Protein and Bacterial Antifouling Behavior of Melt ... - ACS Publications

Apr 4, 2016 - Both bacterial infections and the foreign body response can be attenuated by ... covalent bonds between the two materials for prolonged...
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Protein and Bacterial Antifouling Behavior of Melt-Coextruded Nanofiber Mats Si-Eun Kim, Cong Zhang, Abigail A. Advincula, Eric Baer, and Jonathan K. Pokorski* Department of Macromolecular Science & Engineering, Case Western Reserve University, 2100 Adelbert Road, Cleveland, Ohio 44106, United States S Supporting Information *

ABSTRACT: Antifouling surfaces are important for biomedical devices to prevent secondary infections and mitigate the effects of the foreign body response. Herein, we describe meltcoextruded poly(ε-caprolactone) (PCL) nanofiber mats grafted with antifouling polymers. Nonwoven PCL fiber mats are produced using a multilayered melt coextrusion process followed by high-pressure hydroentanglement to yield porous patches. The resulting fiber mats show submicrometer cross-sectional fiber dimensions and yield pore sizes that were nearly uniform, with a mean pore size of 1.6 ± 0.9 μm. Several antifouling polymers, including hydrophilic, zwitterionic, and amphipathic molecules, are grafted to the surface of the mats using a two-step procedure that includes photochemistry followed by the copper-catalyzed azide−alkyne cycloaddition reaction. Fiber mats are evaluated using separate adsorption tests for serum proteins and E. coli. The results indicate that poly(oligo(ethylene glycol) methyl ether methacrylate)-co-(trifluoroethyl methacrylate) (poly(OEGMEMA-co-TFEMA)) grafted mats exhibit approximately 85% less protein adhesion and 97% less E. coli adsorption when compared to unmodified PCL fibermats. In dynamic antifouling testing, the amphiphilic fluorous polymer surface shows the highest flux and highest rejection value of foulants. The work presented within has implications on the highthroughput production of antifouling microporous patches for medical applications. KEYWORDS: antifouling, nanofibers, polymer brushes, coextrusion, zwitterionic polymers surface roughness or manipulating surface topology.17−19 Serrano et al. introduced nanopatterned suturing on commercially available threads using oxidative plasma treatment to increase surface roughness and prevent bacterial attachment.10 The results showed that this simple method provided topological changes that were significant enough to produce a bactericidal surface. An alternative approach to modify the surface is to coat antifouling polymers onto a material.20 This method is facile, rapid, energy efficient, and widely utilized in industry.21 However, physical adsorption between the material surface and the antifouling coating is not strong enough to endure over time causing peeling or desorption of the coating layer.22 Hence, chemical grafting of antifouling polymers onto the material surface has been introduced to provide stable covalent bonds between the two materials for prolonged antifouling behavior.23,24 Photochemistry offers a convenient solution for covalent immobilization and allows two molecules to form a new chemical bond with simple ultraviolet (UV) irradiation.25 Photochemical methods provide additional advantages including spatial control of surface modification with photomasks, and varying degrees of functionalization by

1. INTRODUCTION Medically related devices that come into contact with the human body have seen an increase in device-associated infections, which are rapidly becoming a major clinical problem.1,2 These devices are also known to trigger “foreign body responses” or encapsulation by the immune system, rendering the device ineffective.3,4 The foreign body response is characterized by serum protein adhesion, followed by macrophage activation, and finally encapsulation of the substrate.5 Both bacterial infections and the foreign body response can be attenuated by developing antifouling surfaces, or surfaces that repel biological entities that promote infection or protein adhesion.6,7 A recent report indicated that about 270 bacteria/ cm2 fall into a wound site during a surgical procedure per hour.8 Therefore, antifouling surfaces have been of interest for clinical applications (e.g., sutures, surgical products, and wound healing patches) to prevent infections when the material comes into contact with wounds or blood.1,9,10 In general, antifouling surfaces aim to minimize the interaction between biomolecules and the surface of a biomaterial.11,12 To minimize pathogenic microbial adhesion, various modifications have been studied to impart antifouling behavior onto polymeric surfaces.13−16 These methods can be classified as chemical, for instance, hydrophobic, hydrophilic, or charged surface coatings, or physical, by way of increasing © XXXX American Chemical Society

Received: January 5, 2016 Accepted: March 23, 2016

A

DOI: 10.1021/acsami.6b00093 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces tuning the intensity of UV irradiation.26,27 Recently, immobilizing brush polymers using surface initiated atom-transfer radical polymerization (SI-ATRP) has garnered attention as a useful means for chemically modifying surfaces with antifouling polymers.28,29 For example, Stevens et al. described poly(εcaprolactone) (PCL) bilayers that can selectively adhere cells on one face when functionalized with arginine-glycine-aspartic acid (RGD) peptides and resist cells on the opposite face when grafted with poly(oligo(ethylene glycol) methyl ether methacrylate) (poly(OEGMEMA)) brushes. This antifouling surface was processed by SI-ATRP from PCL scaffolds that were prefunctionalized with an ATRP initiator (2-bromoisobutyryl bromide) followed by electrospinning.30 The results concluded that while a high cell density and bovine serum albumin (BSA) adhered on the RGD peptide layer, the poly(OEGMEMA)grafted PCL fiber layer showed significantly less cell density and BSA adherence. This stabilized chemically grafted poly(OEGMEMA) provides a selective antifouling surface. Several classes of surface-grafted polymers have been studied as candidates for improving antifouling properties. Hydrophobic polymers, such as fluorinated polymers, have a low surface energy, which minimizes the intermolecular interactions between the fluorine surface and fouling materials.31 For hydrophilic polymers, the mechanism of antifouling is a result of water molecules binding to the polymer, creating a protective hydrated surface, reducing interactions between the foulant and the surface.32,33 A common hydrophilic polymer, poly(ethylene glycol) (PEG), for example, has shown good antifouling properties in biomedical applications.34 Zwitterionic polymers have also been of recent interest as surface antifoulants because of their exceptional hydrophilic properties and excellent resistance to protein and bacterial adhesion.22,35,36 A wellknown zwitterionic polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)), is blood-compatible and superhydrophilic and creates a low friction coating when grafted onto surfaces.37 Recently, amphiphilic polymers, which are a combination of hydrophilic and hydrophobic polymers, have shown effective antifouling properties.38,39 Typically, the hydrophobic segment prevents biofoulants from adhering, while the hydrophilic segment is able to release biofoulants that may have been adsorbed.38,40 For example, Wooley et al. highlighted amphiphilic zwitterionic phosphorylcholine to improve antibiofouling properties.41 They found higher antibiofouling efficiency with this amphiphilic surface, resisting both protein and marine organisms. Therefore, we sought to design a highly antifouling, nonwoven fiber mat, bearing in mind the need for a relatively hydrophilic surface to maintain a moist environment between the body and the nonwoven fiber mat. We also incorporated a highly microporous design into our system to afford a high fluxa key component for biomedical applications.42 Previously, we reported meltcoextruded PCL nano- and microfibers, wherein fibers are produced at a high production rate when compared to electrospun fibers and can be densely chemically functionalized on the surface.43−45 The polymers, PCL and poly(ethylene oxide) (PEO), that are utilized are inexpensive and FDA approved in several applications providing a biocompatible system.8,23,24 Herein, the processing method is described to prepare nonwoven PCL nanofiber mats that are surfacemodified with antifouling polymer brushes. In this study, poly(OEGMEMA) (hydrophilic polymer), poly(MPC) (zwitterionic polymer), and poly(OEGMEMA-co-TFEMA) (amphiphilic polymer) were chosen as antifouling candidates. Since

poly(TFEMA) has poor water solubility and amphiphilic polymers drastically repel foulant materials, the combination of hydrophilic and hydrophobic segments of poly(OEGMEMA-co-TFEMA) was chosen for their combined effect.

2. EXPERIMENTAL SECTION 2.1. Materials. α-Bromoisobutyryl bromide (98%), 2,2′-bipyridine (bpy, ≥99%), monomer oligo(ethylene glycol) methyl ether methacrylate (OEGMEMA, Mn 475 g/mol), 2-methacryloyloxyethyl phosphorylcholine (MPC, 97%), and 2,2,2-trifluoroethyl methacrylate (TFEMA, 98%) were purchased from Sigma-Aldrich. L-Ascorbic acid sodium salt (99%) and copper(II) sulfate pentahydrate (99%) were purchased from ACROS Organics. Bovine serum albumin (BSA, powder) was obtained from Invitrogen. Escherichia coli (E. coli, BL21) cells were obtained from NEB. Viability/cytotoxicity assay kit for bacterial live and dead cell staining was purchased from Biotium. Fluorescein isothiocyanate (FITC) was purchased from Chem. Impex. Deionized (DI) water (Milli-Q, 18.2 MΩ cm) was used for filtration tests. 2.2. Instrumentation. Multilayer coextrusion was performed using a two-component coextrusion system with a series of identical multiplication units. Pore size was measured by Quantachrome Porometer 3G. Surface analysis was investigated on a PHI Versaprobe 5000 scanning X-ray photoelectron spectrometer (XPS) with an Al Kα X-ray source (1486.6 eV photons). Scanning electron microscopy (SEM, JSM 6510LV) was performed using a JEOL SEM under an emission voltage of 20 kV. A high-intensity UV lamp (Bluepoint 4 Ecocure from Honle UV America Inc.) was used for surface modification of the PCL fibers with propargyl benzophenone (PrBz). The molecular weight of the synthesized BSA-FITC was measured on a Bruker Autoflex III MALDI-TOF/TOF mass spectrometer using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. Fluorescent images of BSA-FITC and E. coli cells were taken via laser scanning fluorescence confocal microscopy using a Leica TCS SPE confocal microscope. Water contact angle (WCA) measurements were tested on a CAM 200 optical contact angle meter (KSV Instruments Ltd.). Tensile testing was performed on a Zwick/Roell mechanical testing instrument at room temperature at a rate of 50%/ min with a 100 N load cell. 2.3. Methods. 2.3.1. Melt Coextrusion of PCL Fibers and Preparation of Nonwoven PCL Nanofiber Mats. The melt coextrusion process began with PCL (CAPA 6800 pellets, MW = 80 kg/mol) and PEO. In order to match the rheology of PCL and PEO for the melt extrusion processing, two different molecular weights of PEO (Dow Chemical, POLYOX N80 (MW = 200 kg/mol) and POLYOX N10 (MW = 100 kg/mol)) were used with a ratio of 30:70 (200 kg/mol:100 kg/mol, N80:N10). The mixture of two molecular weights of PEO was used to ensure a viscosity match between PEO and PCL, critical to maintaining fiber uniformity. The two grades of PEO were premixed using a Haake Rheodrive 5000 twin screw extruder and pelletized. The viscosities of the obtained PEO blend and PCL melt matched at 180 °C which was chosen as the extrusion temperature. PEO and PCL were completely dried at 40 °C under high vacuum for 48 h in advance of coextrusion to prevent void volumes from residual moisture. PCL fiber domains embedded in a PEO matrix were fabricated via multilayer coextrusion at 180 °C. Eighteen vertical multipliers and 5 horizontal multipliers were utilized in this process. Finally, this structure went through a 3 in. exit die, and the extruded tape contained 8192 × 32 fiber domains. The extruded tape was collected on a chill roll at room temperature with a speed of 15 rpm. 2.3.2. Preparation of Nonwoven PCL Nanofiber Mats. To remove the separating PEO domains, the composite tape was cut (10 cm length) and placed in a water bath while stirring (12 h). After prolonged immersion the majority of the PEO was removed from the PCL/PEO composite tape yielding PCL fiber bundles. Two strips of PCL fiber bundles were stacked in a cross-ply (90°) on a metal plate and covered by an aluminum grid of mesh size 250 μm. A highB

DOI: 10.1021/acsami.6b00093 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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2.3.6. Conjugating Antifouling Polymers to the Alkyne-PCL Fiber Mats. Alkyne-decorated fiber mats were soaked in one of three different polymer solutions: poly(OEGMEMA), poly(MPC), and poly(OEGMEMA-co-TFEMA) (100 mg/mL, 3 mL) in DI water. CuSO4 (50 mM, 40 μL) and tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) (50 mM, 200 μL) were premixed and the catalyst complex was added to the polymer solution. Sodium ascorbate (100 mM, 200 μL) was added and then mixed. The reaction mixture was incubated for 2 h at 37 °C. Residual reagents were removed by washing with DI water and MeOH sequentially. All samples were dried under vacuum. 2.3.7. Surface Characterization of PCL Fiber Mats. Highresolution XPS and WCA were used to investigate the surfacemodified fiber mats. Polymer-modified fiber mats were scanned by XPS with specific attention paid to atoms unique to the grafted polymer (i.e., fluorine, phosphorus, or nitrogen). WCA was measured using standard static water contact angle measurements with ultrapure water in order to determine the surface wettability of each sample. The water contact angle was measured with the mean values from the right and left sides of the droplet (n = 3). 2.3.8. BSA-FITC Conjugation and Protein Absorption Test. To evaluate protein adsorption, BSA (10 mL, 0.3 μmol) was labeled with FITC (1 mL, 1 mmol) in 100 mM sodium carbonate buffer (pH 9.5) at 4 °C for 12 h. After the reaction, BSA solutions were dialyzed with cellulose membranes (cutoff 3 kDa) against 2 L of 10 mM phosphate buffered saline (PBS) at pH 7.4 and at 4 °C. The dialysis buffer was changed 3 times to completely remove free FITC. The molecular weight of BSA-FITC was confirmed by MALDI-TOF and SDS-PAGE. The ratio of FITC functionalization to BSA was determined by MALDI-TOF. Protein concentration was measured by Nanodrop using the BSA extinction coefficient (ε = 43 824 M−1cm−1) with λmax at 280 nm. The BSA adsorption test was performed to evaluate the antifouling nature of the fiber mats. Each polymer-functionalized fiber mat (control PCL, poly(OEGMEMA), poly(MPC), and poly(OEGMEMA-co-TFEMA) fiber mat, 0.5 cm × 0.5 cm square) was fully soaked in the 2.0 mL of BSA-FITC solution (9.2 mg/mL of PBS, pH 7.4) in a 12 well plate and then incubated for 2 h while shaking at 60 rpm. 2.3.9. Bacterial Culture and Viability/Cytotoxicity Assay for Live and Dead Cells. E. coli (BL21) bacteria were cultured in super optimal broth (SOB). When an optical density indicated 0.9 absorbance units at 600 nm, corresponding to a concentration of 7.53 × 108 colonies forming units per milliliter (CFU/mL), a 1 cm × 1 cm square of fiber mat (either control PCL, poly(OEGMEMA), poly(MPC), or poly(OEGMEMA-co-TFEMA)) was incubated in 2.0 mL of the SOB media at 37 °C on a shaker at 60 rpm for 24 h. After the fiber mats were taken out from the bacterial solution, each fiber mat was washed 3 times in water. Ten microliters of bacterial live and dead assay stain in 80 μL of 0.85% NaCl solution was added onto each fiber mat and incubated in the dark for 15 min. Each sample was mounted on a slide glass and then covered with a square coverslip and sealed with nail polish for confocal microscopy imaging. Using two fluorescent channels, live cells were scanned with DMAO (green fluorescence) indicating the presence of nucleic acids and EthD-III (red fluorescence) indicating the compromised membranes of dead cells at 488 and 522 nm laser excitations, respectively. 2.3.10. Dynamic Fouling Filtration Tests. Polymer-grafted nonwoven fiber mats were prepared in 0.85 cm diameter disks to evaluate the antifouling properties. A dead-end filtration (Millipore XX7104712) was setup including a solution reservoir with a vacuum filtering flask, a filter holder, and a vacuum pump. The fiber mat was precompacted by Milli-Q water at a pressure of 61 kPa for 10 min to obtain a steady flux, then pressure-driven filtration was performed. BSA solution (1 g/L in pure Milli-Q water) was utilized as a fouling agent. The permeation flux of pure water, Jw1, and permeation flux of protein solution, Jp, are determined by eqs 2 and 3

pressure waterjet, through a 0.010 in. diameter nozzle, was swept across the fibers parallel to the directions of the fibers on the top and bottom of the fiber mat for 5 min each with a 500 psi rotator pressure. This process removed the remaining residual PEO and produced a nonwoven fiber mat. To evaluate the mechanical properties of the PCL fibermat, tensile tests were performed in both the horizontal and vertical directions of the fibermat ply. 2.3.3. Porosity and Pore Size Distribution. The pore size of the fiber mats was determined by a porometer. Porosity was calculated by eq 1 ⎛ ⎞ ρ Porosity(%) = ⎜⎜1 − fibermat ⎟⎟ × 100 ρpolymer ⎠ ⎝

(1)

where the density of the fiber mat is ρfibermat and the density of polymer PCL is ρpolymer. The ρpolymer was obtained from averaging the original polymer densities (PCL; CAPA 6800 provided by Dow Chemical) at 25 °C. To determine ρfibermat the thickness was measured according to the ASTM standard D5729-97 by using an Instron 5565 device in compression mode. The fiber mat thickness was defined as the Instron plate distance under a pressure of 4.14 ± 0.21 kPa and was found to be 0.042 ± 0.011 cm (n = 4). The PCL nonwoven fiber mat was punched with metal rounds (diameter 2.5 cm) yielding a fiber mat volume of 0.21 ± 0.04 cm3. The pore size was measured by a Porometer 3G Macro instrument from Quantachrome (Boynton Beach, FL) in accordance with ASTM F316 standard for the through-pore characterization.43 The porometer wetting fluid was applied on the surface of the PCL fiber mat until completely wet. 2.3.4. Photochemistry. For alkyne surface functionalization of the PCL fiber mats, photochemistry was carried out using previously reported methods.46,47 Briefly, a 0.5 cm × 0.5 cm of fiber mat was dip coated in a solution of Pr-Bz (10 mg/mL in MeOH) and sat at room temperature until all of the methanol was evaporated. The PCL fiber mats were exposed to a UV light source for 10 min on both sides (33.5 mW/cm2). PCL nonwoven fiber mats were washed in methanol 3 times to completely remove excess Pr-Bz. The alkyne-decorated fiber mats were vacuum-dried overnight. 2.3.5. ATRP Synthesis of Antifouling Polymers. All monomers were purified prior to ATRP. To purify MPC, the monomer was stirred in acetonitrile at 80 °C for 7 h. After cooling to room temperature, the MPC solution was placed at −20 °C. The white crystalline MPC was filtered and vacuum-dried. OEGMEMA monomers were purified by basic alumina column to remove inhibitors. 2,2,2-Trifluoromethacrylate (TFEMA) was used as purchased. Copper(I) bromide (CuIBr) was purified by stirring in glacial acetic acid to remove the oxidized product, and the white powder of CuIBr was filtered, washed with MeOH, and dried under high vacuum overnight. 2,2′-Bipyridine (bpy; 30 mg, 0.22 mmol) and purified CuIBr (8 mg, 0.056 mmol) were placed in a Schlenk flask with degassed methanol under N2 gas, and the flask was quickly sealed with a rubber septum and parafilm. Either pure MPC (1 g, 3.4 mmol), OEGMEMA monomer (2.2 mmol), or the mixture of OEGMEMA (0.8 g, 1.7 mmol)/TFEMA (0.28 g, 1.7 mmol) (1:1 equiv) monomers and the initiator (0.056 mmol) were dissolved in methanol, placed in a Schlenk flask, and degassed using N2. ATRP was performed under N2(g) for 24 h at room temperature after combining the catalyst into the reaction. The reaction was quenched by exposure to air, and the solvent was removed by rotary evaporation. The crude material was redissolved in 10 mL of THF (poly(OEGMEMA) and poly(OEGMEMA-co-TFEMA)) or in 10 mL of MeOH (poly(MPC)). This final green mixture was passed through an alumina column (Al2O3) in order to remove the copper catalyst complex, yielding a clear polymer solution. This solution was precipitated in 90 mL of cold diethyl ether and collected by centrifugation. The final polymers were dried under high vacuum overnight. Gel permeation chromatography (GPC) analysis was carried out by a Shimadzu Prominence GPC instrument equipped with a Shimadzu RID10A differential refractometer detector, at a flow rate of 1.0 mL/min at 40 °C with THF as the eluent. To investigate the molecular weight of the synthesized polymers, 1H NMR spectrum was recorded utilizing a 600 MHz Varian Inova NMR spectrometer.

Jw = C

Vw Δt × A

(2) DOI: 10.1021/acsami.6b00093 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Schematic of melt coextrusion of PCL/PEO tape within the barrel of the extruder (left) and water treatment to release PCL nanofibers by removing PEO (right).

Jp =

Vp

Coextrusion was carried out by layering PCL and PEO melt flows horizontally followed by vertical rotation in the multiplier (Figure 1A); it should be noted that a blend of two molecular weights of PEO was used to ensure a rheological match between PCL and PEO at the extrusion temperature. Rheologically mismatched polymer melts result in encapsulation during processing and nonuniform fiber domains.48,49 In step B, layers were repeatedly split horizontally and recombined vertically to form 2n vertical layers of alternating PCL and PEO, where n was the number of vertical multipliers (18) in the melt coextrusion line. After the vertical multiplication, a thin surface layer of PEO was placed on top and bottom as shown in step C. Finally, the melt flow was split by m (5) horizontal multipliers. These horizontal multipliers split the polymer flow vertically and recombined them horizontally to form an organized sea− island structure (Figure 1D) with 2n−m × 2m islands. A final PCL/PEO composite fibrous tape was created with 213 × 25 PCL domains embedded in a PEO matrix as shown in Figure 1. Melt coextrusion with 23 multipliers (1.36 kg/h production rate) produced the multilayered composite PCL/PEO tape (Figure 2A). PEO was removed in a two-step process: (1) a 12 h immersion in a water bath to swell and remove a large portion of the PEO, followed by (2) two PCL fiber bundles that were cross-plied and stacked, then mats were consolidated utilizing a high-pressure water jet (Figure 2B). The latter step served to remove all traces of PEO, a requisite for obtaining high-porosity fiber mats. The high-pressure wash served to occasionally break individual fibers and entangle them within the adjacent ply. This hydroentanglement process reduces concerns regarding the use of organic solvents, and pure PEO

(3)

Δt × A

⎛ Jp ⎞ ⎟⎟ × 100 DR (t)(%) = ⎜⎜1 − Jw1 ⎠ ⎝

(4)

⎛ Jw2 − Jp ⎞ DR (r)(%) = ⎜⎜ ⎟⎟ × 100 ⎝ Jw1 ⎠

(5)

⎛J − J ⎞ w2 ⎟ DR (ir)(%) = ⎜⎜ w1 ⎟ × 100 ⎝ Jw1 ⎠

(6)

⎛J ⎞ FRR(%) = ⎜⎜ w2 ⎟⎟ × 100 ⎝ Jw1 ⎠

(7)

The pure water flux (Jw1) (mL/(cm ·min)) was measured for 10 min and then the flux Jp was measured for 20 min with the fouling solution. All samples were washed with pure water for 10 min to remove the BSA foulant from all fiber mats, and then pure water fluxes (Jw2) were recorded for 10 min. Based on the time-dependent fouling test, the fiber mats were evaluated by total flux decline ratio (DRt), reversible flux decline ratio (DRr), irreversible flux decline ratio (DRir), and the flux recovery ratio (FRR) for further investigation of antifouling properties following from eqs 4−7. 2

3. RESULTS AND DISCUSSION 3.1. Melt Processing of Fiber Mats. Melt coextrusion provides polymeric nanofibers with high surface area and possesses the advantages of continuous processing, high production rates, and a solvent-free processing system. D

DOI: 10.1021/acsami.6b00093 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. (A) Digital image of the PCL/PEO multilayer tape as collected from the extruder, (B) digital image of as-extruded PCL/PEO multilayer tape prior to mat consolidation, (C) digital image of consolidated nonwoven PCL nanofibrous mat, (D) SEM micrographs of the surface of a nanofiber mat, (E) SEM micrograph of the cross section of a mat, (F) histogram representing widths of PCL fibers, and (G) histogram representing thickness of PCL fibers.

Figure 3. (A) 1H NMR spectra of composite (top) and PCL fiber mat (bottom). The proton peak at 3.6 ppm represents PEO and is completely removed following hydroentanglement. (B) Pore size distribution as measured via porometer; mean pore size is 1.6 ± 0.9 μm (n = 4).

spinning.43,50 The fiber morphology was investigated by SEM revealing a mean thickness of 0.31 ± 0.07 μm and a mean width

can be recovered from the aqueous wash, mitigating matrix waste as compared to alternative processes, such as electroE

DOI: 10.1021/acsami.6b00093 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. Schematic to prepare antifouling surfaces (top). Polymers indicated below are candidates for antifouling polymers.

of 0.83 ± 0.19 μm (Figures 2D−G). The 23 and 24% variances in fiber cross sections are typical for the coextrusion process and show similar dispersity as that seen with other fiber processing technology.51 It can be noted that the size of fibrous domains can be increased or decreased by changing the number of multipliers in the coextrusion line. In addition, we performed tensile testing in both directions on the fiber mats to evaluate mechanical properties. The mean value of the Young’s modulus of the fiber mat was 19.98 ± 1.49 MPa in the horizontal direction and 20.34 ± 0.68 MPa in the vertical direction (Figure S1). Finally, the mat thickness was found to be 0.042 ± 0.011 cm; this value is the likely minimum limit, however, increasing the ply number would presumably increase the mat thickness. The nonwoven fiber mat was investigated using 1H NMR spectroscopy to determine whether PEO was completely removed after water jetting (Figure 3A). Any remaining PEO would compromise the high levels of porosity that are typical of nanofibrous mats. PEO has a characteristic peak at 3.6 ppm from the composite tape, which was no longer present, indicating complete removal of PEO from the PCL fiber mat (Figure 3A). The porosity can be calculated by eq 1 (see Methods), where pure PCL is used as the polymer density, showing 68.9% ± 7.4% porosity. This high porosity interconnects pores in the fiber mat, which may allow for gas exchange and nutrient influx, while providing a means for efflux of waste products if used for biomedical applications. The pore size was measured by a porometer, plotting two flow rate− pressure curves. The determined pore size of the PCL fiber mat shows a narrow distribution of pores, and the mean diameter of pore size was 1.6 ± 0.9 μm (n = 4) (Figure 3B). Additionally, porometer data indicate minimal pore size distribution between 2 and 4 μm and no pore sizes greater than 5 μm. This narrow distribution of pore sizes indicates a well-controlled processing of porous fiber mats. 3.2. Grafting of Antifouling Polymers to PCL Nanofiber Mats. The scheme for designing antifouling mats utilizing two-step chemistry is depicted in Figure 4. First, the bare PCL nanofiber mats were decorated with alkyne groups via photochemistry using Pr-Bz. The photochemistry provides a handle to perform the copper-catalyzed azide−alkyne cycloaddition (CuAAC) reaction upon. After photochemistry, the presence of Pr-Bz was confirmed by Fourier transform-infrared

spectroscopy (FT-IR) with a horizontal ATR accessory by monitoring aromatic signals at 1606 and 1508 cm−1 where C C and CH are observed, respectively (Figure S2). The surface density of Pr-Bz can be controlled by varying the intensity of UV irradiation and provides a straightforward method to control the surface density of alkyne moieties.46 Our previous work indicates that this method maintains the mechanical properties of PCL nanofibers and leads to negligible chain scission and cross-linking.47 In order to synthesize azideterminated polymers, an azide-functionalized ATRP initiator was synthesized to facilitate CuAAC (Supporting Information). From this initiator, three antifouling polymer candidates, poly(OEGMEMA), poly(MPC), and poly(OEGMEMA-coTFEMA), were synthesized using standard ATRP conditions. The chain length is an important factor for antifouling properties, where a balance must be met between chain length and surface coverage. For instance, a long chain reduces the grafting density due to the excluded-volume effect. Therefore, we limited the degree of polymerization (DP) to approximately 10 kDa polymers.52,53 In order to confirm the molecular weight and investigate the chemical composition, 1H NMR, 19F NMR, and GPC were utilized. The number-average molecular weight (Mn) of poly(OEGMEMA) was measured by GPC using a polystyrene standard, indicating 13.4 kDa and a PDI (Mw/Mn) of 1.12. The Mn of poly(OEGMEMA-co-TFEMA) was 13.6 kDa with a PDI of 1.07. 1H NMR shows the ratio of poly(OEGMEMA) vs poly(TFEMA) (1.7:1) with two distinct peaks at 4.33 ppm and at 4.07 ppm. The molecular weight of the poly(MPC) was calculated by end group analysis using 1H NMR spectroscopy (integrated ratio, the initiator vs the polymer (1:50), 15.1 kDa) (Figure S3). The fluorine signal from the poly(OEGMEMA-co-TFEMA) shows a broadened peak at 73.36 ppm by 19F NMR spectroscopy (Figure S4). These well-controlled polymerizations of comparable molecular weight and narrow polydispersity allow for an accurate comparison of the antifouling properties among the different samples. After obtaining the antifouling candidates, “click” chemistry was employed to graft the polymers onto PCL fibers by forming a triazole from the azide-polymer and alkyne-fiber mat, using ligand accelerated “click” conditions for biological substrates.54,55 3.3. Surface Characterization of Antifouling Polymer Mats. To confirm the presence of polymers grafted onto the F

DOI: 10.1021/acsami.6b00093 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 5. Surface characterization with XPS and WCA. (A−C) High-resolution XPS spectra of atoms unique to antifouling polymers, (A) N1s, (B) P2p, and (C) F1s. The table below indicates elemental percentages and water contact angle of grafted samples.

CuAAC reaction. Poly(OEGMEMA-co-TFEMA)-grafted mats are the only samples to display an F1s peak, which contributes to 4.5% of the grafted surface. Surface grafting density of poly(OEGMEMA-co-TFEMA) was calculated by using an F/C ratio and indicated 17 chains of poly(OEGMEMA-co-TFEMA) were attached per PCL polymer chain. The control PCL surface has the highest water contact angle, 117.9 ± 6.0°, as a result of the intrinsic hydrophobicity of PCL. The contact angle of poly(OEGMEMA) was determined to be 81.4 ± 9.2° confirming the presence of conjugated hydrophilic moieties. However, the poly(MPC) surface demonstrated the lowest surface energy indicated by its water contact angle of only 67.7 ± 7.4°, as tabulated in Figure 5. This decrease in water contact angle is attributed to the super hydrophilic nature of zwitterionic polymers. Because poly(MPC) induces electrostatic interactions with water molecules, this leads to a surface hydration layer, which allows for a reduced water contact angle despite its lower grafting density. Poly(OEGMEMA-coTFEMA) shows a lower water contact angle, 106.0 ± 3.3°, than that of bare PCL. This is because the copolymer includes both hydrophobic poly(TFEMA) and hydrophilic poly(OEGMEMA). This water contact angle also supports the 1H NMR data that the ratio of poly(OEGMEMA) to poly(TFEMA) is 1.7:1; thus, the amphiphilicity of the copolymer tends toward a more hydrophilic balance. 3.4. Surface Adsorption Test of BSA-FITC. A BSA-FITC adsorption test was implemented to assess the antifouling nature of the three polymer brushes. BSA-FITC adsorption was used to evaluate how effective individual polymer brushes were at minimizing the adsorption of proteins onto the surface of the fiber mat. In order to visualize BSA adherence, BSA-FITC was synthesized and purified via dialysis. MALDI-TOF was used to determine the molecular weight of BSA and BSA-FITC resulting in 66,377 and 67,550 m/z, respectively (Figure S5). Thus, the molar ratio of FITC to BSA was calculated to be 3:1. After immersing all samples in the BSA-FITC protein solution

surface of PCL nanofiber mats, surface characterization was performed using XPS and WCA to determine chemical composition and surface energy, respectively. First, XPS was used to track surface immobilization when unique atoms were grafted to the fibers (Figure 5). The surface chemical composition for N1s at 401 eV, P2p at 131 eV, and F1s at 687.0 eV indicated signature signals from the three grafted polymers (poly(OEGMEMA), poly(MPC), and poly(OEGMEMA-co-TFEMA)) that are not present in the control PCL nanofiber mat (Figure 5). The XPS analysis of the unmodified PCL fiber mat shows no nitrogen signal, while the elemental compositions of carbon and oxygen indicate 74.7 and 25.3% of each element, respectively. The C/O ratio from the PCL fiber mat matched the measured ratio from neat PCL showing an approximate ratio of 3 (Figure 5). However, both poly(OEGMEMA)- and poly(OEGMEMAco-TFEMA)-grafted mats show 0.3% N1s, supporting triazole formation and successful CuAAC reaction. Grafting density was calculated using XPS by comparing unique atoms from the grafted polymer component (i.e., N, P, and F) as a ratio to carbon content, while adjusting for molecular weight.46 This method is able to approximate the number of modifications per surface-exposed PCL chain in the mat. The nitrogen signal for surface-grafted poly(OEGMEMA) was used, as there are no unique atoms in the polymer backbone, and indicated 22 poly(OEGMEMA) chains attached per PCL chain (DPPCL = 701). Poly(MPC)-grafted mats exhibit the highest N1s content and a P2p signal with 0.6 and 0.5%, respectively. Moreover, the control PCL, poly(OEGMEMA), and poly(OEGMEMA-coTFEMA) mats do not exhibit P2p signals, while the poly(MPC)-grafted mat clearly demonstrates the presence of phosphorus at 131 eV, indicating successful grafting of poly(MPC). The percent composition of P2p is low and corresponds to a grafting density of ∼0.7 chains per PCL molecule. This may be explained by interaction of the zwitterionic polymers with the copper catalyst, hindering the G

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surface shows both features of hydrophilic repulsion of BSA proteins and hydrophobic prevention of BSA adsorption. 3.5. Bacterial Viability and Antifouling Testing of Fiber Mats. To further investigate the antifouling properties of the nanofiber mats, bacterial adhesion tests were conducted with E. coli (BL21) cells as a model microorganism due to their tendency to adhere onto surfaces. After 24 h of bacterial incubation with each fiber mat, the cells were assayed with live and dead stains, and visualized via confocal microscopy (Figure 7). The confocal micrographs show the control PCL fiber mat

for 2 h, confocal microscopy was used to visualize the relative adsorption of fluorescently labeled BSA onto the surface of the samples. Confocal micrographs demonstrate the adsorption of BSA-FITC onto the surface of both the control and polymergrafted fiber mats (Figure 6 A-D). Adsorbed BSA-FITC was

Figure 7. Antifouling surface testing with E. coli. After 24 h incubation in bacterial solution, bacterial live (green) and dead (red) stains for bacteria were visualized by confocal microscopy. (A) PCL, (B) poly(OEGMEMA), (C) poly(MPC), and (D) poly(OEGMEMA-coTFEMA).

(A) and three different samples (B−D) exhibit very few dead bacteria cells (red), as would be expected as none of the grafted polymers are known to be cytotoxic. The number of bacteria on the control PCL nanofiber mats were 615 ± 180 cells, within the micrograph (n = 3, Figure 7A). The poly(OEGMEMA) polymer surfaces showed 229 ± 49 cells indicating a 63% reduction in the number of bacteria adhered in comparison to the control PCL fiber mat (Figure 7B); this corresponds with the result of BSA-FITC adsorption test demonstrating the least biofoulant repulsion. The poly(MPC) polymer surface shows a dramatic decrease in the number of bacterial cells adsorbing, only 32 ± 3 cells, approximately 95% less than the control (Figure 7C). However, the poly(OEGMEMA) surface is an effective antifoulant when compared to the PCL control fiber mat, however it is less efficient at biofouling with regards to the poly(MPC). Although the grafting density of poly(MPC) is lower than other polymers, its strong hydrophilicity due to its charged nature causes high chain mobility and hydration, which in turn repels bacteria in an aqueous environment. The least amount of bacterial adhesion was displayed by the poly(OEGMEMA-co-TFEMA) mat, with only 19 ± 8 bacteria. This result indicates the amphiphilic surface possesses the most effective antifouling and repelling properties exhibiting a 97% decrease in bacteria compared to the control PCL fiber mat. In

Figure 6. (A−D) BSA-FITC absorption tests using confocal fluorescence microscopy. (A) PCL fiber mat, (B) poly(OEGMEMA) fiber mat, (C) poly(MPC) fiber mat, and (D) poly(OEGMEMA-coTFEMA) fiber mat. (E) Quantification of BSA absorption onto fiber mats as determined by molar absorptivity of released proteins (n = 3).

released by sonicating fibermats in buffer and quantified by UV/vis absorbance based on the BSA-FITC extinction coefficient. (Figure 6E and S6). The PCL control fiber mat exhibits the most adhered BSA-FITC (n = 3) 1.46 ± 0.13 mg. The poly(OEGMEMA) surface shows 0.66 ± 0.13 mg of adsorbed BSA-FITC which is an approximate 55% reduction in BSA-FITC adhesion compared to the control. Poly(MPC) and poly(OEGMEMA-co-TFEMA) surfaces show similar protein adhesion, resulting in 0.27 ± 0.12 mg and 0.22 ± 0.03 mg of adsorbed protein, respectively. Poly(MPC) shows an approximate 82% reduction, and poly(OEGMEMA-co-TFEMA) shows an approximate 85% reduction of BSA-FITC adsorption. The poly(MPC) and poly(OEGMEMA-co-TFEMA) surfaces demonstrate an effective resistance against protein adsorption. The poly(MPC) behavior is associated with the super hydrated surface. As for poly(OEGMEMA-co-TFEMA), the polymer H

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Figure 8. Time-dependent filtration relative flux of the PCL fiber mat, poly(OEGMEMA)-grafted PCL fiber mat, poly(MPC)-grafted PCL fiber mat, and poly(OEGMEMA-co-TFEMA). Three steps were conducted under pure DI water initial filtration, foulant filtration of BSA solution, and pure water filtration of pure DI water recovering flux. The table (below) shows antifouling indexes (DR(t), DR(r), DR(ir), and FRR) percentage (%) of control PCL fiber mat (PCL) and three different polymers-grafted fiber mats.

reversible fouling ratio (DR(r)) can be defined as the ratio of foulant BSA that can be removed when a strong shear force is applied. To evaluate the reversible performance, reversible fouling percentage (DR(r)/DR(t)) was calculated. The PCL fiber mat shows the lowest reversible fouling percentage of 69.2% when compared to other samples. This indicates the foulant BSA on the control PCL fiber mat is hard to remove by simple washing. In contrast, the poly(OEGMEMA) surface shows 83.3% of DR(t) and 13.4% of DR(ir), which indicates that poly(OEGMEMA) surface has improved antifouling properties compared to the control. The poly(OEGMEMA) fiber mat also shows high reversible fouling percentage (83.9%) due to weak interactions between the poly(OEGMEMA) surface and BSA allowing easy removal of BSA from the fiber mat. The lower DR(t) and higher FRR (75.0% and 86.5%) of the poly(MPC) fiber mat show improved antifouling properties, consistent with previous observations. The high reversible fouling percentage of 82.0% and the decreased value of Dr(ir) of 13.5% indicate that the BSA proteins would be highly removable from the MPC surface and trap less BSA protein. Finally, the poly(OEGMEMA-co-TFEMA) fiber mat shows the best antifouling behavior as indicated by the minimum value DR(t) 66.8% corresponding to the least fouling over the time; as well as, the highest FRR 89.1%, which indicates easily removable foulant proteins with a simple hydraulic washing. This indicates that poly(OEGMEMA-co-TFEMA)-grafted PCL nanofiber mats perform as excellent antifouling substrates over time as shown by a high reversible fouling percentage of 83.6% and the lowest DR(ir) of 10.9%. These antifouling properties are

brief, the protein adsorption and bacterial culture tests indicate that poly(MPC) and poly(OEGMEMA-co-TFEMA) fiber mats were significantly better at resisting biofoulants than PCL or poly(OEGMEMA) coated surfaces. 3.6. Dynamic Antifouling Testing. To evaluate the effect of grafted polymers on antifouling performance, a dynamic antifouling test was conducted. Using pure water and a BSA solution, the permeability of these solutions through fiber mats was investigated. First, the pure water was perfused through each sample (PCL, poly(OEGMEMA), poly(MPC), and poly(OEGMEMA-co-TFEMA) fiber mats) for 10 min. The pure water flux (Jw1) showed no significant changes compared to initial flux (J0) on all samples (Figure 8). When the BSA solution was passed through each fiber mat for 20 min, the flux results (Jp) showed a dramatic decrease over time. The fluxes varied due to the different amounts of adsorption and deposition of BSA on each sample; however, when compared to the control PCL fiber mat, all of the polymer-modified fiber mats showed less of a decrease in flux. It is clear that polymergrafted fiber mats exhibit enhanced antifouling behavior. When the BSA solution was introduced, the control PCL fiber mat showed severe flux decline and poor flux recovery indicated by the highest total flux decline ratio DR(t) and irreversible flux decline ratio DR(ir) which were 96.9 and 29.8%, respectively. It is speculated that formation of deposited BSA decreases the flux over time by blocking pores on the surface of the fiber mat. The highest DR(t) and the lowest flux recovery ratio (FRR) 70.2% indicate BSA proteins adsorbed onto the surface or inside of pores of the control PCL fiber mat. The I

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(6) Yuan, S.; Li, Y.; Luan, S.; Shi, H.; Yan, S.; Yin, J. InfectionResistant Styrenic Thermoplastic Elastomers That Can Switch from Bactericidal Capability to Anti-Adhesion. J. Mater. Chem. B 2016, 4, 1081−1089. (7) Cao, B.; Lee, C.-J.; Zeng, Z.; Cheng, F.; Xu, F.; Cong, H.; Cheng, G. Electroactive Poly(sulfobetaine-3,4-Ethylenedioxythiophene) (PSBEDOT) with Controllable Antifouling and Antimicrobial Properties. Chem. Sci. 2016, 7, 1976−1981. (8) Busscher, H. J.; van der Mei, H. C.; Subbiahdoss, G.; Jutte, P. C.; van den Dungen, J. J. A. M.; Zaat, S. A. J.; Schultz, M. J.; Grainger, D. W. Biomaterial-Associated Infection: Locating the Finish Line in the Race for the Surface. Sci. Transl. Med. 2012, 4, 153rv10. (9) Banerjee, I.; Pangule, R. C.; Kane, R. S. Antifouling Coatings: Recent Developments in the Design of Surfaces That Prevent Fouling by Proteins, Bacteria, and Marine Organisms. Adv. Mater. 2011, 23, 690−718. (10) Serrano, C.; García-Fernández, L.; Fernández-Blázquez, J. P.; Barbeck, M.; Ghanaati, S.; Unger, R.; Kirkpatrick, J.; Arzt, E.; Funk, L.; Turón, P.; del Campo, A. Nanostructured Medical Sutures with Antibacterial Properties. Biomaterials 2015, 52, 291−300. (11) Krishnan, S.; Weinman, C. J.; Ober, C. K. Advances in Polymers for Anti-Biofouling Surfaces. J. Mater. Chem. 2008, 18, 3405. (12) Venault, A.; Yang, H.-S.; Chiang, Y.-C.; Lee, B.-S.; Ruaan, R.-C.; Chang, Y. Bacterial Resistance Control on Mineral Surfaces of Hydroxyapatite and Human Teeth via Surface Charge-Driven Antifouling Coatings. ACS Appl. Mater. Interfaces 2014, 6, 3201−3210. (13) Kim, S.; Gim, T.; Kang, S. M. Versatile, Tannic Acid-Mediated Surface PEGylation for Marine Antifouling Applications. ACS Appl. Mater. Interfaces 2015, 7, 6412−6416. (14) Zhu, X.; Jańczewski, D.; Guo, S.; Lee, S. S. C.; Parra Velandia, F. J.; Teo, S. L.-M.; He, T.; Puniredd, S. R.; Vancso, G. J. Polyion Multilayers with Precise Surface Charge Control for Antifouling. ACS Appl. Mater. Interfaces 2015, 7, 852−861. (15) Zhao, X.; He, C. Efficient Preparation of Super Antifouling PVDF Ultrafiltration Membrane with One Step Fabricated Zwitterionic Surface. ACS Appl. Mater. Interfaces 2015, 7, 17947−17953. (16) Xue, C.-H.; Guo, X.-J.; Ma, J.-Z.; Jia, S.-T. Fabrication of Robust and Antifouling Superhydrophobic Surfaces via Surface-Initiated Atom Transfer Radical Polymerization. ACS Appl. Mater. Interfaces 2015, 7, 8251−8259. (17) Sun, W.; Liu, J.; Chu, H.; Dong, B. Pretreatment and Membrane Hydrophilic Modification to Reduce Membrane Fouling. Membranes 2013, 3, 226−241. (18) Van Wagner, E. M.; Sagle, A. C.; Sharma, M. M.; La, Y.-H.; Freeman, B. D. Surface Modification of Commercial Polyamide Desalination Membranes Using Poly(ethylene Glycol) Diglycidyl Ether to Enhance Membrane Fouling Resistance. J. Membr. Sci. 2011, 367, 273−287. (19) Cheng, Q.; Zheng, Y.; Yu, S.; Zhu, H.; Peng, X.; Liu, J.; Liu, J.; Liu, M.; Gao, C. Surface Modification of a Commercial Thin-Film Composite Polyamide Reverse Osmosis Membrane through Graft Polymerization of N-Isopropylacrylamide Followed by Acrylic Acid. J. Membr. Sci. 2013, 447, 236−245. (20) Voo, Z. X.; Khan, M.; Narayanan, K.; Seah, D.; Hedrick, J. L.; Yang, Y. Y. Antimicrobial/Antifouling Polycarbonate Coatings: Role of Block Copolymer Architecture. Macromolecules 2015, 48, 1055−1064. (21) Demirel, Y. K.; Khorasanchi, M.; Turan, O.; Incecik, A. On the Importance of Antifouling Coatings Regarding Ship Resistance and Powering. In 3rd International Conference on Technologies, Operations, Logistics and Modelling for Low Carbon Shipping; London, 2013. (22) Li, X.; Cao, Y.; Kang, G.; Yu, H.; Jie, X.; Yuan, Q. Surface Modification of Polyamide Nanofiltration Membrane by Grafting Zwitterionic Polymers to Improve the Antifouling Property. J. Appl. Polym. Sci. 2014, 131, 41144. (23) Ma, J.; Luan, S.; Song, L.; Jin, J.; Yuan, S.; Yan, S.; Yang, H.; Shi, H.; Yin, J. Fabricating a Cycloolefin Polymer Immunoassay Platform with a Dual-Function Polymer Brush via a Surface-Initiated Photoiniferter-Mediated Polymerization Strategy. ACS Appl. Mater. Interfaces 2014, 6, 1971−1978.

attributed to the hydrophilic poly(OEGMEMA) releasing foulant materials and hydrophobic poly(TFEMA) segment preventing adhesion of BSA.

4. CONCLUSIONS We have successfully developed antifouling PCL fiber mats from extruded PCL fibers produced by melt coextrusion. This unique method can fabricate bulk polymeric nanofibrous mats with a high production rate on an industrial scale while minimizing environmental waste. We successfully obtained nanoscale fiber mats with a narrow pore size distribution and a high porosity. These attributes allow for potential applications such as microfiltration, wound healing patches, and membrane filtration. Antifouling surface properties have been demonstrated by rejection of protein and bacteria, with several candidate antifouling polymers grafted to the mats. The results indicate that poly(OEGMEMA-co-TFEMA) polymer surfaces have the most effective antifouling capability and showed a relatively steady high flux. We speculated that this amphiphilic copolymer has the best antifouling performance due to the enhanced effects of the joint hydrophilic and hydrophobic segments. In future work, these fiber mats will be extended for biomedical applications providing high antifouling ability, simple modification at low cost, and excellent biocompatibility.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00093. Detailed synthetic methods for ATRP and additional Figures S1−S6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected]. Phone: +1-216-368-6373. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J. K. P., C. Z., E. B., and S. E. K. acknowledge the NSF Center for Layered Polymeric Systems (CLiPS) for financial support (DMR 0423914). Melanie Hutnick is acknowledged for helpful discussions.

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REFERENCES

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