), and after DTT treatment to release the protein from hydrogel patterns (f and g).

Attachment and Release of Cells. The efficiency of the PDS functional hydrogels on cell attachment and their release was investigated (Figure 9a). The hydrogel coated glass slides were treated with a thiol containing cell adhesion peptide (RGD-SH) to promote the adhesion of human umbilical vein endothelial cells (HUVEC). The cells were stained with calcein-AM and 23 ACS Paragon Plus Environment


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visualized under fluorescence microscope to confirm their spreading and adherence on the hydrogel surface (Figure 9b). Without the modification of hydrogel with the cell adhesive peptide, cells did not adhere and spread on the otherwise bioinert surface (Figure S10). HUVECs were also seeded on a RGD-SH-attached maleimide-containing micropatterned hydrogel40 through the non-cleavable maleimide thiol linkage and visualized by fluorescence microscopy (Figure S12a). Upon exposure to DTT solution, cells on the PDS-containing micropatterned hydrogel became globular and detached from surface (Figure S13). It is proposed that the detachment of the cells from the hydrogel surface was a consequence of the scissoring of the cell adhesion peptide RGD-SH. Contrarily, the surface-bound cells on the maleimide-containing hydrogel (Figure S11) remained unaffected (Figure S12b). This observation is expected since in these hydrogels the cell adhesion peptide is attached to the hydrogel via a non-cleavable thioether bond. Cell viability after the detachment is of crucial importance for adaptation of this platform for potential applications. To test this, surface-detached cells were transferred into a well plate and stained with a calcein-AM and propidium iodide (PI) mixture. Under these conditions, live cells emit green fluorescence due to the calcein generated from calcein-AM by esterase, and dead cells emit red fluorescence since PI can only pass through damaged areas of dead cell membrane and then intercalates with the DNA. Merged fluorescence microscopy images revealed that the majority of detached cells were alive after their detachment from surface (Figure 9c).


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Figure 9. (a) Scheme of immobilization and detachment of HUVEC cells from surfacemicropatterned hydrogel. (b) Fluorescence microscopy images of cells immobilized on RGD attached hydrogel layer. (c) Merged fluorescence microscopy images showing viable (green) and dead (red) cells after detachment and removal from hydrogel.

CONCLUSIONS Facile fabrication of a novel ‘catch and release’ hydrogel platform based upon the thiol-disulfide exchange

reaction

is

disclosed.

The

thiol-reactive

hydrogels

were

obtained

by

photopolymerization of a pyridyl disulfide functional group containing monomer and a PEG based crosslinker. Equilibrium swelling in water, morphology and mechanical properties of these hydrogels could be tuned by varying the comonomer composition. Straightforward functionalization of these hydrogels under reagent-free mild conditions was demonstrated through attachment of a thiol-containing tripeptide, namely, glutathione. Micropatterned hydrogels were fabricated on silicon oxide surfaces using micromolding in capillaries. Efficient immobilization and release of a thiol-containing fluorescent dye was demonstrated. Additionally,



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selective attachment of protein was realized through ligand-directed protein immobilization on biotinylated hydrogels. The immobilized protein could be recovered for further analysis by efficient detachment upon treatment of hydrogels with DTT. Finally, these platforms could be easily functionalized with cell adhesive peptides to enable attachment of cells, which through simple treatment with DTT enables their harvesting with high viability. One can envision that the facile fabrication, modular functionalization and the dynamic nature of linkage chemistry will make this class of hydrogels an attractive platform for various biomedical applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Further characterization of the bulk hydrogels; fluorescence microscopy images of the dye conjugated micropatterns and surface-bound cells, gel electrophoresis results of the protein released from hydrogel. AUTHOR INFORMATION Corresponding Author *Email: [email protected], Tel: +902123597613 ORCID Amitav Sanyal: 0000-0001-5122-8329 Rana Sanyal: 0000-0003-4803-5811 26 ACS Paragon Plus Environment

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TABLE OF CONTENT GRAPHIC


Facile Fabrication of a Modular 'Catch and Release' Hydrogel

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