Low Fouling Electrospun Scaffolds with Clicked Bioactive Peptides

Thickness measurements (Filmetrics F20 thin film analyzer, fitted using the optical properties of polystyrene) suggested this formed a layer of approx...
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Low Fouling Electrospun Scaffolds with Clicked Bioactive Peptides for Specific Cell Attachment Andrew E. Rodda,†,‡,§ Francesca Ercole,†,∥ Veronica Glattauer,‡ James Gardiner,‡ David R. Nisbet,⊥ Kevin E. Healy,# John S. Forsythe,† and Laurence Meagher*,†,‡,§ †

Department of Materials Science and Engineering & Monash Institute of Medical Engineering, Monash University, Wellington Road, Clayton 3800, Victoria, Australia ‡ CSIRO Manufacturing Flagship, Bayview Avenue, Clayton 3168, Victoria, Australia § Cooperative Research Centre for Polymers, 8 Redwood Drive, Notting Hill 3168, Victoria, Australia ∥ ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Victoria, Australia ⊥ School of Engineering, The Australian National University, Canberra 0200, Australian Capital Territory, Australia # Departments of Bioengineering and Materials Science and Engineering, University of California at Berkeley, Berkeley, California, United States S Supporting Information *

ABSTRACT: While electrospun fibers are of interest as scaffolds for tissue engineering applications, nonspecific surface interactions such as protein adsorption often prevent researchers from controlling the exact interactions between cells and the underlying material. In this study we prepared electrospun fibers from a polystyrene-based macroinitiator, which were then grafted with polymer brushes using surfaceinitiated atom transfer radical polymerization (SI-ATRP). These brush coatings incorporated a trimethylsilyl-protected PEG-alkyne monomer, allowing azide functional molecules to be covalently attached, while simultaneously reducing nonspecific protein adsorption on the fibers. Cells were able to attach and spread on fibrous substrates functionalized with a pendant RGD-containing peptide, while spreading was significantly reduced on nonfunctionalized fibers and those with the equivalent RGE control peptide. This effect was observed both in the presence and absence of serum in the culture media, indicating that protein adsorption on the fibers was minimal and cell adhesion within the fibrous scaffold was mediated almost entirely through the cell-adhesive RGD-containing peptide.

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INTRODUCTION

Several different strategies have been investigated that reduce unwanted nonspecific interactions between a substrate and biological molecules. These include various coatings on flat surfaces, such as self-assembled monolayers,8,9 grafted polymer brushes,10−13 interpenetrating polymer networks,14 plasma polymer coatings,15,16 and layer-by-layer coatings,17 and threedimensional (3D) hydrogels made from synthetic18−20 or natural polymers.21 These substrates act as a “blank canvas” on which the desired pattern of biological signals may be created, while preventing the adsorption of other biological signals from the surrounding environment. However, the vast majority of studies on electrospun fiber scaffolds have not used a lowfouling fiber substrate. An exception to this was the work of Grafahrend et al.,22,23 who used a blended polymer to simultaneously control protein adsorption (via poly(ethylene glycol-co-propylene glycol) star

Electrospun fibers are used in a variety of applications where their surface properties are of prime importance, including as scaffolds for tissue engineering. However, nonspecific surface interactions such as protein adsorption typically prevent precise control of cell−material interactions. Previous work has examined electrospun fibers that were modified with a range of receptor-binding biological signals, including extracellular matrix proteins, growth factors, and short peptides,1−6 in an attempt to influence cell survival, proliferation, migration, or differentiation. While many of these studies report changes in cell behavior, both the resulting cell response and the analysis of these changes are complicated by poorly controlled microenvironments emerging from the scaffold’s surface. Various interactions, including nonspecific protein adsorption at the fiber surface and the attachment of signaling molecules with varying orientations and conformations, can lead to the cells’ local microenvironment becoming unpredictable and heterogeneous.7 © XXXX American Chemical Society

Received: April 20, 2015 Revised: May 25, 2015

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polymers) and provide functional groups (e.g., isocyanates) for specific functionalization of fibers with RGD-containing peptides. Cells were able to attach to fibers modified with an RGD peptide, but not to the unmodified fibers or to those with an attached control peptide. This could potentially allow further studies to stimulate the cells within electrospun fiber scaffolds in a more specific, targeted manner than has previously been possible, leading to a deeper understanding of cell signaling within 3D scaffolds. In this study we looked to extend this previous work so that the surface properties of the fibers (in this case the chemical reactive groups that are present for attachment of biomolecules and the low protein fouling behavior) could be systematically changed without affecting bulk properties such as fiber alignment and diameter, bulk mechanical properties, and degradation behavior. We and other groups have shown that surface-initiated polymerization via techniques such as atom transfer radical polymerization (ATRP) can be used to change the surface properties of electrospun fibers. ATRP initiating groups may be present within the structure of a polymer macroinitiator prior to electrospinning (as either side or end functionalities),24−29 or introduced in a second step (via covalent bonds or electrostatic attractions) after the electrospinning process is completed.30−33 In this way, we can produce a blank canvas of electrospun “bottlebrush” fibers, which can then be given tailored surface properties to suit a range of specific applications. In addition, different surface properties can be created on a single sample of fibers, reducing variability between test groups and removing any need to reoptimize the electrospinning process for different samples. We prepared electrospun fibers from a polystyrene-based macroinitiator, similar to those we have previously reported.24,34 We used surface-initiated atom transfer radical polymerization (SI-ATRP) to create low-fouling, functionalizable electrospun fibers via the copolymerization of monomers containing alkyne functional groups. Surface polymerization of a functional monomer allows a high density of reactive groups to be achieved on the fiber surface, particularly compared to approaches that utilize the relatively low-density end-chain halogen for coupling. While previous studies have attempted to polymerize functional alkynes via SI-ATRP, analysis of the resulting coatings showed that only thin or sparse coatings were formed.24,35 To circumvent these problems, we predominantly examined SI-ATRP of a PEG-alkyne monomer that was protected via a trimethylsilyl (TMS) protecting group. The alkynes within these coatings could be deprotected postgrafting using a mild treatment with potassium fluoride, and reacted with functional azides via the highly efficient, bio-orthogonal copper-mediated azide alkyne click (CuAAC) reaction. While silyl-protected alkyne monomers have been used for polymerizations in solution36,37 and from a swollen, cross-linked resin,38 this study marks the first successful use of ATRP to polymerize a protected alkyne from a solid surface. Results presented in this study showed that the bottlebrush fibers greatly decreased the level of nonspecific adsorption of proteins, allowing cells grown on the fibrous scaffolds to be specifically stimulated using a covalently attached peptide. This study demonstrates the effectiveness of applying a modular design to electrospun scaffolds, and opens up a range of possibilities for creating electrospun fibers with well-controlled surface interactions.

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MATERIALS AND METHODS

Materials and Instrumental Methods. The RAFT agent, Sdodecyl S-(2-cyano-4-carboxy)but-2-yl trithiocarbonate, was synthesized according to literature procedures39 and kindly donated by Dr. San Thang. The monomer trimethylsilyl-propargyl-hexaethylene glycol methacrylate (TMS-Prg-HEGMA) was synthesized as described in the Supporting Information. A fluorinated small-molecule label, 2,2,2trifluoroethyl 4-(azidomethyl)benzoate (TFAB) and a water-soluble ligand for use in the click reaction, tris((1- (4-carboxybenzyl)-1H1,2,3-triazol-4-yl)methyl)amine) (TBTA-COOH) were synthesized as previously reported.24,40 An azide-functional fluorescent RGDcontaining peptide (azidopentanoic acid-GGNGEPRGDTYRAYK(FITC)GG, based on the bsp-RGD(15) sequence used in previous studies14,41) was synthesized as described in the Supporting Information, while an equivalent RGE control peptide (lacking the FITC label) was obtained from Mimotopes (Melbourne, Australia). The enzyme α-chymotrypsin from bovine pancreas (type II, >40 units/mg) was acquired from Sigma-Aldrich. Full details of other reagents and the instrumental techniques used for characterization are reported in the Supporting Information. Preparation of Substrates from p(S-co-VBC) Copolymer via Spin Coating and Electrospinning. Spin coating of the macroinitiator was performed on glass coverslips (14 mm diameter, ProSciTech, Australia) and gold-coated QCM crystals (Q-Sense) similarly to previously reported methods.34 Briefly, polymer solutions (2% w/v) in toluene were pipetted onto the substrate, which was then rotated at 6000 rpm for 20 s. Thickness measurements (Filmetrics F20 thin film analyzer, fitted using the optical properties of polystyrene) suggested this formed a layer of approximately 250 ± 50 nm. To prepare mats of electrospun fibers, a PEG underlayer was first prepared to facilitate easy removal of the macroinitiator from the foil backing. Poly(ethylene glycol) (1 MDa) was dissolved at 4% (w/v) in water, while p(S-co-VBC) was dissolved to 60% (w/v) in DMF. The PEG solution was electrospun onto a slowly rotating mandrel with the following conditions: needle voltage +10.5 kV; collector voltage −1.5 kV; working distance 18 cm; pump speed 0.4 mL/h; time 1 h. A thin white film was formed on the foil. A second layer of macroinitiator was then spun onto the PEG underlayer using the following conditions: needle voltage +15 kV; collector voltage −2 kV; working distance 12.5 cm; pump speed 1.5 mL/h; time 30 min, at which point a thick mat had formed. Fiber mats were annealed in an oven at 100 °C for 1 h. Circular fiber samples (10 mm diameter) were cut from the mats using a punch. Fibers were removed from the foil backing by immersion in water, which dissolved the PEG underlayer, and placed in 48-well plates. Fibers were imaged via SEM, alignment, and diameter were measured and fitted to a continuous distribution (further details and MATLAB code for this procedure are available in the Supporting Information). Grafting of Poly(TMS-Prg-HEGMA-co-OEGMA) Brushes from Styrene-Based Copolymer Surfaces. One typical procedure is described here, with all reagents scaled linearly for different batch sizes. MQ water and ethanol were transferred into a glovebox and deoxygenated via nitrogen sparging for 30 min each. A stock solution of CuCl2 (16 mg/mL in MQ) was also prepared and similarly deoxygenated. Purified CuCl (49.5 mg) and HMTETA (400 μL) were weighed into a glass vial, while OEGMA 475 (4.28 g) and TMS-PrgHEGMA (460 mg) were weighed into a separate vial. These were then transferred to the glovebox. The monomer solution was sparged with nitrogen for 20 min. Substrates (in 48-well plates) were prepared by rinsing once in ethanol (5 min) and twice in MQ. The monomers were dissolved in ethanol, while CuCl2 stock (810 μL) was added to the CuCl/ligand. These two solutions were then added together. Final concentrations were 25, 5, and 75 mM for CuCl, CuCl2, and HMTETA, respectively, in a 40:60 ethanol/water solution. This was mixed well and added to the substrates, whereupon the plates were sealed with parafilm and left to shake within the glovebox for 20 h. Substrates were then removed from the glovebox to quench the reaction, washed in 50 mM Na2EDTA solution for 15 min and 4× in B

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Biomacromolecules MQ, before being dried under vacuum. Grafting of pOEGMA-only brushes was performed using a similar, previously reported protocol.34 Deprotection of Surface Coatings Containing TMS-PrgHEGMA. Samples for XPS analysis were cut in half prior to deprotection, with half kept untreated as a control. Grafted substrates (both spin-coated and fibrous samples) were immersed in a solution of 0.5 M KF in methanol. Trays were sealed with parafilm to prevent evaporation of the solvent and shaken at room temperature for 16 h. Substrates were the rinsed twice in methanol and twice in MQ water and then dried under vacuum. Immobilization of Azide-Functional Molecules via CopperMediated Click Reaction. To attach the FITC-labeled peptide (azRGD-FITC), HEPES buffer was made up to 0.1 M (pH 8.5), and a 4 mM stock solution of CuSO4 in this buffer was prepared. Fiber substrates with either grafted pOEGMA or deprotected p(TMS-PrgHEGMA-co-OEGMA) coatings were prepared in 48-well plates (3 repeats per condition). TBTA-COOH was combined with CuSO4 stock solution and left to dissolve for approximately 10 min. Fluorescent peptide was serially diluted in buffer. The two solutions were combined, and 225 μL of this solution was then added to each substrate. The reaction was started by the addition of 75 μL of ascorbic acid solution (20 mM in HEPES buffer). Final concentrations were thus 1 mM CuSO4, 2 mM TBTA-COOH, 5 mM ascorbic acid, and either 500, 100, or 10 or 1 μM peptide. The plate was sealed with parafilm, covered with aluminum foil and shaken at 25 °C for 3 h. The substrates were then rinsed in water, 50 mM EDTA solution, and then 4× in water. Further rinsing was performed over the next 4 days, with the water being changed twice daily until negligible fluorescence was detected in the supernatant. Attachment of nonfluorescent RGD/RGE peptides was performed similarly, without including pOEGMA-grafted (nonalkyne) substrates. Attachment of TFAB was performed similarly using a 1 mM TFAB concentration, in a 1:4 solution of DMSO/buffer. Quantification of Peptide Density. Following attachment of a fluorescently labeled peptide, substrates were rinsed in buffer for 2 h at 30 °C to ensure that rinsing had been adequate and that negligible fluorescence was measured. Measurements of peptide density were made by adapting the procedure of Barber et al.42 Chymotrypsin was dissolved to 6 mg/mL in Tris-HCl buffer (10 mM, pH 8.0), supplemented with 10 mM CaCl2. This solution was added to the wells (0.5 mL), which were covered with aluminum foil and allowed to react for 2 h at 30 °C. Samples were then examined for fluorescence using a plate reader as described above. Readings were compared to standards produced from known concentrations of digested peptide. Cell Culture. Stock cultures of L929 murine fibroblasts (cell line ATCC-CCL-1, Rockville, MD, U.S.A.) were cultured in minimum essential medium containing 10% fetal bovine serum (FBS) and 1% nonessential amino acids. Cells were incubated at 37 °C in a 5% CO2 atmosphere. Coated glass coverslips and electrospun fiber substrates were transferred to a new 48-well plate and sterilized by soaking in 2× Antibiotic-Antimycotic (Lifetech, U.S.A.) for 1 h. Prior to seeding, the substrates were washed 3× in sterile PBS. Where prestaining of cells was performed to visualize cells on the materials, a confluent L929 cell layer on a T75 cell culture flask (Nunc, Denmark) was incubated with a fluorescent DiLC12(3) membrane stain (BD Biosciences) at 1 μg/ mL in media for 1 h at 37 °C. This was followed with a 10 mL wash of PBS and cells were then harvested with TrypLE Express (Gibco) and washed twice in 30 mL media. Cells were then seeded at 2.5 × 104 cells/cm2 and samples were incubated at 37 °C and 5% CO2 in air for 24 h. Cells on coverslips were first imaged in situ, after which the substrates were gently removed from the wells and placed into new wells (prefilled with cell culture medium) for further imaging, simultaneously removing nonadhered cells. Cells on fibrous substrates were either prestained with DiLC12(3) as described above, or stained in situ following culture with a calcein/ ethidium live/dead stain (Life Technologies). Samples were photographed using a Nikon TE 2000 Eclipse fluorescent inverted microscope. Image analysis on fibrous substrates was performed by

applying a threshold to the live/dead stained images, followed by automated analysis using the ImageJ Particle Analysis tool. Particles at the edge of the image and clusters of multiple particles were excluded from analysis. Statistical analysis was performed in Graphpad Prism software, with groups being compared using a nonparametric Kruskal− Wallis test followed by Dunn’s multiple comparison test.

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RESULTS AND DISCUSSION Macroinitiator Synthesis and Substrate Preparation. A poly(styrene-co-VBC) macroinitiator was synthesized as illustrated in Table 1 (see Supporting Information for full Table 1. Polymerization Scheme and NMR/GPC Characterization for the p(S-co-VBC) Macroinitiator

VBC conva (%; NMR)

styrene conva (%; NMR)

Mn theob (kDa; NMR)

%VBCc (NMR)

Mnd (kDa; GPC)

Mwd (kDa; GPC)

Đd (GPC)

85

84

151

16.3

79.8

106

1.33

a

Calculated from ratio of integrals of monomer peaks to broad polymer peaks. Separate estimates for the individual monomers were made by examining peaks at 5.25 ppm, where one VBC monomer peak can be separated from the remaining monomer peaks. b Calculated from the conversion of monomers, using the equation Mn,theo = MWRAFT + [monomer]/[RAFT] × MWmon. × conversion). c Calculated from 1H NMR of the 3× precipitated polymer by comparing the ratio of the integrals of the chloromethyl peak (∼4.5 ppm) with those of the aromatic protons (6−7.5 ppm). dGPC Mn, Mw, and Đ were determined by analysis in THF against PS standards and calculated using Waters Millennium/Empower software.

experimental procedure and discussion). Fibers were electrospun from the copolymer (Figure 1A) to produce fibers with a smooth morphology, which were then annealed; this was found to stabilize the structure of the fiber matrix during later procedures. No change was seen in fiber morphology following annealing (Figure 1B). XPS analysis of the resulting fibers confirmed that chlorine was present on the surface in similar quantities (see Supporting Information) to those previously observed for related polymers.24,34 Fiber orientation and diameter were sampled via SEM and were fitted to a probability distribution function. This method provides a superior measurement of these variables compared to commonly used procedures (e.g., binning of data into discrete sets) that are often lossy. The best-fit distribution of fiber diameters is shown in Figure 1C, illustrating a slightly skewed distribution with a median fiber diameter of 890 nm, and a 95% confidence interval on the median of 840 to 970 nm. Fiber orientation was not statistically different from a random distribution (Figure 1D). Larger fiber sizes are known to lead to increased interfiber pore sizes,43 and thus the relatively large fibers produced here were expected to allow improved cell infiltration into the scaffold. Synthesis of Clickable Compounds. An alkyne-functional PEG methacrylate was synthesized for use in SI-ATRP grafting of the electrospun fibers, while a range of azidefunctional molecules were either synthesized or purchased. This monomer represented an improvement from a previous HEMA-based alkyne-methacrylate monomer,24 with simpler synthesis and potentially improved resistance to protein fouling. C

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Figure 2. (Top) Artistic impression of cells growing within the functionalized grafted fiber scaffolds examined in this study. (Center) The azide-functional, fluorescently labeled RGD-containing peptide (azRGD-FITC) that was used to quantify peptide density, showing the site where enzymatic cleavage is used to release a fluorescent label. (Bottom) Schematic of the different surface reactions used to prepare the bottlebrush scaffolds.

Figure 1. Low resolution SEM of fibers (A) prior to and (B) following heat treatment. (C) Cumulative and probability distribution functions describing the measured diameter of fibers. (D) Fiber alignment showed no statistical deviation from a random distribution using a Komogorov−Smirnov goodness-of-fit test, even with significance assessed at P < 0.1. A significant difference in distribution would lead to points falling outside the dotted lines.

Grafting of Alkyne Functional Brush Coatings via SIATRP. Grafted brushes were prepared in an identical fashion on both spin coated and fibrous substrate, with a 1:9 molar ratio of TMS-Prg-HEGMA:OEGMA475 being used. While previous studies had found that grafting of OEGMA from this macroinitiator was relatively poor in solvent systems that contained alcohols,34 the addition of ethanol was required to dissolve the more hydrophobic-protected monomer. Attempts to copolymerize unprotected Prg-HEGMA often resulted in extensive cross-linking during the reaction, due to side reactions with the copper catalyst.36,46 These side reactions were often more extensive than previously noted in the solution polymerization literature, resulting in insoluble cross-linked polymers, presumably due to the very high concentration of monomer. While these interactions would seem to preclude the use of unprotected alkyne monomers for effective SI-ATRP, protected alkyne monomers have been polymerized in a controlled fashion via solution ATRP.37,47 TMS-Prg-HEGMA was consistently copolymerized with OEGMA475 using a modified protocol, leading to the formation of a polymer brush coating on the fiber surface. These coatings

A further synthetic step protected the alkyne group with a trimethylsilyl (TMS) protecting group to form TMS-PrgHEGMA (see Figure 2). Several silyl-based protecting groups are available for the protection of alkynes, all of which may be deprotected under well-known conditions by TBAF (tetra-nbutylammonium fluoride) in THF.44 However, since the TMS group is the most labile of these protecting groups, it can also be removed using milder techniques.44,45 TMS protection was thus chosen due to the solubility of the macroinitiator surfaces in most solvents, including THF. In order to confirm that the alkyne monomers could be deprotected without damaging the underlying polymer fibers, several mild techniques were examined as replacements for the widely used TBAF/THF protocol. These were first tested for their ability to deprotect the monomer in solution, with the reactions being performed in deuterated methanol and monitored via NMR. It was found that treatment with a 0.5 M solution of KF resulted in deprotection of the monomer with high yield and without detectable side-reactions within 16 h (see Supporting Information). D

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easily be dismissed as being due to silicon contamination, which is commonly seen in XPS spectra, several pieces of secondary evidence support the idea that the detected silicon was due to the presence of TMS groups. The Si 2p peaks appeared at a shift of 99 eV, rather than the higher shifts (approximately 102 eV) that would be expected from silicon oil or grease containing Si−O covalent bonds.48 Furthermore, these peaks were almost completely lost following treatment of the surfaces with KF, which would not be expected for a silicon-containing contaminant. A concordant decrease was observed in the hydrocarbon component of the C 1s peak; the carbons in the TMS moiety would be expected to appear at this shift also. Further analysis of the C 1s peak showed that the ether carbon:hydrocarbon ratio obtained for the grafted substrates was much lower than would be expected from a copolymer that contained 10 mol % of TMS-Prg-HEGMA. Finally, the abundances of fluorine and nitrogen that were observed on TFAB-labeled fibers are much higher than would be expected for a copolymer brush that contained only 10 mol % of PrgHEGMA (the calculated yields would be well over 100%). SEM was used to examine the morphology of fibers following grafting and deprotection, confirming that these reactions did not significantly alter the fiber structure. Further investigations into the structure of the brush were pursued via sectioning of the fibers via cryomicrotomy and imaging via TEM, however, minimal contrast between the solid electrospun core and the polymer brush coating was observed, and neither staining protocols nor attachment of azide-functional gold nanoparticles was able to improve this (see Supporting Information for details). As far as we are aware, this study represents the first published use of a protected alkyne monomer in SI-ATRP. We observed that the side reactions in this study when attempting to graft an unprotected alkyne were even more problematic than has been described in the literature for solution polymerization. These side reactions were observed only in solutions that contained both the Cu(I) catalyst and the alkyne. As such, this study demonstrates an effective method to incorporate alkyne moieties into polymer brush coatings via SIATRP, while maintaining the desired functional properties of the coating. Characterization of Brush Functional Properties. Fibers were exposed to a 10% solution of FBS in PBS to investigate the level of nonspecific protein adsorption in a manner relevant to cell culture. The N 1s XPS peak was used to compare levels of protein adsorption in a semiquantitative manner (Figure 4). We found that grafted fibers adsorbed significantly less protein than nongrafted fibers, in some cases at levels below the detection limit of XPS. Quantification of the protein that was adsorbed to the grafted brush coating was performed using QCM crystals that were spin coated with macroinitiator thin films (i.e., nonfibrous surfaces). According to the fitted model, the macroinitiator-only surface adsorbed 29 ng/cm2 of protein, and retained 24 ng/cm2 of adsorbed protein following rinsing with buffer solution. No adsorption was detected on the grafted surfaces. While this could indicate that the coating is highly resistant to protein adsorption, it should be noted that the presence of a thick, presumably viscous polymer brush coating might decrease the sensitivity of the technique. Hence, while detection limits in QCM can be as low as a few ng/cm2, we believe that under these conditions it would be more difficult to detect small quantities of adsorbed protein.

could be deprotected using the KF/methanol solution investigated earlier and were reacted with a fluorinated azide label to demonstrate the activity of the deprotected alkyne. The resulting coated fibers were characterized extensively using XPS at each stage (Figure 3). XPS analysis of the grafted substrates

Figure 3. Summary of typical XPS spectra of fibers: (A) Nongrafted fibers; (B) Fibers grafted with poly((TMS-Prg-HEGMA)-co-OEGMA) brushes (increases in O 1s and Si 2p peaks, along with ether- and ester-carbon, aromatic shakeup component disappears from C 1s peak); (C) Grafted fibers following removal of the TMS protective group (Si 2p peaks disappear); (D) Deprotected fibers that had been reacted with a fluorinated azide label (F 1s, N 1s, and CF3 peaks appear).

showed increases in oxygen, ether carbon and silicon, commensurate with a thick coating incorporating these monomers. Following deprotection of the substrates, the silicon peak was significantly reduced in size (from 2.2 to 0.2 atomic percent), indicating the loss of the TMS group in high yield (see Supporting Information for full XPS results). Reaction of TFAB with the surface resulted in peaks for both fluorine and nitrogen being observed in XPS spectra of the fibers, in abundances indicating that high yields (approximately 70%) were obtained. Interestingly, analysis of XPS spectra at several different stages indicated that TMS-Prg-HEGMA made up approximately 30% of total monomer residues, despite only being present in the feed solution at 10%. Primary evidence comes from the silicon content of the coated fibers, which was much higher than would be expected for a copolymer coating that contained 10% TMS-protected monomer. While this might E

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paramount importance in creating reproducible, well-defined microenvironments. A range of interactions (discussed in detail elsewhere7) can cause the density and bioactivity of the attached signaling molecules to be inconsistent, preventing quantitative analysis of the resulting cell-material interactions. The CuAAC bioconjugation chemistry and the particular peptide ligand used in this study were chosen to minimize these issues as far as possible. The low fouling brush coating reduces interference from nonspecifically adsorbed proteins and other molecules. However, it also acts to keep the attached signals from interacting with the substrate to which they are attached. The CuAAC reaction is highly specific, restricting the reaction to a single reaction site on each peptide, ensuring consistent orientation and conformation of the attached ligand. This reaction also proceeds to high yields under aqueous conditions, and produces a linkage that is insensitive to hydrolysis or other degradation mechanisms, both of which are highly advantageous in these studies. The assay used here (fluorescence-release via enzymatic digestion) was adapted from previous studies42,49 and has several advantages in the materials described here. The fluorescence release format allows very high sensitivity to be achieved without the need for specialized equipment and extra safety procedures that are required for radiometric approaches. Releasing the fluorescent label, followed by measurement in solution, allows comparison with soluble standards and prevents inaccuracies that stem from fluorescence quenching at surfaces.42 Furthermore, it is ideal for use in a porous anisotropic 3D system such as this, in which many assay techniques cannot be applied. In order to normalize the peptide density to the surface area of the fibers, BET gas adsorption was used to directly measure this surface area. Measurements were made on nongrafted fibers for a variety of reasons, which are discussed fully in the Supporting Information, resulting in a BET surface area of 5.8 m2/g being recorded. A theoretical surface area calculation, made by recording a large number of fiber diameters from SEM images and modeling fibers as perfect, solid cylinders (as used in previous studies22), resulted in a smaller surface area of 4.8 m2/g, which may have been due to nanoscale topography or flattened cross sections that were not discernible in the SEM images. Internal pores have also been observed in electrospun polystyrene fibers, which could also affect the accuracy of calculations based on the assumption of a cylindrical morphology.50 Quantification of the peptide attached to fibrous substrates showed that a well-controlled density of peptide could be coupled to the substrate by simply adjusting the peptide concentration in the coupling reaction (Figure 5). Nonspecific interactions of the peptide with pOEGMA brushes (no alkyne) under the same conditions also increased with concentration, but the concentration of peptide on these substrates was consistently an order of magnitude lower than was seen on the alkyne-functional brushes. These nonspecific interactions potentially include adsorption at the brush−liquid interface and entrapment or entanglement of the peptide within the brush coating or nanopores, with potential for later release, although it should be noted that substrates were washed extensively prior to the assay and the levels of passive release measured prior to the assay and found to be insignificant compared to the measured concentrations of attached peptide. The use of an enzyme to successfully release the fluorescent label implies that the peptide molecule is accessible to large

Figure 4. (A) XPS detection of nitrogen (acting as a marker for adsorbed protein) on grafted vs nongrafted fibers following exposure to FBS solution. (B) Monitoring of flat surfaces (grafted and nongrafted) via QCM during exposure to 10% FBS solution. Error bars show one standard deviation.

Finally, we prepared flat films via spin coating of the macroinitiator, and measured the static water contact angle on grafted and nongrafted surfaces. As may be observed in Table 2, Table 2. Static Water Contact Angle Measurements of Spin Coated Surfaces Showed That Their Hydrophilicity Increased Significantly Following Grafting, and May Have Increased Further Following Deprotection of the TmsProtected Monomer Residuesa surface

static water contact angle

bare macroinitiator macroinitiator-graf t-p(OEGMA-co-TMS-Prg-HEGMA) macroinitiator-graf t-p(OEGMA-co-TMS-PrgHEGMA), deprotected macroinitiator-graf t-pOEGMA (previous study34) a

79 ± 1 25 ± 4 18 ± 2 19 ± 6

Values are presented as mean ± standard deviation.

grafting of the PEG-type bottlebrush coating led to a significant decrease in contact angle; the contact angle of the deprotected alkyne brushes was almost identical to that observed on poly(oligoethylene glycol methacrylate) brushes in a previous study.34 A slightly higher contact angle (no statistically significant difference) was measured on TMS-protected alkyne brushes. Attachment and Quantification of Cell Adhesion Peptides via Copper-Mediated Click Chemistry. Controlling the density and bioactivity of molecules, particularly proteins and peptides attached to scaffold materials, is of F

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It was found that cells attached and spread strongly on surfaces with 8 pmol/cm2 of available RGD peptide, with a decreasing response at lower surface densities (Figure 6).

Figure 5. Quantification of the surface concentration of azRGD-FITC vs feed concentration into the reaction, performed on grafted fiber substrates with/without alkynes present in the brush coating.

proteins when coupled to the brush coating. Previous studies have shown that the penetration of a molecule into a surface polymer brush coating and its subsequent immobilization within the coating is highly dependent on the size of that molecule. For example, Schuh and Ruhe showed that penetration of PEG molecules of defined sizes into a brush coating exponentially decayed for larger PEGs.51 Furthermore, Barbey et al. showed that a small amine-functional tag molecule could react throughout the depth of an epoxy-functionalized brush, however bovine serum albumin (a 66 kDa protein) was localized at the surface.52 These results suggest that the RGDcontaining peptide and the integrin receptor protein could penetrate the brush coating to different degrees. As the assay relies on chymotrypsin (another protein, 25 kDa) to release peptide fragments for quantification, these measurements are more likely to correspond to peptide that is available for cell binding, rather than peptide which is trapped within the brush and inaccessible, in effect using the enzyme as a proxy for receptor integrins to test ligand availability. It should be noted that fluorescence was still observed following digestion when the fiber mats were directly read inside the plate reader, however a subsequent digestion did not release any further fluorescent label; this suggests that peptide was still present within the brush coating or in nanopores in the coating, but was not accessible to the enzyme (nor for later integrin binding). This potentially gives the assay an advantage over many other assays with similar sensitivity, such as radioiodination, which cannot discriminate between peptide molecules that are available to proteins and cells at the surface of the brush and those within the coating that are not accessible. Cell Attachment to Peptide-Modified Substrates. Initial testing of cell responses to varying peptide surface densities was carried out on flat substrates, in order to determine the surface density of peptide required for effective cell attachment. Grafted surfaces with varying densities of attached RGD peptide were seeded with L929 murine fibroblasts in the presence of 10% FBS, which were allowed to attach for 24 h. Surfaces without peptide served as a negative control, while nongrafted macroinitiator and TCPS surfaces acted as positive controls. It was assumed that surface peptide density was identical on both flat substrates and fibers, due to the large excess of peptide used in the CuAAC reaction.

Figure 6. Growth of L929 fibroblasts on flat surfaces with varying concentrations of attached azRGD peptide. (A) Spin coated macroinitiator. (B) Macroinitiator grafted with poly(TMS-PrgHEGMA-co-OEGMA) brushes, followed by deprotection with KF. (C) Grafted macroinitiator with 0.3 pmol/cm2 covalently attached azRGD peptide. (D) Grafted macroinitiator with 2 pmol/cm2 azRGD. (E) Grafted macroinitiator with 8 pmol/cm2 azRGD.

Attachment and some spreading was observed on surfaces with 2 pmol/cm2 peptide, while lower levels of attachment and no spreading were observed on surfaces with 0.3 pmol/cm2. Notably, cells were more spread (increased apparent surface area) on the 8 pmol/cm2 surfaces than on the bare macroinitiator (with adsorbed FBS proteins), with the cell morphology being qualitatively similar to TCPS surfaces. Almost no cells were observed on polymer brush surface containing no peptide, as the unattached cells on these surfaces were removed during the transfer of surfaces into new wells for imaging. L929 cells were then seeded on brush-coated fiber substrates that bore 8 pmol/cm2 of the covalently attached RGD peptide, to examine cell attachment on and within the 3D structure. An RGE control peptide, which differs from the RGD by a single methyl group but lacks biological activity, was used to create otherwise identical control substrates, in addition to controls that contained no peptide (i.e., polymer brush coating only). Cell culture was performed in both serum-free and serumcontaining media, to account for any residual protein adsorption that might be mediated by the attached peptide. Cells were stained prior to culture in order to visualize their morphology alongside the fiber structure. As this can affect cell survival and proliferation, nonprestained cells were also examined for viability and morphology using calcein/ethidium live−dead staining. Cell counts were performed, and cell shape G

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Figure 7. Live/dead staining of L929 fibroblasts grown on fibrous substrates for 24 h in (A−C) media containing 10% FBS and (D−F) serum-free media: (A, D) Grafted fibers only; (B, E) Grafted fibers with attached azRGE control peptide; (C, F) Grafted fibers with attached azRGD peptide. Inserts show wide-field low magnification images. Higher magnification images (G−I) show prestained cells grown in serum-free media on fibers with no attached peptide (G, rounded morphology), attached azRGE (H, rounded morphology), and azRGD (I, spread morphology). Cell counts for each of the conditions are given above the inset (live = green, dead = red).

was analyzed using measurements of roundness and aspect ratio in order to quantify cell attachment characteristics on the different surfaces. Under no-serum conditions, cells seeded onto RGDfunctionalized fiber substrates were able to survive well, attach to the fibers in large numbers and spread (Figure 7F), often along the length of the fiber (Figure 7I). Conversely, limited attachment was observed on RGE surfaces, and cells were alive but rounded in shape (Figure 7E). A large proportion of cells grown on the brush-only surfaces were dead, and the remaining live cells were rounded in morphology (Figure 7D). There was no evidence that the copper catalysts used in SI-ATRP and CuAAC were present (in agreement with XPS results) or contributing to any cytotoxicity; cell death was only observed on substrates where neither serum nor peptides were present. Little difference was observed in cultures performed on RGD-functionalized substrates with/without 10% FBS, although cell numbers were higher when FBS was present. Cells grown on RGE control surfaces were able to attach when serum was present, while only small numbers of cells attached in the absence of serum. However, spread cells were significantly less numerous under these conditions than on RGD-functionalized fibers, although small numbers of spread cells were observed on RGE-functionalized fibers when serum was present. Cells grown on brush-only substrates were similarly rounded in serum/no serum cultures, but no significant cell death was observed in those that contained serum. Images of cells were converted to black and white by choosing a threshold, followed by statistical analysis of cell shape using ImageJ. This showed that cells on RGD/FBS substrates were significantly less round (roundness = (4 × area)/(π × (major axis)2) and had significantly higher aspect ratios (major axis/minor axis) than RGE/FBS (Figure 8). In fact, average cell shape was not significantly different between

Figure 8. Automated shape analysis of L929 fibroblasts grown on grafted fibrous substrates with attached cell adhesion (RGD) and negative control (RGE) peptides. Cells grown in both the presence and absence of fetal bovine serum were analyzed. Plots that do not share a letter are significantly different (p < 0.0001).

RGE cultures with/without serum, despite the observation of a number of well-spread cells when serum was present. While significant differences were also seen in circularity of the cells (C = 4π × A/P2), this parameter is sensitive to edge roughness, such as can appear artifactually in the thresholded images or could result from cells that are rounded, but have numerous small processes extended that do not generate tension nor allow the cell to spread. These results are therefore not reported here. In summary, the analysis of cell cultures showed that the combination of RGD peptide with low fouling brush coatings H

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was able to mediate cell spreading in a peptide-specific manner, even in the presence of serum proteins. RGE control surfaces did not mediate attachment in large amounts without serum proteins being present, and mediated cell attachment, but not spreading, in the presence of serum. It is well-known that cell adhesion can occur at relatively low adhesion ligand densities, while cell spreading requires higher ligand densities.53 These results indicated that the large 19-mer peptides sequence may have provided sites for protein adsorption, but that the predominant mode of interaction was through specific RGDintegrin interactions. The small amount of residual attachment seen in serum-free cultures could potentially be due to the cells producing their own matrix proteins, which could then adsorb weakly to the RGE peptide; potentially smaller ligands such as cyclic RGDs could be used in future studies to further decrease nonspecific interactions.

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ASSOCIATED CONTENT

REFERENCES

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This work describes the modification of electrospun fibers with a low-fouling polymer brush coating, which simultaneously allows the conjugation of cell signaling peptides (via CuAAC chemistry) while reducing nonspecific interactions. The principal advantage of this system is that it decouples the surface and bulk properties of the resulting fibers, allowing these to be independently processed and optimized for specific applications, allowing much greater flexibility and a wider range of scaffold properties that can be explored. In a proof of concept, cells were able to spread on RGD functionalized fiber matrices, while cell−material adhesion interactions on the same substrate were minimal when functionalized with a control RGE sequence. This occurred even in the presence of serum proteins, demonstrating this system’s ability to stimulate cells in a highly specific manner.

S Supporting Information *

Detailed information on materials, instrumental methods, synthesis procedures, further supporting results and discussion, and MATLAB code for the fitting of continuous distributions. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00483.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.E.R. was supported in this work by an Australian Postgraduate Award and by the Cooperative Research Centre for Polymers. D.R.N. was supported by an NHMRC CDA Fellowship. The authors would like to thank Thomas Gengenbach and Chris Easton for assistance with XPS analysis, Xinghai Ning for assistance with monomer synthesis, Ben Fairbanks and Suzie Pereira for synthesis of TBTA-COOH and TFAB molecules, respectively, and Tae-Hyun Bae for BET measurements. I

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NOTE ADDED AFTER ASAP PUBLICATION This paper was publised ASAP on June 10, 2015, with an error to Figure 8. The corrected version was reposted on June 11, 2015.

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DOI: 10.1021/acs.biomac.5b00483 Biomacromolecules XXXX, XXX, XXX−XXX