Preparation and Characterization of Amphiphilic Triblock Terpolymer

Apr 4, 2012 - Alex Khang , Prashanth Ravishankar , Aditya Krishnaswamy , Patrick K. Anderson , Stephanie G. Cone , Zizhao Liu , Xianghong Qian , Karti...
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Preparation and Characterization of Amphiphilic Triblock Terpolymer-Based Nanofibers as Antifouling Biomaterials Youngjin Cho,† Daehwan Cho,*,‡,§ Jay Hoon Park,‡ Margaret W. Frey,§ Christopher K. Ober,† and Yong Lak Joo‡ †

Department of Materials Science and Engineering, ‡School of Chemical and Biomolecular Engineering, and §Department of Fiber Science and Apparel Design, Cornell University, Ithaca, New York 14853, United States S Supporting Information *

ABSTRACT: Antifouling surfaces are critical for the good performance of functional materials in various applications including water filtration, medical implants, and biosensors. In this study, we synthesized amphiphilic triblock terpolymers (tri-BCPs, coded as KB) and fabricated amphiphilic nanofibers by electrospinning of solutions prepared by mixing the KB with poly(lactic acid) (PLA) polymer. The resulting fibers with amphiphilic polymer groups exhibited superior antifouling performance to the fibers without such groups. The adsorption of bovine serum albumin (BSA) on the amphiphilic fibers was about 10-fold less than that on the control surfaces from PLA and PET fibers. With the increase of the KB content in the amphiphilic fibers, the resistance to adsorption of BSA was increased. BSA was released more easily from the surface of the amphiphilic fibers than from the surface of hydrophobic PLA or PET fibers. We have also investigated the structural conformation of KB in fibers before and after annealing by contact angle measurements, transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), and coarse-grained molecular dynamics (CGMD) simulation to probe the effect of amphiphilic chain conformation on antifouling. The results reveal that the amphiphilic KB was evenly distributed within as-spun hybrid fibers, while migrated toward the core from the fiber surface during thermal treatment, leading to the reduction in antifouling. This suggests that the antifouling effect of the amphiphilic fibers is greatly influenced by the arrangement of amphiphilic groups in the fibers.

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INTRODUCTION Biofouling is an undesirable building up of various proteinbased living microorganisms, plants, and algae on a material surface. The fouling is caused by a complex interaction between the material surface and the aformentioned foulants. Preventing the accumulation of protein foulants is one of the essential processes to minimize the protein adsorption on a material surface. A fouling surface especially hampers membrane systems used in pressure-driven processes such as reverse osmosis and nanofiltration.1 Therefore, fouling of surfaces has drawn much interest in many academic studies and industrial research due to its effect on various applications such as marine and industrial systems,2−6 protective apparel,7−9 medical implants,10−12 biosensors,13,14 textiles,15−17 food packaging and storage,18,19 and water purification systems.20 Various strategies have been investigated in developing good antifouling materials.21,22 In the marine industry, the most effective methods have used an ablative-biocidal coating with tributyltin and copper. However, the biocidal coating has been gradually banned because of environmental issues. Consequently, many studies are ongoing to find alternative eco-friendly approaches for achieving antifouling surfaces.23 © 2012 American Chemical Society

Based on knowledge from extensive previous research, it has been agreed that an increase in hydrophilicity on a material surface offers better resistance to nonspecific protein.22 Poly(ethylene glycol) (PEG) for a hydrophilic coating has been mainly used to provide exceptional resistance to protein and cell adhesion.24,25 Hydrophilic and zwitterionic material has also demonstrated good antifouling performance.21 Therefore, a great number of recent studies have been focused on the control of the wettability of coatings in order to impart hydrophilic features and protein resistances to a material surface through surface chemistry treatments such as plasma treatment,26,27 graft-to-surface method,28,29 and spin-coating.30 Li et al. fabricated an antifouling ultrafiltration membrane using a PEG-conjugated dope and found that the membrane was desirable in the treatment of oily wastewater.31 Fan et al. blended hydrophilic polyaniline nanofibers with polysulfonate membrane to produce antifouling properties, which achieved both hydrophilic properties and permeability of the blended membrane.32 A recent study reported a limitation in pursuing a Received: March 1, 2012 Revised: March 28, 2012 Published: April 4, 2012 1606

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polymers (6201 D, Mw = 143000) were supplied by Cargill Dow (Minnetonka, MN). Chloroform and acetone were purchased from Mallinckrodt Laboratory Chemicals (Phillipsburg, NJ). PET nonwoven substrate was obtained from Dupont (Wilmington, DE). Synthesis of Triblock Terpolymer. We have studied polystyrenebased diblock copolymer systems for antifouling and fouling-release applications in our previous work.33,34 However, diblock would be less capable of optimizing the surface segregation of side chain functional moieties than triblock, which led us design a new triblock terpolymer such as a polystyrene-b-poly(ethylene-r-butylene)-b-polyisoprene. The procedure and characterization of the triblock terpolymers with grafted Brij 76 side chain was described in detail in a previous work.35 The PS8K-b-P(E/B)25K-b-PI10K precursor polymer (5 g, 14.5 mmol of reactive isoprene sites) was dissolved in cyclohexane (100 mL) in a round-bottomed flask. 3-Chloroperoxybenzoic acid (m-CPBA, 3.9 g, 17.4 mmol) was added to the mixture, and the solution was stirred vigorously for 5 h at room temperature. Subsequently, the polymer was precipitated in methanol, collected by filtration, and reprecipitated from dichloromethane to remove residual m-CPBA and its respective byproducts. The white, rubbery product was dried at room temperature under reduced pressure for 48 h to remove remaining solvent. To produce ether-linked side chain surface active block copolymers, epoxidized PS8K-b-P(E/B)25K-b-PI10K (2.1 g, 5.8 mmol of epoxide) was taken in a round-bottomed flask in conjunction with a 3fold molar excess (17.4 mmol) of nonionic Brij 76 alcohol. The reactants were purged with argon and subsequently dissolved in anhydrous chloroform (150 mL). Activated molecular sieves were added to the reaction mixture and it was allowed to sit for 12 h to optimize the adsorption of water. Etherification was performed through the addition of boron trifluoride diethyl etherate catalyst (0.345 g, 2.4 mmol) followed by vigorous stirring at room temperature for 48 h. Following the reaction, 6.25 N sodium hydroxide aqueous solutions (1 mL) was added to quench any residual boron catalyst and the reaction mixture was concentrated under reduced pressure using a rotary evaporator. The resultant surface active triblock terpolymer was precipitated into methanol. The fluorine-free, amphiphilic, nonionic surface active free block copolymer was collected by filtration and subsequently reprecipitated twice into methanol from chloroform to remove additional residual surface active side-chain alcohol precursors. Finally, the finished compounds were dried under reduced pressure at room temperature for 48 h to fully remove residual solvent. Preparation of Polymeric Elecrospun Fibers. To fabricate the pure PLA and the PLA/KB hybrid-amphiphilic fibers, PLA polymers, and KB were dissolved in chloroform and acetone (3:1, v/v%). The pure PLA fibers were electrospun by adjusting the concentration of PLA polymers to 9.0 wt % in the solvent and agitating it with an armforced shaker. The spinning dopes for the hybrid-amphiphilic fibers were prepared by adjusting the ratio of PLA to KB and total concentrations (Table 1S in Supporting Information). The PLA and KB were separately dissolved in the mixture solvent system for 24 h with an arm-forced shaker. Each polymer solution was blended together and then vigorously agitated for 3 min with a vortex to make a homogeneous spinning dope. For electrospinning, the spinning dope was put into a 5 mL syringe (VWR Scientific, West Chester, PA). The syringe was attached with a metal needle (ID = 0.60 mm, Hamilton). A positive high voltage of 15 kV was applied to the needle using a high voltage power supply (Gamma High Voltage Research, Ormon Beach, FL). The solution was fed at a 0.03 mL/min feed rate driven by a programmable syringe micropump (Harvard Apparatus, MA). A copper collector covered with a PET substrate was grounded. The fibers were accumulated to form a fiber membrane on the PET substrate for 5 min. Protein Adsorption Tests. BSA labeled with fluorescein isothiocyanate (FITC-BSA, 0.5 mg) was dissolved in a PBS buffer solution (5 mL). The fiber samples were then incubated in a FITCBSA solution in a dark room for 2 h and rinsed with deionized water thoroughly afterward. Then the fluorescence intensities on these fibers were measured with fluorescence microscopy. Protein Detachment Tests. After measuring the fluorescence intensities on the fiber samples, protein detachment experiments were

hydrophilic approach to achieving antifouling performance on a hydrophilic material.33 More recently, several amphiphilic block copolymers (BCPs) have been developed to generate an active function on a material surface.34 In our previous work, we reported the synthesis, characterization, and antifouling performance of active KB derived from the grafting of selected nonionic side chains, which have amphiphilic features of fluorine-free and nonionic side chains. Thin films were made using the syntheized KB and their antifouling and foulingrelease properties against marine organisms were evaulated.35 To date, several studies have reported some methods for developing antifouling fibers: dipping substrate fibers into a hydrophilic polymer dope,31 blending hydrophilic fibers with substrate fibers,32 or modifying substrate fibers with hydrophilic BCPs.36 Recently, a great deal of work has focused on developing polymeric nanofibrous membranes because such membranes show extremely good performance in the application of water filtration and in biomedical devices.37,38 Water filtration, in particular, needs an extended use-time without pressure drop. To avoid pressure drops, a filtration membrane is required to alleviate fouling on the membrane surface.37 Li et al. worked on a specific incorporation of biotin into nanofibers to prepare a membrane substrate for biosensors based on biotin−streptavidin specific binding.38 Electrospinning is a key process for the fabrication of polymeric fibers in a range of diameters from micrometers to nanometers.39,40 These polymeric nanofibers are excellent candidates to improve targeted properties for various applications due to their intrinsically larger specific surface-tovolume ratio and smaller pore size.41,42 Although electrospinning has proven to be an effective way of fabricating nanofibrous structures, antifouling application of electrospun nanofibers has not been studied systematically. In addition, various polymeric groups (hydrophilic, hydrophobic, and amphiphilic) of fiber-based materials are needed to analyze the effect of antifouling performance on the fibers with each group. In the present study, we report the first fabrication of amphiphilic fibers by electrospinning of solutions prepared by blending synthesized KB with a PLA polymer. The synthesized KB was used to make conjugated spinning dopes with a hydrophobic polymer, PLA.43 The antifouling properties of various amphiphilic fibers where the ratio of PLA to KB were adjusted, were characterized by protein adsorption and release test. We have also extensively studied the structural conformation of the amphiphilic groups in the polymeric nanofibers before and after annealing with contact angle, EDX, and TEM. A CGMD simulation was used to give further insights into experimentally observed reduction in antifouling after annealing.

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EXPERIMENTAL SECTION

Materials. The polystyrene8K-block-poly(ethylene-ran-butylene)25K-block-polyisoprene10K (PS8K-b-P(E/B)25K-b-PI10K) triblock precursor terpolymer was produced using anionic polymerization and subsequent catalytic hydrogenation by Kraton Polymers. 3-Chloroperoxybenzoic acid (m-CPBA, ClC6H4COOOH, FW 172.57, 77%), boron trifluoride diethyl etherate (BF3·Et2O, BF3·O(CH2CH3)2, FW 141.93, 99.9%), Brij 76 alcohol (C18H37(OCH2CH2)nOH, n ∼ 10), anhydrous chloroform (CHCl3), phosphate buffered saline (PBS), and BSA with fluorescein isothiocyanate were purchased from Sigma Aldrich and used without further purification. Cyclohexane, dichloromethane (CH2Cl2), methanol (CH3OH), 6.25 N sodium hydroxide (NaOH), and all other reagents were used as received. The PLA 1607

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conducted with a water-jet sprayer. To determine the strength of attachment, a fiber surface was exposed to a flowing buffer solution with an incident angle of 15° and an incoming pressure at 100 psi for 10 min. The fluorescence intensities on these fibers were measured with fluorescence microscopy. The percentage of BSA removal was calculated by comparing the fluorescence intensity on a fiber surface not exposed to a flowing buffer solution with that on an exposed fiber. Coarse-Grained Molecular Dynamics (CGMD) Simulation. A CGMD simulation was used to simulate the self-assembly of the block copolymer/homopolymer blend used in the experiment. Within the polymer chains, the neighboring monomers are connected by a finitely extensible nonlinear elastic (FENE) potential,

⎡ ⎛ r ⎞⎤ 1 2 u FENE(r ) = − kR max ln⎢1 − ⎜ ⎟⎥ 2 ⎝ R max ⎠⎥⎦ ⎣⎢

the thermostat used is Gaussian instead of DPD. We use the SLLOD equations of motion to implement elongational flow,

dri , v dt dpi , v dt

where the spring constant k is 30, and the maximum extensibility Rmax is 1.5.44 For a homopolymer, the polymer chain consists of one type of spherical monomers. For the triblock terpolymer, each polymer chain consists of A, B, and C blocks of monomers. These monomers have excluded volume interactions among them and they are modeled by the Weeks−Chandler−Anderson (WCA) potential,45

u REP(r ) = 0,

r ≤ 21/6

r > 21/6 (2)

where r is the separation distance between beads and σ and ε are the Lennard-Jones parameters. An attractive potential between the same type of monomers (i.e., A−A, B−B, C−C) was used to incorporate the physics of microphase separation between the different blocks. The attractive potential, as described by Horsch et al.,46 to model the equilibrium properties of diblock copolymer melts, is again an LJ potential, but now it is cut and shifted at values that differ from those presented in eq 2.

⎡⎛ σ ⎞12 ⎛ σ ⎞6 ⎤ u ATT(r ) = 4ε⎢⎜ ⎟ − ⎜ ⎟ ⎥ + u LJ (2.5), ⎝r⎠ ⎦ ⎣⎝ r ⎠ u ATT(r ) = 0,

r > 2.5

pi , v mv

+ rj , v

= Fi , v − pj , v

∂ui ∂xj

(4)

∂ui − αpi , v ∂xj

(5)

where ri,v, pi,v, and mv are the position vector, peculiar momentum, and mass of the vth particle, respectively. δui/δxj is the velocity gradient and α is the Gaussian thermostat multiplier. To learn more detail about the simulation mechanics, we refer the readers to our previous simulation of diblock copolymer/nanoparticle melt under planar elonagational flow.50 Microtoming Nanofibers. The internal structure of an electrospun fiber was studied by examining microtomed fibers using TEM (Tecnai T-12 transmission electron microscope, Hillsboro, OR). First, the electrospun fibers were embedded in an epoxy matrix using Epofix. The embedded fibers were then microtomed with Dupont Sorvall Ultramicrotome at room temperature using a diamond knife. The sections were cut at a thickness of around 70 nm. The microtomed samples were then stained with osmium tetroxide to make the KB darker for clear distinction between the KB and PLA matrix under TEM. To characterize the elemental composition of the microtomed fiber, EDX was used to detect the characteristic X-rays generated by the electron beam under TEM. The built-in software can analyze the signals collected to quantify the amount of elements within the spot of interest. Fiber Characterization. The morphology of the electrospun fibers was evaluated with a Leica 440 scanning electron microscope (SEM) after the fibers were coated with Au−Pd. Image analysis software (ImageJ 1.41) was used to evaluate the morphology of the fibers. The electrospun fibers samples were characterized using Fourier transform infrared spectroscopy (ATR-FTIR, Vertex 80v, Bruker Optics) and found to be 800 to 3800 cm−1 with a 4 cm−1 resolution and the coaddition of 64 scans. Water contact angles of the fibers were measured with an Advanced Goniometer/Tensiometer (Model 500, Ramé-Hart Instrument Co., Netcong, NJ). Fluorescence microscopy was performed using an Olympus BX51 upright microscope with a 40× UPlan Fluorite 40× dry objective (N.A. 0.75). Images were acquired using a Roper Cool Snap HQ CCD camera and Image Pro image acquisition. Fluorescein and FITC were observed with a 450 nm excitation and 550 nm emission filter set. False color fluorescence images subsequently reported here were also processed using the ImageJ. TEM images were taken using a Tecnai T-12 at an accelerating voltage of 120 kV.

(1)

⎡⎛ σ ⎞12 ⎛ σ ⎞6 ⎤ u REP(r ) = 4ε⎢⎜ ⎟ − ⎜ ⎟ ⎥ + ε = u LJ (r ) + ε , ⎝r⎠ ⎦ ⎣⎝ r ⎠

=

r ≤ 2.5, (3)

The higher cutoff means that this is not purely repulsive and that monomers of the same type are attracted to each other. Because the blocks A and C both simulate hydrophobic blocks of the BCPs, the pairwise interaction between A−C is given the attractive potential as well. The thermostat used was a dissipative particle dynamics (DPD) thermostat.47 The velocity Verlet algorithm was used to integrate the equations of motion. The MD integration time step size, Δt, was fixed at 0.01. A cell list algorithm was used to make the code efficient.48 To prevent undue periodic boundary artifacts, we varied the system’s radius and height (z-axis length) until the system attained stacked disk morphology symmetrical diblock copolymer. When the periodic height of the box size, Z, is set at 9.54, the system attains the most stable form of stacked disks at equilibrium. The simulations were run for a sufficiently long time until variables such as pressure, potential energy, radius of gyration, and mean squared end-to-end distance remained constant. The parameter was reaching equilibrium when the MD time was approximately 2000, which is about 4000000 time steps.47 To learn more about the simulation system used, we point the readers to our most recent simulation study with block-copolymer self-assembly under cylindrical confinement.49 Additionally, planar elongational flow was applied to the system in order to demonstrate how flow can affect the microphase separation of the block copolymer/homopolymer blend, which can be directly compared with as-spun nanofibers. Most parameters are the same as stated before except for the polymer model that uses a bead-rod model instead of bead-spring FENE model, and

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RESULTS AND DISCUSSION Chemical Structure of KB. The chemical structure of the amphiphilic triblock terpolymer (KB) and PLA is depicted in Figure 1. We have previously reported the synthesis and characterization of the KB systems in detail.35 Functionalization of the triblock precursor, PS8K-b-P(E/B)25K-b-PI10K, was achieved through epoxidation of the residual alkene groups followed by subsequent catalytic ring-opening etherification

Figure 1. Chemical structures of amphiphilic triblock terpolymer and PLA. 1608

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reactions using nonionic Brij 76 alcohol carrying amphiphilic functionality. Each block of triblock terpolymer used in the present study has its own function. In the case of the polyisoprene block, it is necessary to perform the grafting of the amphiphilic surfactant. The polystyrene and poly(ethylene-rbutylene) blocks were closely related to the fouling-release performance due to their low surface energy, which was described in our previous study.34 In the present study, these blocks need to control the fouling-release of protein and to interact with hydrophobic PLA polymer chains. Especially the poly(ethylene-r-butylene) block as a molecular spacer is used to optimize the surface segregation of a side chain as a functional group. Amphiphilic side chains play an important role in antifouling properties. It is also a good alternative antifouling material to replace any fluorinated counterpart because previous fluorinated materials are potential environmental hazards. Properties of Electrospun Fibers. As shown in Figure 2, a porous array of smooth and uniform fibers with good

Figure 3. FTIR spectra of the electrospun fibers and KB (tri-BCPs).

To study the effect of the KB in the amphiphilic fiber on antifouling, we annealed the PET substrate, pure PLA fiber, and PLA/KB (70/30) fiber in a 120 °C oven for 6 h. The heat treatment induced a slight change in the fiber morphology, in which the fibers were shrunken and became thicker (Figure 1S, Supporting Information). To measure the wettability of the as-spun and annealed fibers, the dynamic water contact angles were measured, as shown in Table 1. PET fibers are generally known to have an Table 1. Compositions and Water Contact Angles of the Prepared Fibers fiber

θw,ad (deg)

θw,re (deg)

PET substrate PLA PLA/KB (70/30) PLA/KB (40/60) PLA/KB (20/80) annealed PET substrate annealed PLA annealed PLA/KB (70/30) annealed PLA/KB (60/40) annealed PLA/KB (20/80)

N/A1 127 ± 2 104 ± 2 96 ± 2 91 ± 2 N/A 134 ± 3 142 ± 2 127 ± 2 112 ± 2

N/A 77 ± 3 74 ± 1 49 ± 1 42 ± 2 N/A 82 ± 3 100 ± 3 71 ± 3 63 ± 3

Figure 2. SEM images of as-spun fibers: (A) pure PLA, (B) PLA/KB (70/30), (C) PLA/KB (40/60), and PLA/KB (20/80); scale bar represents 10 μm.

1

morphology was formed. All the as-spun fibers except for PLA/KB (20/80) fibers have approximately submicrometer scale diameters (average 0.8 μm), and the PLA/KB (20/80) fibers show bigger diameters (average 1.0 μm). A PET membrane was used as a substrate for all four cases. The PET substrate membrane was made of thicker fibers (mean diameter: 17 μm) and had much larger pores than the as-spun fibers. The KB incorporation in the hybrid fibers was examined using FTIR spectroscopy. Figure 3 presents the FTIR spectra of the KB and two asspun fibers. FTIR spectroscopy of the KB shows two broad peaks for cis-C−H (out of plane bend) of the alkene group at 700 and 716 cm−1, which corresponded well with the peaks of the PLA/KB fibers. The carbonyl compound peak appears at the region around 1750 cm−1 due to the stretching vibration of the CO bond. Two characteristic peaks at 2930 cm−1 and 2850 cm−1 represent the groups of −CH2 and −CH for the all samples except for the PLA fibers that do not have groups of −CH2 and −CH. As a result, these findings of FTIR spectra demonstrated that the KB was well incorporated into PLA polymers to form the amphiphilic fibers.

intrinsic feature of hydrophobicity.51 The contact angle of the PET substrate could not be measured because a droplet was absorbed on the coarse substrate surface due to its large pore size. Therefore, the PET substrate was applied to an alternative method for evaluating the wettability before and after the heat treatment. The PET substrate (3 cm by 0.5 cm) above a dish of water was brought near to water until contact was made. The sample was weighed during the water contact and after the detachment of the sample from the water. As a result, the annealed PET substrate showed much lower adsorption of water than the non-heat-treated PET substrate, indicating that the annealed PET substrate became more hydrophobic (Figure 2S, Supporting Information). The contact angles of the PLA/KB hybrid fibers were gradually lowered with the increase of the KB contents in the fibers. The KB on the fiber surface imparted a specific contact angle with θw,advancing, ranging from 104 to 91°, and θw,receding, ranging from 74 to 42°, according to the KB contents. The PLA/KB hybrid fibers resulted in a much lower contact angle than the pure PLA fibers. With the annealed PLA/KB (70/30)

Not available; quick absorption of a droplet of water on the sample surface.

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samples, their contact angles of advancing and receding were increased to 142 and 100°, respectively. In the case of the PLA fibers, the hydrophobicity before and after annealing shows less difference than that of the PLA/KB fibers. Consequently, the PLA/KB hybrid fibers demonstrated a large difference in the hydrophilic characteristics before and after annealing. It is well-known that block copolymers undergo phase separation at equilibrium, that is, the like monomers attract each other while the unlike monomers repel from each other, to form ordered structure.52,53 In this case, the hydrophilic groups in KB form their own ordered domain within the hydrophobic PLA matrices. However, due to the extensive elongation and rapid evaporation during electrospinning, such microphase separation does not occur because it is in kinetically metastable state, as observed from irregular block copolymer morphologies formed in as-spun fibers.54,55 By annealing the as-spun nanofibers, they may recover the equilibrium morphologies as seen from other studies.56,57 Thus, the KB blocks undergo phase separation and form more ordered structure after annealing. Kinetically, it is more favorable to form regular KB structures with even spacing. Thus, the KB blocks were evenly placed across the fiber in the as-spun state due to the extensive flow in electrospinning. During annealing, the KB blocks may self-assemble to form their own domains at the center of the PLA matrix in order to reach more kinetically favored state because there is more space available within the fiber rather than just along the surface. The effects of the KB contents in the as-spun fibers on the hydrophobicity were clearly displayed, and thus, it appears that the polymer chains of the KB were strongly related to the fiber wettability. From the large difference of contact angles on the PLA/KB fibers, the morphological structures of the KB on the fiber surfaces may be subjected to change with annealing. The KB containing amphiphilic side chains was randomly distributed to place its hydrophilic PEO side chains on the fiber surface during electrospinning. Polymer jets from a nozzle were subjected to a vigorous whipping motion due to the electric charges on the jet surface which led the low molecular side chains to migrate to the fiber surface.58,59 As heat was applied to the fibers, the two phases of the PLA and KB polymers were stabilized and they self-assembled, which will be verified by TEM, EDX, and coarse-grained MD simulation in a later section. The advancing contact angle of the annealed PLA/KB fibers was increased to 140°, which was much higher than that of its counterpart as-spun fibers (around 100°). Therefore, the annealed fibers resulted in an increase in hydrophobicity, suggesting that the hydrophobic groups (PS8kb-P(E/B)25k-b-PI10k) and the hydrophilic groups (PEO chains (-CH2-CH2-O-)n n = 10) in the fibers were self-assembled, and thus, the PEO chains had less impact on the hydrophilicity of the fibers. Protein Adsorption on Electrospun Fibers. For the protein adsorption test, the prepared fibers were systematically treated with BSA. Figure 4 shows the fluorescence intensities and images on the PET substrate and electrospun fibers (PLA and PLA/KB). As depicted in Figure 4, PET and PLA show high protein adsorption on their surfaces whose hydrophobicity is consistent with the fouling tendency. The amphiphilic fibers whose surfaces were structured with the KB were found to strongly resist BSA adsorption, especially when compared to the control fiber samples (PLA and PET substrate). The BSA fluorescence intensity on the amphiphilic fibers was about 10-fold less than that on the control samples.

Figure 4. Fluorescence intensity and fluorescence microscopy images of the prepared samples at FITC-BSA 0.1 mg/mL in PBS after incubation for 2 h (error bar represents a standard deviation, number of samples = 5).

With the increase of the KB contents in the hybrid fibers, the antifouling effects were improved. In the case of the amphiphilic fibers with a high KB content, barely any BSA protein was adsorbed on the surface. This observation indicates that the PLA/KB (20/80) fibers exhibited very similar fluorescence intensity with that of a blank sample (a blank means that the sample surface has no BSA). These results clearly demonstrate the large difference in protein adsorption properties between the hydrophobic fibers and the fibers with amphiphilic KB. The amphiphilic groups on the fiber surface interfered with a binding activity of the BSA. Figure 5 shows the adsorption test results of BSA on the fiber surfaces before/after annealing. In this study, we dealt with only

Figure 5. Adsorption of protein on the prepared samples before and after annealing at 120 °C for 6 h (inset images are the annealed samples, error bar represents a standard deviation, number of samples = 5).

PLA/KB (70/30) fiber to analyze the antifouling effects before/ after annealing with the TEM images because it was difficult to obtain good images with higher KB content in the fibers. After annealing, the adsorption of BSA on each fiber surface was increased possibly due to the morphological changes on the fiber surface. We note that, although the difference was less drastic, we did observe the increase in the adsorption of BSA for PLA/KB (70/30) fiber after annealing, possibly due to the 1610

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changes in KB chain conformation. This supposition was supported by TEM and EDX studies and CGMD simulation experiments, which are presented in the following section. The KB incorporated within the fiber in the CGMD simulation experiments explains why the as-spun fibers exhibited better antifouling characteristics when compared with their annealed counterparts, for the as-spun fiber has more KB near the fiber surface. To analyze the differences in antifouling performance before/after annealing, the statistically significant difference in protein adsorption was evaluated using a one-way analysis of variance (ANOVA). A 0.05 significance level (95% confidence interval) was adopted in this analysis. As the results show (Table 2S in Supporting Information), the p-values for all the samples are lower than 0.05, indicating that there are statistical significances according to the annealing treatment. All the fibers show a significant difference in the BSA test results before/after annealing. It was noted that all the fibers after annealing were more hydrophobic than as-spun fibers, which may also have caused the increase in BSA adsorption. Protein Release from Electrospun Fibers. As the protein-adsorbed fibers were exposed to a flowing buffer solution at an incoming pressure of 100 psi, some BSA was detached from the fiber surfaces, as depicted in Figure 6.

Figure 7. TEM images of the PLA and PLA/KB (70/30) fiber: a cross-section and longitudinal section of as-spun pure PLA (A and B), a cross-section of as-spun PLA/KB fiber (C), and the annealed PLA/ KB fiber at 120 °C for 6 h (D). The darker contrast represents KB, while the lighter contrast represents PLA (inset of C and D mimicked distribution of KB in each fiber cross section).

nanofiber. The pure PLA fiber illustrates a uniform white image, but the hybrid fiber has two-colored regions (black and white). The darker-colored nanostructure within the fiber represents the KB, while the lighter one represents PLA. The bright circle spots found within the fiber are holes caused by fibers torn during microtoming. The inset is a schematic drawing to show the distribution of the KB in the fibers based on the TEM images. As the fiber was annealed, more KB domains were seen near the center of the fiber. This evolution indicates KB phase separation takes place toward the core from the fiber surface with prolonged thermal treatment of the fiber. This phenomenon is in good agreement with the results of the contact angles. Then we quantified and compared the elemental composition of the as-spun PLA/KB fiber with annealed PLA/KB fiber using EDX. Figure 8 shows the elemental signals picked up at the core of the as-spun and annealed PLA/KB fibers. Because the osmium used to stain the PLA/KB fibers would prefer the KB phase over the PLA, higher peaks of osmium would indicate a higher concentration of the KB in that region. With the as-spun fiber, the osmium peaks are lower than carbon peaks in the core, indicating that the KB are well dispersed throughout the fiber surface and core. However, the annealed fiber clearly shows higher peaks of osmium than carbon in the core, which is a strong evidence of phase separation of the KB toward the core from the fiber surface. Coarse-Grained Molecular Dynamics Simulation. We used a CGMD to simulate the annealed PLA/KB blend selfassembly. Note that the equilibrium state reached from the simulation is equivalent to the annealed experimental result. Figure 9 A shows the simulation results of the self-assembled blend. As can be seen from the results, the KB components colored in red, green, and sky blue are found more readily in the core rather than in the wall. The color schemes used in Figure 9 A were then changed to black and white, and the three-dimensional snapshot was cut diagonally and perpendicularly to the fiber axis in Figure 9 B to resemble the TEM image represented in Figure 9 C. Between the simulation and experiment, the self-assembled nanostructure shows good similarity in both cross sections. This qualitative agreement points to the KB gathering at the center of the fiber after annealing, again consistent with the

Figure 6. Detachment of protein on the sample surface exposed to flowing buffer solution.

Because the content of BSA was drastically lowered with the increase of the KB content in the fibers, the fraction of BSA for each case was probed. It is worth noting that BSA was easily released on the fiber surface that had more amphiphilic polymer groups. Meanwhile, the fibers made with both PLA and PET released tiny amounts of BSA during the washing-off process, indicating that BSA was strongly attached to the hydrophobic polymer surfaces such as PET and PLA. This result clearly indicates that the amphiphilic nonionic side chain structure has a much greater effect on fouling-release behavior than the hydrophobic polymer structure does. Fiber Structure Analysis by TEM and EDX. To observe the nanostructure inside the spun fibers, the fibers were microtomed at room temperature and observed under TEM. Figure 7 shows the cross sections of the as-spun PLA, as-spun PLA/KB (70/30) fibers, and the annealed PLA/KB fibers. We chose to microtome the PLA/KB (70/30) fiber sample because a higher KB content appears too dark under TEM, obstructing clear visualization of PLA/KB phase separation within the 1611

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Figure 8. EDX spectra for the as-spun PLA/KB fiber and annealed PLA/KB fiber at 120 °C for 6 h are shown in the left and right panels, respectively. The electron beam was shot in the core region (hundreds of nm3) of the sample. The signals detected show the counts of carbon, oxygen, and osmium. Because the osmium signals are found heavily among the KB, higher osmium signals refer to higher concentration of the KB in the core area in the right panel than in the left panel.

Figure 9. Snapshots of the simulation results compared with equivalent experimental TEM. The simulation results of the equilibrium structure in (A) is shown in axial view in top and crosssectional view in bottom (red = main chains of KB, light blue = hydrophilic side chains, green = hydrophobic side chains, blue = homo PLA polymers). In (B), both main chains and the side chains of KB are all colored black, while the homopolymer is colored white. The cylinder was cut diagonally in the top and perpendicular to the fiber axis in the bottom. The matching TEM images of annealed PLA/KB (70/30) are shown in (C).

Figure 10. Radial concentration profile of KB chains under elongational flow (green solid line) and at equilibrium (red dashed line). The former represents the as-spun state, while the latter represents the annealed state. As absolute value of the normalized distance increases to 1, the further away it is from the center of the fiber.

inferior antifouling characteristics of the annealed fiber. The EDX results from Figure 8B and the morphologies of the annealed PLA/KB predicted with CGMD in Figure 9 are also in good agreement, indicating high concentration of the KB in the core. As we have quantitatively examined the KB chains movement toward the center by annealing, we have compared the radial concentration profile of the KB chains under elongational flow and equilibrium in Figure 10. Under elongational flow, which essentially represents the as-spun fiber, the KB chains are found relatively evenly throughout. However, when the system is at equilibrium without any external flow, the fractions of KB chains show phase separation behavior where more KB chains are found in specific regions, especially away from the wall regions. Here, the difference in the highest and the lowest peak is about 0.05, which is five times more than that of the simulated as-spun case. Thus, the simulated annealed case clearly demonstrates more regional concentration differences, or KB

phase separation. The comparison between two plots shows that the annealed homopolymer/KB blend has lower fractions of KB away from the center and higher fractions of KB overall in some specific core regions than the as-spun case (Figure 3S in Supporting Information), which is consistent with the experimental results. From this comparison, we can confirm that the extensive flow that occurs in electrospinning causes the even distribution of the polymers in the as-spun fiber. However, the KB chains undergo microphase separation after annealing to form their own fixed domains in core areas away from the fiber surface.

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CONCLUSIONS We have described the first fabrication of the amphiphilic triblock terpolymer-based fibers by electrospinning of solution of PLA polymer and the synthesized KB. The polymer structural and protein antifouling properties of the amphiphilic fibers in the different ratios of PLA to KB were characterized. 1612

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We have also extensively studied the structural conformation of the polymeric nanofibers before and after annealing with TEM, as well as the surface chemistry of the amphiphilic groups in the fibers by measuring the contact angles. To interpret the novel KB phase separation from the experiment, a CGMD simulation was used and compared with the experimental result. The comparison showed that the antifouling effects of amphiphilic fibers were influenced by the conformation of amphiphilic groups on the fiber surface. It should be noted that the protein antifouling properties of the annealed fibers show a reduction due to the migration of the KB toward the center of the fiber after annealing. The fibers with amphiphilic triblock terpolymer groups showed superior protein antifouling performance to the fibers without them. With the increase of the KB content in the hybrid fibers, the amphiphilic fibers were found to strongly resist protein adsorption and reduce the adhesion strength of the protein. Therefore, nanofibers made with amphiphilic triblock terpolymers are good candidates for the development of a cost-effective protein-resistant membrane.

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ASSOCIATED CONTENT

S Supporting Information *

SEM images of annealed samples, wettability behavior, snapshots of the simulation results, preparation of spinning dopes, and one-way ANOVA results of each fiber regarding antifouling results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support from NSF CBET-0852900 and the National Textile Center. This work was partially supported by the Office of Naval Research (ONR) through Award #N00014-02-1-0170, as well as through the KAUST CU Center for Energy and Sustainability.

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