Sequential Nucleophilic Substitutions Permit Orthogonal Click

Aug 13, 2013 - The novel application of propargylamine-mediated substitution ... for ligand immobilization via versatile copper-free click reactions, ...
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Sequential Nucleophilic Substitutions Permit Orthogonal Click Functionalization of Multicomponent PEG Brushes Jin Sha,†,‡ Ethan S. Lippmann,†,§ Jason McNulty,†,∥ Yulu Ma,*,‡ and Randolph S. Ashton*,†,§ †

Wisconsin Institute for Discovery, §Department of Biomedical Engineering, and ∥Department of Mechanical Engineering, University of Wisconsin−Madison, Madison, Wisconsin, United States ‡ School of Mechanical and Power Engineering, East China University of Science and Technology, Shanghai, China S Supporting Information *

ABSTRACT: Multicomponent poly(ethylene glycol) (PEG) brushes (i.e., ≥2 adjacent PEG brushes) can be used to engineer culture substrates with microscale, nonfouling regions decorated with covalently immobilized ligands that mediate biospecific interactions. However, synthesizing such brushes with orthogonal immobilization chemistries to permit differential biofunctionalization is nontrivial and often requires synthesis of PEG-co-polymers. To simplify synthesis and enhance the versatility of such substrates, we developed a protocol for generating orthogonal click-functionalized multicomponent PEG brushes using sequential nucleophilic substitutions by sodium azide, ethanolamine, and propargylamine. The novel application of propargylamine-mediated substitution functionalizes PEG brushes with acetylene groups, and for the first time, ethanolamine-mediated substitution is shown to be sufficient for passivating the “living” polymer chain ends between brush synthesis steps. Thus, our multicomponent PEG brushes present dual orthogonal chemistries (i.e., azido and acetylene groups) for ligand immobilization via versatile copper-free click reactions, which are useful for in situ surface modifications during cell culture.

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INTRODUCTION Polyethylene glycol (PEG)-grafted materials are ubiquitous in biotechnology due in large part to the polymer’s nonfouling properties.1,2 Of particular interest to tissue and bioengineers, PEG-grafted materials can serve as the basis of tailored culture substrates that mediate biospecific interactions between adherent cells and covalently conjugated ligands, thereby enabling exploration of the specific effects of immobilized biochemical cues on cell signaling and fate.3−7 Monolayers and micropatterns of PEG have been grafted onto surfaces by adsorption,8−12 covalent coupling via a reactive terminal group,13−15 and by photoinitiated or controlled radical polymerization reactions.16−19 For facile spatial control, microcontact printing (μCP) followed by backfilling with oligo(ethylene glycol)-terminated alkanethiolates onto gold (Au)-coated substrates can be used to generate microscale oligo(ethylene glycol)-presenting self-assembled monolayers (SAMs).20,21 These are nonfouling and can be modified with bioactive ligands using photo-4 or electrochemistry22 or Diels− Alder reactions.5,6 However, the stability of alkanethiol SAMs is limited (i.e., approximately ≤1 week) in culture due to the thiol groups’ susceptibility to autoxidation by oxygen and transition metals typically present in cell cultures.23,24 To address this issue, the Chilkoti group combined μCP of an alkanethiol polymerization initiator, ω-mercaptoundecyl bromoisobutyrate, with atom transfer radical polymerization (ATRP)25 of oligo(ethylene glycol) methyl ether methacrylate (OEGME© 2013 American Chemical Society

MA) monomers to synthesize surfaces with micropatterned poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) brushes, which are stable for over a month in high serum cell culture.19 Such surface-initiated ATRP (SI-ATRP) has now become an established method for modifying culture substrates due to the ease and flexibility of μCP and ATRP reactions.2,26−28 Plus, a variety of chemistries, including “click” chemistry, can be used in conjunction to immobilize bioactive ligands onto grafted PEG brushes.7,28−31 While several chemistries exist to functionalize a single SIATRP synthesized PEG brush with bioactive ligands, limited methods exist to orthogonally functionalize multicomponent PEG brushes (i.e., ≥2 adjacent PEG brushes) to create tailored culture substrates with microscale and differential bioactive regions. Huck and co-workers pioneered the synthesis of multicomponent PEG brushes using sequential rounds of μCP and SI-ATRP interspersed with sodium azide-mediated nucleophilic substitutions to passivate the “living” ends of newly polymerized brushes.28 Lutz et al.32 further demonstrated that conversion of the terminal bromine group on an ATRP synthesized PEG polymer to an azide creates a “clickable” handle for bio-orthogonal 1,3-dipolar cycloaddition of alkynepresenting ligands.33 Still, this only provides a single Received: June 19, 2013 Revised: August 8, 2013 Published: August 13, 2013 3294

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Scheme 1. Fabrication of Orthogonal Multicomponent PEGMEMA Brushes by Nucleophilic Substitutionsa

(i) Microcontact printed ω-mercaptoundecyl bromoisobutyrate molecules are used for (ii) SI-activators generated by electron transfer (AGET) ATRP of OEGMEMA. The terminal bromines on resulting PEGMEMA brushes can be substituted using (iii) ethanolamine, (iv) propargylamine, or (vi) sodium azide. Substitution by propargylamine can be detected via (v) click cycloaddition of azide-PEG4-biotin, and substitution by sodium azide can be detected via (vii) click cycloaddition of acetylene-PEG4-biotin.

a

biospecific culture substrates with microscale regions displaying differential bioactive functionalities.

immobilization chemistry for surfaces grafted with PEG brushes, and the use of complex UV photodegradation schemes that generate fouling and thus nonbiospecific areas would be required to create additional differential bioactive regions.34 Here, we present a novel sequential nucleophilic substitution protocol that facilitates fabrication of multicomponent PEG brushes with two orthogonal and clickable functionalities. As shown in Scheme 1, we demonstrate for the first time that the terminal bromine groups on SI-ATRP synthesized PEG brushes can be passivated between multiple synthesis steps by (iii) nucleophilic substitution with ethanolamine. Also, nucleophilic substitution with (vi) sodium azide can provide one clickreactive handle, and the novel application of (iv) propargylamine-mediated substitution can provide an orthogonal click immobilization chemistry. Further, we show that the degree of azide or acetylene functionalization, and thereby, the surface density of immobilized bioactive ligands, can be tuned by varying the duration of sodium azide and propargylamine substitution reactions. Lastly, we use cell culture assays to prove that sodium azide-, propargylamine-, or ethanolamine-reacted PEG brushes remain cell adhesion resistant, even at the highest level of functionalization examined. Thus, our novel sequential nucleophilic substitution protocol is suitable for engineering

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EXPERIMENTAL SECTION

Materials. Milli-Q water was generated with a Millipore Simplicity 185 system. Copper(I) bromide (99.999% trace metals basis), Copper(II) bromide (99.999% trace metals basis), 2,2′-bipyridine (BiPy; ≥99%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99%), L-ascorbic acid, oligo(ethylene glycol) methyl ether methacrylate (OEGMEMA, Mn = 475), potassium hydroxide, sodium azide, zinc (purum, ≥99%, powder), silicon gel, ethyl 2bromoisobutyrate (EBiB), tributyltin hydride (99%), 2,2′-azobis(2methylpropionitrile) (AIBN, 98%), triethylamine (Et3N, ≥99%), copper(II) sulfate (CuSO4, ≥99%), tris[(1-benzyl-1H-1,2,3-triazol-4yl)methyl]amine (97%), propargylamine (98%), donkey serum, methanol (for HPLC ≥99%), N,N-dimethylformamide (DMF, anhydrous, 99.8%), and dimethyl sulfoxide (DMSO, anhydrous, ≥99.9%) were purchased from Sigma-Aldrich (Milwaukee, WI). Streptavidin-Alexa Fluor 488 conjugate, Streptavidin-Alexa Fluor 546 conjugate, mouse laminin, and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Life Technologies. CuBr catalyst was activated by stirring overnight in glacial acetic acid, rinsed twice with ethanol, twice with diethyl ether, and dried under vacuum. OEGMEMA monomer was purified with basic Al2O3 column to remove the inhibitor. The initiator, ω-mercaptoundecyl bromoisobu3295

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mmol) was injected into the flasks over a period of 5 min, and the reaction was continued for 16 h. After the reaction, the slides were cleaned with water and ethanol, and dried under nitrogen. As an alternative, tributyltin hydride dehalogenation reaction chemistry, micropatterned PEGMEMA slides were submerged in DMF (10 mL), AIBN (26.3 mg, 0.16 mmol), EBiB (45 μL, 0.31 mmol), and tributyltin hydride (0.25 mL, 0.93 mmol) for 16 h at 40 °C. Then the resulting slides were cleaned with water and ethanol, and dried under nitrogen. For zinc powder dehalogenation reactions, micropatterned PEGMEMA slides were submerged in 0.4 g zinc powder, 0.2 g silica gel, 3 mL of acetate acid, and 2 mL of H2O at room temperature for 8 h. The resulting slides were cleaned with water and ethanol, and dried under nitrogen. For ethanolamine dehalogenation reactions, micropatterned PEGMEMA slides were submerged in 5 mL of DMSO, ethanolamine (30.5 mg, 0.5 mmol), and triethylamine (Et3N, 1.5 mmol) at 40 °C for specified reaction times (12−72 h), followed by rinsing with water and ethanol, and drying under nitrogen. “Click” Immobilization of Biotinylated Conjugates on Functionalized PEGMEMA Brushes. A total of 20 μL of azidePEG4-biotin conjugate/DMF solution (25 mg/mL), 120 μL of CuSO4/TBTA complex solution (15 mM CuSO4, 30 mM TBTA, 1:1 v/v water/DMF), and 1.46 mL of water were vortexed for 1 min prior to the addition of an acetylene-functionalized micropatterned PEGMEMA slide. Then, 400 μL of aqueous L-ascorbic acid solution (0.15M) was added to initiate reaction, which proceeded for 8 h at room temperature. After removal, the slide was washed thoroughly with water and ethanol, and dried using nitrogen. Light was avoided during the entire procedure. Azide-functionalized micropatterned PEGMEMA slides were reacted in the same manner, except an acetylene-PEG4-biotin conjugate was used. Immunofluorescent Detection of Biotin. After immobilization onto micropatterned PEGMEMA slides using copper-catalyzed click chemistry, biotin conjugates were detected by immunostaining using streptavidin-Alexa Fluor conjugates. To block nonspecific binding, the slides were submerged in 3% donkey serum/DPBS and shaken for 1 h. Next, 20 μL of 2 mg/mL, streptavidin-Alexa Fluor 488 or 546 conjugate in 3% donkey serum/DPBS was added, and the slides were shaken for an additional 2 h at room temperature. Then, the substrates were washed with Tris-buffered saline (TBS, pH 7.4) twice for 10 min each, and one last time in 1% donkey serum/DPBS for 1 h. All protocols were performed in minimal light. Cell Seeding and Culture. H9 human embryonic stem cells (H9 hESCs, passages 26−50) were maintained in E8 medium35 on Matrigel-coated plates (BD Biosciences) and differentiated to neuroepithelium by seeding at 1 × 105 cells/cm2 and removing transforming growth factor-β and fibroblast growth factor-2 from the media (i.e., E6 media). Daily media changes were performed for six days, resulting in a pure (>90%) population of neuroepithelial cells (hNECs), as deemed by flow cytometry for Pax6, a neuroectoderm marker, and immunocytochemical (ICC) analysis with Pax6 (Covance) and N-cadherin (BD Biosciences) antibodies (see Supporting Information, Figure S1). As indicated by ICC, the cells uniformly assumed a characteristic neuroepithelial columnar morphology and generated “rosette” structures with apically polarized Ncadherin cell adhesion proteins.36 To seed hNECs onto patterned surfaces, cells were dissociated with accutase (Life Technologies), collected by centrifugation, and seeded at 1 × 105 cells/cm2 in E6 medium containing 10 μM ROCK inhibitor (Y27632; R&D Systems), 25 μg/mL laminin (Life Technologies), and 1× penicillin− streptomycin (Life Technologies). Medium was changed every other day with E6 medium containing penicillin−streptomycin. Human umbilical vein endothelial cells (HUVECs) were maintained in EGM-2 supplemented with growth factors as instructed by the manufacturer (Lonza). HUVECs were seeded onto patterned surfaces at 1 × 104 cells/cm2, and the media was changed every other day. Click Modification of Cell-Seeded Functionalized PEGMEMA Brushes. hNECs were seeded onto slides with azide-functionalized, micropatterned PEGMEMA brushes, as described prior. After 2 days of culture, a media change was used to wash away loosely bound cells, and 2 μL of DBCO-PEG4-biotin/DMF (10 mM) was added to the

tyrate, was synthesized using a previously published procedure.26 (Tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane was purchased from Gelest, Inc. 4-(Trimethylsilyl)-3-butyn-1-amine (TMS-propargylamine) was purchased from Synthonix, Inc. Azide-PEG4-biotin, acetylene-PEG4-biotin, and DBCO-PEG4-biotin conjugates were purchased from Click Chemistry Tools. All other materials and reagents that do not have a source denotation were purchased from Fisher, and used as received.

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METHODS

μCP of ω-Mercaptoundecyl Bromoisobutyrate on AuCoated Slides. Elastomeric stamps used for μCP were generated by standard soft lithographic techniques. Silicon masters were designed using AutoCAD 2012 software, and purchased from the Stanford Microfluidics Foundry. The silicon master was rendered inert by overnight exposure in vapors of (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane. Then, poly(dimethyl siloxane) (PDMS; Slygard 184 Kit) was used to create a replica of the silicon master by curing a 10:1 mixture of PDMS and curing agent overnight at 60 °C. Fisherbrand microscope cover glass (24 × 50 #1) slides were cleaned by sequential washes in toluene and methanol, followed by sonication for 1 min in acetone. After drying, 35 Å of titanium (Ti), followed by 180 Å of gold (Au) were deposited onto cover glass slides using a CHA-600 Telemark focused electron-beam evaporation system (Wisconsin Center for Applied Microelectronics). Au-coated glass substrates were rinsed with absolute ethanol prior to use. To make micropatterned substrates, PDMS stamps were coated with ω-mercaptoundecyl bromoisobutyrate (2 mM in absolute ethanol), dried under nitrogen, and then brought in conformal contact with Ti/Au-coated glass slides. Then, the chemically modified slides were incubated in absolute ethanol for 2 h prior to being dried under nitrogen. SI-AGET ATRP of OEGMEMA. The macromonomer OEGMEMA (8 g, 16.7 mmol), water (7.5 mL), methanol (7.5 mL), copper(II) bromide (0.08 mmol, 17.9 mg), and 2′,2-bipyridine (0.24 mmol, 37.5 mg) were added to a Schlenk flask, sealed, and stirred for 30 min. Then, the flask was degassed with three freeze−vacuum−thaw cycles and backfilled with nitrogen. Next, the mixture was transferred by syringe to a reaction flask containing micropatterned glass slides under vacuum. To start the reaction, L-ascorbic acid (0.8 mmol, 140.9 mg) in deionized water was injected into the flask. The reaction continued for 16 h to generate micropatterned PEGMEMA brushes. Polymerization was stopped by adding air, and the slides were rinsed with ethanol, water, and ethanol before drying under nitrogen. Prior to cell culture, the slides were immersed in a large volume of ethanol for 2 h to ensure complete removal of copper ions. Acetylene Functionalization of Micropatterned PEGMEMA Brushes. Nucleophilic substitution by propargylamine was carried out by reacting micropatterned PEGMEMA brushes in 0.1 M propargylamine/DMSO solution at room temperature with specified reaction times (3−24 h), followed by rinsing with water and ethanol and drying with nitrogen. TMS-propargylamine was also carried out using a 0.1 M TMS-propargylamine/DMSO solution at room temperature for 24 h, and deprotection of the TMS group was executed by immersing the substrate in KOH (30 g) in methanol (6 mL) at ambient temperature for 2 h, followed by rinsing with toluene, methanol, water, and ethanol, and drying with nitrogen. Azide Functionalization of Micropatterned PEGMEMA Brushes. Azide substitution was carried out by reacting micropatterned PEGMEMA brushes in 0.1 M sodium azide/DMF solution at room temperature with specified reaction times (1−24 h), followed by rinsing with water and ethanol, and drying with nitrogen. Dehalogenation of PEGMEMA Brushes. For one tributyltin hydride dehalogenation reaction chemistry, micropatterned PEGMEMA slides were submerged in anisole (10 mL), PMDETA (64.5 μL, 0.31 mmol), and EBiB (45 μL, 0.31 mmol). The flask was sealed with a rubber stopper and degassed by four freeze−vacuum−thaw cycles. During the final cycle, the flask was filled with nitrogen, and CuBr (44.2 mg, 0.31 mmol) was added to the frozen mixture. The flask was sealed, evacuated, and backfilled with nitrogen four times before immersion in a 40 °C oil bath. Tributyltin hydride (0.25 mL, 0.93 3296

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Figure 1. Characterization of micropatterned PEGMEMA brushes. (A) Single beam FT-IR spectrum of μCP/SI-AGET ATRP fabricated PEGMEMA brush showing characteristic peaks at 2750−3100, 1730, 1240 and 1110, and 1460 and 1390 cm−1 due to deformation of indicated chemical groups. Insets present a SEM image and a 3D intensity distribution FT-IR mapping of the −CH2/−CH3 peak. (B) Plot of PEGMEMA brush thickness vs polymerization time based on data obtained by AFM. 3D AFM images of PEGMEMA brushes at each time point are presented as insets. (C) PEGMEMA brushes fabricated using a welled PDMS stamp (i.e., opposite of stamp in part A) were functionally nonfouling as demonstrated by long-term resistance to hNEC and HUVEC adhesion. Scale bars are 300 μm. slides’ culture wells, which contained 2 mL of E6 media. After a 1 h incubation time, the media was removed, and the wells were washed with PBS three times. Then, a LIVE/DEAD Viability/Cytotoxicity Kit was used as instructed by the manufacturer (Life Technologies) to stain for cell viability post-copper-free click modification. Following the assay, the cells were fixed using a 4% paraformaldehyde solution in DPBS for 15 min, washed with DPBS three times, and immunostained with streptavidin-Alexa Fluor 546 conjugate, as described prior. Acetylene-functionalized PEGMEMA substrates were fixed and stained with DAPI before copper-catalyzed click modification with the azidePEG4-biotin conjugate. Immobilized biotin molecules were detected by immunostaining with streptavidin-Alexa Fluor 488 conjugate, as described prior. Characterization. The surface topography of micropatterned PEGMEMA brushes was observed by atomic force microscopy (AFM) using a Bruker (Veeco/DI) Nanoscope III Multimode SPM equipped with an atomic head of 100 × 100 μm2 scan range. The AFM was operated in tapping mode, under dry ambient conditions, and using silicon tips with a spring constant of 42 N/m and a frequency of 230 kHz to map the surface morphology. AFM data was processed with Gwyddion Software. To demonstrate the spatial distribution of micropatterned PEGMEMA brushes, FT-IR mapping was performed using a Thermo Scientific Nicolet iN10 MX FT-IR microscope with a mercury cadmium telluride (MCT) detector array in reflection mode. A total of 64 scans were taken for each spectrum at a resolution of 8 cm−1 over an area of 30 × 25 mm, with an optical resolution of 25 × 25 μm and a spatial resolution power of 10 × 10 μm. OMNIC Picta software was used to extract chemical images, size of sample features, and relative distribution in percentage. A Hitachi S-570 LaB6 scanning

electron microscope (SEM) was also used to image micropatterned PEGMEMA brushes at a 20° grazing angle to enhance contrast. PMIRRAS spectra of functionalized PEGMEMA brushes were obtained using a Nicolet Manga-IR 860 FT-IR spectrometer with a photoelastic modulator (PEM-90, Hinds Instruments, Hillsboro, OR), a synchronous sampling demodulator (SSD-100,GWC Technologies, Madison, WI), and a liquid nitrogen cooled MCT detector. All spectra were obtained at an incident angle of 83°, with modulation centered at 2500 cm−1 to produce a spectrum with a range of 1000−3000 cm−1. For each sample, 512 scans were taken using a resolution of 4 cm−1 per modulation center. Data were acquired as transmittance versus wavenumber, and spectra were normalized and converted to absorbance units versus wavenumber via methods outlined by Frey et al.37 Optical images of micropatterned slides were taken on a Nikon microscope (TS100 with ECLIPSE ME600L camera), and fluorescent images were taken using a Nikon A1R confocal microscope. Average fluorescence data was acquired using the “ROI” and “Measurement” functions in Nikon NIS Elements software.

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RESULTS AND DISCUSSION Fabrication and Characterization of Micropatterned PEGMEMA Brushes. To micropattern PEGMEMA brushes, PDMS stamps featuring an array of 300 μm diameter posts were used to microcontact print circular patterns of ATRP initiator ω-mercaptoundecyl bromoisobutyrate (Scheme 1, i) on a Au-coated glass slide. OEGMEMA monomer was polymerized from the bromine-terminated SAM using SI3297

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Figure 2. Azide and acetylene functionalization of PEGMEMA brushes using nucleophilic substitution. (A) Confocal images of (i) naive PEGMEMA brushes (ii) reacted with sodium azide and (iii) click-modified with acetylene-PEG4-biotin after immunostaining with streptavidin-Alexa Fluor 546. (B) Analogously, PEGMEMA brushes (i) reacted with propargylamine, click-modified with azide-PEG4-biotin, and stained with streptavidin-Alexa Fluor 488 also fluoresce. (ii) Similarly treated brushes reacted with TMS-propargylamine were converted to a clickable state by (iii) deprotecting the acetylene group prior to the biotin immobilization reaction. Scale bars are 300 μm. (C, D) PM-IRRAS spectra centered around (i) the azide or acetylene peak position (∼2100 cm−1) and (ii) the secondary amine peak (∼1650 cm−1) further confirmed the presence of (C) azide-functionalized PEGMEMA (solid black line) and acetylene-PEG4-biotin conjugated PEGMEMA (gray dashed line) vs naive PEGMEMA control samples (gray dotted line). Similarly, spectra confirmed the presence of (D) acetylene-functionalized PEGMEMA (solid black line) and azide-PEG4-biotin conjugated PEGMEMA (gray dashed line) vs naive PEGMEMA control samples (gray dotted line).

AGET ATRP,38,39 which is a controlled reaction known to produce polymer products with narrow polydispersity.39 Imaging of the resulting substrates by SEM revealed successful grafting of micropatterned PEGMEMA brushes, and the chemical composition and spatial distribution of PEGMEMA brushes were further confirmed using Fourier transform infrared imaging (FT-IR; Figure 1A). Spectral analysis (aperture size of 25 × 25 μm) of a random micropatterned PEGMEMA brush (300 μm diameter) produced signatures indicative of PEGMEMA,40,41 and 3D FT-IR mapping of the −CH2/−CH3 peak also confirmed a micropatterned PEGMEMA brush arrangement. Prior publications have shown that grafted PEGMEMA brushes of ≥10 nm in thickness can produce nonfouling surfaces, and ∼50 nm thick PEGMEMA brushes can be grafted from a SAM of ω-mercaptoundecyl bromoisobutyrate using SIATRP.19,42 Thus, we used atomic force microscopy (AFM) to characterize our SI-AGET ATRP grafting reaction, and determine whether our PEGMEMA brushes were thick enough (i.e., ≥10 nm) to possess nonfouling properties. Dry AFM measurements were performed at the edge of micropatterned PEGMEMA brushes by scanning a 50 × 50 μm area under ambient conditions, and section analysis of AFM images for multiple PEGMEMA brushes from a single slide yielded an average brush thickness per synthesis reaction time (Figure 1B). A highly correlated exponential fit of the data demonstrates our ability to predictably tune the thickness of micropatterned PEGMEMA brushes. Moreover, our grafting reaction produced PEGMEMA brushes of ∼70 nm in thickness after extended polymerization (16 h), which are of sufficient thickness to possess nonfouling properties. Cell culture assays were used to functionally verify the nonfouling properties of grafted PEGMEMA brushes. Such brushes have previously been shown to resist protein adsorption and thereby cell adhesion for approximately one month in culture.19,43 We generated micropatterned PEGMEMA substrates using PDMS stamps featuring a reverse pattern from prior experiments (i.e., 300 μm diameter wells instead of posts) and tested their ability to restrict the adhesion and growth of human embryonic stem cell (hESC)-derived

neuroepithelial cells (hNECs)44 (see Supporting Information, Figure 1) and human umbilical vein endothelial cells (HUVECs) to within 300 μm diameter circles (Figure 1C). PEGMEMA brushes were able to restrict the adhesion and growth of both hNECs, which were seeded in chemically defined media with 25 μg/mL mouse laminin, and HUVECs, which were seeded and cultured in media containing 10% fetal bovine serum. Patterning of hNEC cultures persisted through day 10 when cell proliferation resulted in the formation of neurospheres that detached from the substrates as spherical aggregates. Similarly, HUVEC adhesion was restricted for up to 1 month before the experiment was aborted. Thus, our SIAGET ATRP synthesized PEGMEMA brushes functionally resist cell adhesion due to their nonfouling properties. Orthogonal Click Functionalization of PEGMEMA Brushes. The inert properties of PEGMEMA brushes are attractive for engineering biospecific culture substrates; however, their chemical composition offers limited reactive groups for bioconjugation. Fortunately, the ATRP reaction mechanism maintains the presence of a terminal alkyl halide (e.g., bromine) at the propagating end of “living” polymer chains45 that can be readily converted into other functionalities using nucleophilic substitution reactions.28,46 Due to the bioorthogonality and efficiency of “click” reactions,47 conversion of the terminal halides on ATRP synthesized PEG chains to azido groups via sodium azide-mediated nucleophilic substitution has become a standard method for generating clickable and biocompatible polymers.32 Moreover, this reaction has been extended to functionalize grafted PEG brushes.28,48 To determine whether our micropatterned PEGMEMA brushes can be similarly functionalized with azido groups, we reacted patterned substrates with sodium azide for 12 h and, subsequently, tested whether acetylene-PEG4-biotin could be immobilized on the brushes using the 1,3-dipolar cycloaddition click reaction (Figure 2A). After the reaction, the slides were immunostained with a streptavidin-Alexa Fluor 546 conjugate, which binds to biotin molecules, and imaged using confocal microscopy. No fluorescence was detected on naive brushes (Figure 2A(i)) or those that were only reacted with sodium azide (Figure 2A(ii)). However, fluorescence was detected on 3298

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Figure 3. Passivation of micropatterned PEGMEMA brushes. (A) Confocal images of (I) bromine-terminated or (II) azide-functionalized PEGMEMA brushes reacted with tributyltin hydride/Cu(I)/PMDETA (in anisole, i, v), tributyltin hydride/AIBN (in DMF, ii, vi), zinc powder (iii, vii), or ethanolamine (in DMSO for 16 h, iv, viii) preceding click-immobilization of acetylene-PEG4-biotin and streptavidin-Alexa Flour 546 staining. Post-reaction, group (I) brushes were first azide-functionalized prior to further postprocessing. (B) Semiquantitative analysis and confocal images of ̈ bromine-terminated PEGMEMA brushes reacted with ethanolamine for varying durations prior to postprocessing, the fluorescence emitted by naive as in A (*p ≤ 0.001, Student’s t test). Error bars are s.d.; scale bars are 300 μm.

PEGMEMA brushes reacted with sodium azide and then modified using click chemistry (Figure 2A(iii)), thereby indicating the prior presence of terminal bromine groups that were successfully converted to azides for click immobilization of biotin. Moreover, PM-IRRAS analysis of azide-functionalized PEGMEMA brushes detected the absorption of IR light at ∼2110 cm−1, which is indicative of the alkyl azide terminal groups65 (Figure 2C(i)). Also, the secondary amines contained in biotin upon click-modification with acetylene-PEG4-biotin were detected by PM-IRRAS at ∼1630 cm−165 (Figure 2C(ii)). Spectral analysis was validated using similarly functionalized alkanethiol SAMs (see Supporting Information, Figure S2). To create an orthogonal functional group on grafted PEG brushes, we also explored methods for converting the terminal bromines to acetylene-presenting groups. Typically, acetylene groups are conjugated to PEG brushes by modifying hydroxy30,31,49,50 groups at the ends of oligo-ethylene glycol methacrylate (OEGMA) chains. They can also be added by copolymerization with an acetylene-containing monomer51,52 or modification of a monomer’s terminal carboxyl53 or epoxy54 groups. However, because PEGMEMA brushes do not contain hydroxyl, carboxyl, or epoxy groups, we attempted a nucleophilic substitution using propargylamine (Scheme 1(iv)) to replace the brushes’ terminal bromines. Propargylamine-mediated nucleophilic substitutions have never before been demonstrated in this context, and although alkyl halide substitutions via primary amine groups have been previously documented,55 they are typically viewed as an insufficient side reaction for immobilizing molecules to grafted brushes.56−58 After the nucleophilic substitution reaction using propargylamine, the presence of acetylene groups on PEGMEMA brushes was detected by click immobilization of azide-PEG4biotin followed by immunostaining with streptavidin-Alexa ̈ Fluor 488. No fluorescence was detected upon imagining naive brushes (data not shown); however, micropatterned PEGMEMA brushes reacted for 12 h with proparglyamine were acetylene-functionalized as indicated by fluorescence (Figure 2B(i)). Also, PEGMEMA brushes reacted with proparglyamine for 24 h obtained a density of terminal acetylene groups that is significant and mediated biotin/streptavidin fluorophore immobilization equivalent to brushes reacted with sodium azide for a similar duration (see Supporting Information, Figure

S3). To further verify propargylamine’s acetylene group was the site of azide-PEG4-biotin immobilization, a similar reaction was performed using trimethylsilyl (TMS)-protected propargylamine, in which the acetylene group is physically blocked until deprotected with a basic solution (e.g., potassium hydroxide).51,52 Without a deprotection treatment prior to clickmodification no biotin was immobilized on TMS-propargylamine modified PEGMEMA brushes, as evidenced by a lack of fluorescence (Figure 2B(ii)). However, if the sample was deprotected prior to click-modification, then fluorescence was again readily detected on the micropatterned brush although defects in the PEGMEMA brushes were observed due to the harsh deprotection treatment (Figure 2B(iii)). Thus, nucleophilic substitution of the terminal bromine groups using propargylamine is a viable method for acetylene functionalization of grafted PEGMEMA brushes. Moreover, PM-IRRAS analysis of propargylamine-reacted (Figure 2D(i)) and azidePEG4-biotin-conjugated PEGMEMA brushes (Figure 2D(ii)) detected IR light absorption by acetylene groups at ∼2130 cm−1 and by secondary amine groups at ∼1635 cm−1. Passivation of PEGMEMA Brushes. μCP and SI-AGET ATRP can be used repeatedly to generate multiple, chemically distinct brushes with well-defined spatial arrangements on the same substrate, but this requires passivation of the grafted polymers’ living chain ends between each ATRP step. To date, only a few strategies exist to perform such a dehalogenation step.28,59 Tributyltin hydride is the most widely used dehalogenation reagent, and it replaces alkyl halides with a hydrogen atom via a free radical chain mechanism.46 Zinc powder has been shown to perform a similar conversion, and we tested this as an alternative dehalogenation reagent.60 Also, sodium azide has been shown to effectively dehalogenate PEG brushes,28 but the versatile role of the resulting terminal azide groups in click reactions limits further applications. In contrast, nucleophilic substitution with amine-containing alkane molecules, for example, ethanolamine, should be a more attractive passivation method. Thus, we tested the ability of tributyltin hydride, zinc powder, and ethanolamine to dehalogenate PEGMEMA ̈ brominebrushes, and each reaction was tested on naive terminated and azide-functionalized brushes to determine their suitability for multistep μCP/SI-AGET ATRP synthesis 3299

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Figure 4. Orthogonal, click functionalized, multicomponent PEGMEMA brushes. (A) Schematic of protocol to fabricate multicomponent PEGMEMA brushes with orthogonal click functionalities (i.e., acetylene and azide). (B) Confocal images of stained PEGMEMA brushes over a (i) large and (ii) regular scanning area, with (C, i) a select region further magnified to demonstrate minimal cross contamination of fluorescence when substrate is excited at separate (ii) 546 and (iii) 488 nm wavelengths. Scale bars are 300 μm.

protocols. The presence of terminal azide groups was detected using click-immobilization of biotin followed by streptavidinfluorophore staining, and the presence of bromine groups was similarly detected after an initial nucleophilic substitution using sodium azide. For tributyltin hydride reactions, two different radical generator systems were tested (i.e., CuBr/PMDETA46,59 and AIBN46,59), and we attempted to increase dehalogenation efficiency by adding bromine-bearing initiator ethyl 2bromoisobutyrate (EBiB).59 As a second dehalogenation method, micropatterned PEGMEMA brushes were immersed in an aqueous mixture of acetic acid, silica gel, and zinc powder. Third, passivation by nucleophilic substitution of the brushes’ terminal bromines with ethanolamine was attempted in DMSO containing triethylamine. For each reaction condition, the reactions and subsequent imaging of PEGMEMA brushes were executed simultaneously (Figure 3). Therefore, the fluorescent intensities emanating from (I) bromine-terminated and (II) azide-functionalized brushes post processing can be compared within (e.g., i and v, or iv and viii) but not across dehalogenation or substitution reaction conditions. Both tributyltin hydride and zinc powder treatments failed to completely debrominate naive PEGMEMA brushes as indicated by the ability to click-immobilize and fluorescently detect biotin molecules after reacting the brushes with sodium azide (Figure 3A(i−iii)). This might be due to the size-exclusion properties of grafted, hydrophilic, polymeric brushes.61 Additionally, tributyltin hydride/AIBN reaction conditions eliminated the reactivity of azide-functionalized PEGMEMA brushes (Figure 3A(vi)). In stark contrast, ethanolamine was effective at substituting terminal bromine groups, and it did not extinguish the reactivity of azidefunctionalized PEGMEMA brushes (Figure 3A(iv,viii)). Thus, it appears suitable for passivating PEG brushes during sequential SI-AGET ATRP protocols used to synthesize multicomponent PEG brushes. To our knowledge, this is first

demonstrated use of ethanolamine for this purpose. Further, time course analysis of the ethanolamine substitution reaction revealed that an overnight incubation (≥16 h) is sufficient to ensure dehalogenation of grafted PEGMEMA brushes (Figure 3A,B). Fabricating Orthogonal, Click Functionalized, Micropatterned PEGMEMA Brushes. As presented above, nucleophilic substitution reactions can be used for both PEGMEMA functionalization and passivation. Thus, we tested a novel strategy to create orthogonal, click functionalized, micropatterned PEGMEMA brushes using sequential rounds of μCP/SI-AGET ATRP interspersed with nucleophilic substitutions using sodium azide, ethanolamine, and propargylamine (Figure 4A). As proof of concept, two rounds of μCP/SIAGET ATRP were used to synthesize arrayed PEGMEMA brushes with azido or acetylene functionalities, which were indicated by immobilization of either acetylene- or azide-PEG4biotin and stained with streptavidin-Alexa Fluor 546 or 488, respectively (Figure 4B). Due to the constraints of μCP, some secondarily patterned brushes partially overlapped primarily patterned ones, and biotin/streptavidin-fluorophore analysis indicated minimal cross-contamination of functionalities only within these regions (Figure 4C). This occurred despite the high grafting density and thickness of PEGMEMA brushes (∼70 nm, Figure 1B), indicating that the second round of μCP could still deposit initiator alkanethiols within possible defects of the first patterned SAM.24 Therefore, combining this synthesis protocol with high precision μCP techniques62,63 is necessary to prevent overlap during μCP and thereby crosscontamination. Further, this would facilitate the engineering of novel substrates with complex patterns of orthogonally functionalized multicomponent PEG brushes, which could be made bioactive by immobilizing ligands using highly efficient click chemistries. 3300

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Figure 5. Tuning functionalization of nonfouling PEGMEMA brushes. Semiquantitative analysis of the effect of (A) sodium azide and (B) propargylamine nucleophilic substitution reaction times on the density of biotin ligands immobilized to PEGMEMA brushes, as indicated by the intensity of streptavidin-fluorophore staining. (C) Micrographs of (i) naive PEGMEMA brushes maximally substituted (24 h) with (ii) propargylamine, (iii) sodium azide, or (iv) ethanolamine after two days of hNEC culture. (D, i) An image of azide-functionalized brushes in (C(iii)) after in situ immobilization of biotin followed by Live/Dead cell assay, fixation, and Streptavidin-Alex Fluor 546 staining. (D, ii) An image of acetylene-functionalized brushes in (C(ii)) after fixation, followed by DAPI staining of cell nuclei, copper-catalyzed click immobilization of biotin, and streptavidin-Alexa Fluor 488 staining.

Versatile and Biospecific Cell Substrate Engineering Platform. Bioactive ligands have both molecularly specific and concentration-dependent effects.64 Thus, we investigated whether the degree of azido- or acetylene-functionalization of PEGMEMA brushes could be controllably modulated, thereby tuning the surface density of subsequently immobilized molecules. We also tested whether maximally functionalized brushes remained cell adhesion resistant, thereby indicating their suitability to mediate biospecific interactions between cells and immobilized ligands. This is analogous to using mixtures of azido- and oligo(ethylene glycol)-terminated alkanethiols at various ratios to tune click-immobilization of peptides onto mixed SAMs;65 however, grafted PEG brushes form much more stable interfaces.19 We observed that the reaction kinetics for both sodium azide- and propargylamine-mediated nucleophilic substitutions were slow enough to allow the functionalization densities of PEGMEMA brushes to be tuned by simply varying the reaction duration (Figure 5A,B). As indicated by average fluorescent intensity, the functionalization densities increased significantly between PEGMEMA brushes reacted with sodium azide for 2 versus 4 h and 6 versus 12 h and at all tested durations of propargylamine reactions (p ≤ 0.001, Student’s t test). Further, we tested whether PEGMEMA brushes after 24 h of functionalization or ethanolamine-mediated passivation remained cell adhesion resistant. hNECs were seeded in serumfree media containing 25 μg/mL mouse laminin onto both functionalized or passivated micropatterned substrates and cultured for two days to permit cell adhesion and growth. After seeding and through two days of culture, optical imagining

confirmed that all micropatterned PEG brushes remained cell adhesion resistant despite the high-level of chemical substitution at their chain ends with azido, propargylamine, or ethanolamine groups (Figure 5C). Lastly, we verified that the azido- and acetylene-functionalized PEGMEMA brushes in Figure 5C remained reactive using copper-free and copper-mediated click chemistry in situ and postculture, respectively. Dibenzocyclooctyl (DBCO) molecules contain a highly strained alkyne bond that spontaneously reacts with azido-presenting molecules.66 Thus, to verify the reactivity of sodium azide-functionalized PEGMEMA brushes shown in Figure 5C(iii), DBCO-PEG4biotin was added directly to the slide’s culture media. After an hour of incubation, a LIVE/DEAD viability/cytotoxicity kit was used to determine the viability of adherent cells. Then, the cells were fixed, and streptavidin-fluorophore staining was used to detect immobilized biotin molecules. Imaging revealed successful in situ immobilization of biotin onto azide-functionalized, micropatterned PEGMEMA brushes with the vast majority of cells remaining viable (green spots; Figure 5D(i)). Some cell death (small red spots) was also observed, but this could be due to either the DBCO reaction and postreaction processing procedures, thus, optimization of the in situ modification protocols may require further investigation. For the propargylamine-functionalized PEGMEMA brushes shown in Figure 5C(ii), the presence of reactive acetylene groups was verified by first fixing the cells and staining their nuclei using DAPI (blue) and then using copper-mediated click chemistry to immobilize azide-PEG4-biotin. Similar to the azide-presenting brushes, streptavidin-fluorophore staining 3301

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revealed that the acetylene-functionalized PEGMEMA brushes were click-reactive in addition to being cell adhesion resistant (Figure 5D(ii)).

AUTHOR INFORMATION

Corresponding Author

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

CONCLUSION Nonfouling surface modifications that enable biospecific interactions have numerous applications from biosensors to biomedical devices.67 In particular, micropatterned PEG brushes can be used to engineer custom cellular microenvironments that facilitate biospecific interactions between cells and immobilized ligands. Thus, we aimed to increase the utility of this grafted macromolecule by developing a versatile protocol for synthesizing orthogonally functionalized, multicomponent PEGMEMA brushes using facile nucleophilic substitution reactions. As demonstrated previously,28 sodium azide-mediated nucleophilic substitution of chain end bromine groups was used to generate one click immobilization chemistry, and we demonstrated the novel application of propargylaminemediated substitution to functionalize PEGMEMA brushes with an orthogonal clickable group. The success of this reaction further supports the general use of amine-terminated alkane molecules to decorate PEGMEMA brushes with a variety of functional groups including those prevalent in copper-free click chemistries, for example, acetylene groups.68 Additionally, we showed for the first time that ethanolamine can fully passivate PEGMEMA brushes without extinguishing previously generated terminal azido groups, thereby enabling the fabrication of tertiary (i.e., azido-, acetylene-, and nonfunctionalized) multicomponent brush composites. Thus, the combination of sodium azide, ethanolamine, and propargylamine nucleophilic substitution reactions can be used in conjunction with μCP/SIAGET ATRP to engineer substrates with micropatterned, multicomponent, and orthogonally click-functionalized PEGMEMA brushes. The sequential nucleophilic substitution protocol presented in Figure 4 enables simplistic engineering of microscale regions on culture substrates with orthogonal covalent immobilization chemistries. Through subsequent immobilization of bioactive ligands, such substrates will enable facile generation of microscale regions with differential functionalities that could be used to control cell signaling and fate at the nano-tomicroscale.50,65,69,70 Further, the surface density of immobilized ligands can be easily tuned by varying the duration of nucleophilic substitution reactions, while the PEGMEMA brushes remain nonfouling and thereby maintain the specificity of ligand interactions. In addition, the presence of both the azido and the acetylene groups on multicomponent PEG brushes enables in situ ligand immobilization via both the copper-free thiol-yne51 and strain induced click chemistries.47 Thus, cell culture substrates engineered with this protocol will be useful for performing in situ modifications of surface chemistries to facilitate spatiotemporal control of cell signaling and fate.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Prof. Nicholas Abbott for the generous use of his AFM and PM-IRRAS. This work was supported by funds from the University of Wisconsin−Madison and the Wisconsin Institute for Discovery.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Derivation, flow cytometry, and immunohistochemical analysis of hNECs; PM-IRRAS analysis of ω-mercaptoundecyl bromoisobutyrate, acetylene functionalized, azide functionalized, and ethanolamine functionalized SAMs; side-by-side comparison of azide and acetylene functionalization density. This material is available free of charge via the Internet at http://pubs.acs.org. 3302

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