Antifouling Gold Surfaces Grafted with Aspartic Acid and Glutamic Acid

Sep 27, 2014 - water (75 mL) at 0 °C was adjusted to pH 8.5 by 3 N HCl. .... gold surfaces by SI-PIMP of the AspAA or GluAA monomer. While atom trans...
2 downloads 0 Views 5MB Size
Download PDF
Article pubs.acs.org/Langmuir

Antifouling Gold Surfaces Grafted with Aspartic Acid and Glutamic Acid Based Zwitterionic Polymer Brushes Wenchen Li, Qingsheng Liu, and Lingyun Liu* Department of Chemical and Biomolecular Engineering, University of Akron, Akron, Ohio 44325, United States S Supporting Information *

ABSTRACT: We report two new amino acid based antifouling zwitterionic polymers, poly(N4-(2-methacrylamidoethyl)asparagine) (pAspAA) and poly(N5(2-methacrylamidoethyl)glutamine) (pGluAA). The vinyl monomers were developed from aspartic acid and glutamic acid. Surface-initiated photoinifertermediated polymerization was employed to graft polymer brushes from gold surfaces. Different thickness of polymer brushes was controlled by varying UV irradiation time. The nonspecific adsorption from undiluted human blood serum and plasma was studied by surface plasmon resonance (SPR). With the polymer film as thin as 11−12 nm, the adsorption on pAspAA from serum and plasma was as low as 0.75 and 5.18 ng/cm2, respectively, and 1.88 and 10.15 ng/cm2, respectively, for pGluAA. The adsorption amount is comparable to or even better than other amino acid based zwitterionic polymers such as poly(serine methacrylate), poly(lysine methacrylamide), and poly(ornithine methacrylamide) and other widely used antifouling polymers such as poly(sulfobetaine methacrylate), even under thinner polymer film thickness. The pAspAA and pGluAA grafted surfaces also showed strong resistance to endothelial cell attachment. The possession of both zwitterionic structure and hydrophilic amide groups, biomimetic property, and multifunctionality make pAspAA and pGluAA promising candidates for biocompatible antifouling functionalizable materials.

■

INTRODUCTION Materials that can resist protein, cell, or bacterial adhesion play an important role in many applications, such as biomedical devices,1 drug delivery,2 and ship hulls.3 Some adverse consequences may be caused because of proteins adsorbing onto the surfaces. The biomedical sensor may not respond as accurately as expected. For implanted devices, nonspecific protein adsorption may be an instigator in the foreign body reaction, finally resulting in failure of the devices.1 To overcome these issues encountered in applications, a few materials have been developed. Poly(ethylene glycol) (PEG) and oligo(ethylene glycol) (OEG) are the mostly applied antifouling materials. However, they are susceptible to oxidant oxygen and transition metal ions found in most biochemically relevant solutions.4 Other antifouling materials attributed to hydrophilic groups include polyacrylamide,5 polypeptoids,6 and dextran.7 The zwitterionic materials, such as those based on phosphorylcholine,8 sulfobetaine,9,10 and carboxybetaine,10 have drawn much attention recently due to their ultralow fouling properties. The mixed positively and negatively charged groups can bind water molecules via electrostatically induced hydration, which contributes to reducing protein adsorption.11 A common structural feature of amino acids is that they all include a cationic amine and an anionic acid. Such zwitterionic structure makes them potential candidates to suppress protein adsorption. To date, there are not many reports on amino acid based surfaces to resist biofouling. Murthy and co-workers12 © 2014 American Chemical Society

reported the citrate-capped gold nanospheres modified with either lysine or cysteine, by place-exchange reactions, did not change in size upon incubation in fetal bovine serum and was lack of serum protein adsorption. A thin layer of cysteine was used to modify a gold-coated stainless steel substrate through the solution thiol chemistry.13 The cysteine surface showed an albumin fouling of 3.92 μg/cm2, which was 98.5% lower than gold surfaces and could serve as a PEG alternative for long-term implantable bioelectronics. In biomedical field, poly(ethylene terephthalate) (PET) is a vital polymeric material due to its excellent mechanical property and moderate inflammatory response. Cysteine was grafted onto PET surface via polydopamine; the modified surface can effectively resist platelet and cell adhesion, demonstrating good hemocompatibility.14 Recently, Alswieleh modified silicon wafers with poly(cysteine methacrylate) (pCysMA), which exhibited excellent resistance to biofouling and was used to create protein patterns.15 Shiraishi modified poly(methyl methacrylate) microspheres with the copolymer of O-methacryloyl-L-serine (SerMA) and methyl methacrylate (MMA), which effectively suppressed adsorption of proteins such as albumin, globulin, and fibrinogen.16 Glutamic acid (or aspartic acid) and lysine were Received: July 15, 2014 Revised: September 12, 2014 Published: September 27, 2014 12619

dx.doi.org/10.1021/la502789v | Langmuir 2014, 30, 12619−12626

Langmuir

Article

(DMF, 99%) were obtained from Alfa Aesar (Ward Hill, MA). N,N′Carbonyldiimidazole (CDI, reagent grade), trifluoroacetic acid (TFA, 99%), chloroform (99.5%), dichloromethane (99%), methanol (99.8%), tetrahydrofuran (THF, 99%), ether (98%), and phosphatebuffered saline (PBS, pH 7.4, 10 mM, 138 mM NaCl, 2.7 mM KCl) were purchased from Sigma-Aldrich. Pooled human blood plasma (anticoagulated with citrate phosphate dextrose) and pooled human blood serum were purchased from BioChemed Services (Winchester, VA). Water for all experiments was purified by a Millipore system and had a resistivity above 18 MΩ·cm. Bovine aortic endothelial cells (BAECs) were supplied by Prof. Shaoyi Jiang at the University of Washington. Dulbecco’s modified Eagle medium (DMEM, with 4.5 g/L glucose, 4.0 mM L-glutamine, and 110 mg/L sodium pyruvate) was purchased from Thermo Scientific (Waltham, MA). All other cell culture reagents were acquired from Invitrogen (Grand Island, NY). Synthesis of N-(2-Aminoethyl)methacrylamide Hydrochloride (AEMAA·HCl). AEMAA·HCl was synthesized according to a method reported before.20 Ethylenediamine (7.5 mmol, 5.05 mL) in water (75 mL) at 0 °C was adjusted to pH 8.5 by 3 N HCl. Methacryloyl chloride (8.25 mmol, 8.05 mL) in chloroform (50 mL) was then added to the aqueous ethylenediamine solution dropwise over 2 h. The mixed solution was further stirred at 0 °C for 2 h and allowed to rest. After the organic and aqueous phases were separated, the aqueous layer was purified by chloroform extraction three times to remove methacryloyl chloride and then concentrated under reduced pressure to give a white solid. The solid was then washed with methanol repeatedly. The filtrate was finally concentrated to yield AEMAA·HCl as a clear yellow oil (56% yield). 1H NMR (300 MHz, D2O): 1.96 (s, 3H, CH3−CCH2), 3.21 (t, 2H, CONH−CH2), 3.60 (t, 2H, CH2−NH2), 5.53 (s, 1H, vinyl), 5.76 (s, 1H, vinyl). Synthesis of AspAA and GluAA Monomers. N 4 -(2Methacrylamidoethyl)asparagine (AspAA) and N5-(2methacrylamidoethyl)glutamine (GluAA) were synthesized through the reaction of AEMAA·HCl with aspartic acid and glutamic acid, respectively, via the coupling agent CDI. Boc-L-aspartic acid α-tert-butyl ester (2 g, 6.91 mmol) was first dissolved in 20 mL of dichloromethane, followed by adding CDI (1.232 g, 7.69 mmol) to activate the carboxyl group. After 2 h reaction, AEMAA·HCl (1.46 g, 8.84 mmol), dissolved in 3 mL of DMF, and TEA (2.46 mL, 17.65 mmol) were added. The reaction was carried out overnight at room temperature under stirring. The solution was then purified by extraction with water, 0.1 M HCl, and brine three times each. The solvent was finally removed by rotary evaporation to give the white solid of Boc-AspAA (2.25 g, 85% yield). To obtain AspAA, Boc-AspAA was dissolved in 4 mL of CF3COOH and 2 mL of CH2Cl2 and stirred at room temperature for 24 h to remove the protection groups. The solution was then precipitated by excessive ethyl ether. The precipitate was redissolved in a small amount of TEA and precipitated in ethyl ether repeatedly, yielding the final product AspAA (1.22 g, 89% yield). The GluAA monomer was synthesized using the same procedure as that for AspAA. The AspAA and GluAA monomer structures were confirmed by 1H NMR (300 MHz, D2O), as shown in Figure 1. Preparation of the Photoiniferter SAMs on SPR Chips. The photoiniferter 11-mercaptorundecane-1-[4({[(diethylamino)carbonothioyl]thioethyl}phenyl)carbamate] (DTCA) was synthesized according to the procedure reported before.21,22 Structure of the photoiniferter was confirmed by 1H NMR spectroscopy (Figure S1 in the Supporting Information). SPR chips were made by depositing a 2 nm chromium adhesion layer and a 48 nm surface plasmon-active gold layer on glass substrates by e-beam evaporation. The glass slides were cleaned prior to metal deposition, by first being soaked in piranha solution (3:1, H2SO4:H2O2) for 30 min and then rinsed with water and acetone for three times, respectively. The instrument was pumped down to a minimum pressure of 2 × 10−6 Torr. A 2 nm layer of chromium was deposited first at 1 A/s and a 48 nm layer of gold was subsequently deposited at 2 A/s.

alternately polymerized in the polypeptide form and attached onto gold surfaces, leading to ultralow fouling surfaces.17 Apart from the reports above, we have reported three kinds of amino acid based antifouling polymers, including poly(serine methacrylate) (pSerMA),18 poly(lysine methacrylamide) (pLysAA),19 and poly(ornithine methacrylamide) (pOrnAA).19 The minimal adsorption on pSerMA-grafted surfaces from human blood serum and plasma was only 9.2 and 12.9 ng/cm2, respectively, representing at least 90% reduction relative to the unmodified gold surface.18 Compared to pSerMA, pOrnAA and pLysAA have amide group instead of ester group in the backbone. The minimal adsorption on pLysAA was 3.9 ng/cm2 from serum and 5.4 ng/cm2 from plasma, and the lowest adsorption on pOrnAA was only 1.8 and 3.2 ng/cm2, from serum and plasma, respectively.19 The adsorption on pLysAA and pOrnAA is lower than that on pSerMA even with much thinner polymer film. Besides serine, lysine, and ornithine, two other commonly known amino acids with reactive side chain terminal are aspartic acid (Asp) and glutamic acid (Glu), which belong to the category of acidic amino acid. To the best of our knowledge, the zwitterionic homopolymers based on Asp or Glu for the antifouling applications have not been explored previously. Herein, we report the Asp- and Glu-derived zwitterionic polymers and investigate their antifouling properties in detail. In neuroscience, glutamate is an important neurotransmitter that plays a key role in long-term potentiation and is important for learning and memory. Aspartic acid and glutamic acid have similar structure, both having carboxylic terminal in the side chain. The synthesis method for the corresponding antifouling monomers would therefore be different from those derived from amino acids with amine or hydroxyl group in the side chain. We first synthesized two monomers: N4-(2-methacrylamidoethyl)asparagine (AspAA) and N5-(2-methacrylamidoethyl)glutamine (GluAA). The pAspAA and pGluAA polymer brushes were then grafted from gold chips by surface-initiated photoiniferter-mediated polymerization. Protein adsorption from full blood serum and full blood plasma was studied by surface plasmon resonance (SPR) biosensor under different polymer film thicknesses. Resistance of polymer-grafted surfaces to cell adhesion was also studied.

■

MATERIALS AND METHODS

Materials. Boc-L-glutamic acid α-tert-butyl ester (99%) and Boc-Laspartic acid α-tert-butyl ester (98%) were purchased from ChemImpex (Wood Dale, IL). Methacryloyl chloride (97%), ethylenediamine (99%), triethylamine (TEA, 99%), and dimethylformamide

Scheme 1. Structure of the N4-(2Methacrylamidoethyl)asparagine (AspAA) and N5-(2Methacrylamidoethyl)glutamine (GluAA) Monomers

12620

dx.doi.org/10.1021/la502789v | Langmuir 2014, 30, 12619−12626

Langmuir

Article

Figure 1. 1H NMR spectra of the (a) AspAA and (b) GluAA monomers. a quartz tubing. The tube was sealed with a rubber septum and protected under nitrogen. 10 mL of AspAA or GluAA monomer solution (38.2 mg/mL in PBS) was deoxygenated by passing a continuous stream of nitrogen through the liquid for 30 min and then transferred to the quartz reaction tube using a syringe. Samples were then irradiated with 302 nm UV light (UVP, model UVM-57) coupled with a 280 nm cutoff filter. The filter was used to prevent the cleavage of the thiol−gold bond of the photoiniferter SAM. After the desired

To prepare the self-assembled monolayers (SAMs) of the photoiniferter DTCA, SPR chips were first washed by acetone, ethanol, and water sequentially, treated by UV/ozone for 20 min, rinsed by water and ethanol, and dried. The clean chips were then soaked in 1 mM DTCA in THF overnight, rinsed with THF, and dried with an air stream, forming the photoiniferter SAMs on SPR chips. Surface-Initiated Photoiniferter-Mediated Polymerization (SI-PIMP). The substrate with the photoiniferter SAM was placed in 12621

dx.doi.org/10.1021/la502789v | Langmuir 2014, 30, 12619−12626

Langmuir

Article

Scheme 2. Synthesis of the AspAA Monomer

reaction time, the chips were washed with water and PBS and kept in PBS before use. Ellipsometry. The gold-coated glass substrates grafted with pAspAA or pGluAA brushes were washed with water and dried with an air flow. Film thickness of the polymer brushes, prepared with different polymerization time (i.e., UV irradiation time), was measured by an α-SE ellipsometer (J.A. Woollam Co., Lincoln, NE) equipped with a 632.8 nm He−Ne laser at incidence angles of 65°−75°. The refractive index of 1.45 was assigned to the polymer layer. Contact Angle Measurements. Static contact angles of water on the pAspAA- or pGluAA-grafted gold surfaces, with different polymerization time, were measured by the sessile drop technique under ambient conditions, using a Rame-Hart goniometer (model 100−00, Mountain Lakes, NJ). At least three readings from different locations of each film were taken and averaged. Atomic Force Microscopy (AFM). NanoScope IIId AFM in tapping mode (Veeco Inc., Goleta, CA) was used to characterize surface morphology of the DTCA-modified substrate and polymergrafted surfaces at different UV irradiation time. The chips were washed with PBS and water and then dried with filtered air. A commercial Si3N4 cantilever with an elastic modulus of 0.56 N/m was used for characterization in air. Measurements of Nonspecific Adsorption by SPR. A fourchannel SPR sensor (PLASMON-IV, Institute of Photonics and Electronics, Academy of Sciences, Czech Republic) was employed in this work to evaluate protein adsorption. The sensor measures the change in the resonant wavelength induced by surface adsorption at a fixed incident angle of light. The pAspAA- or pGluAA-grafted sensor chip was attached to the base of the SPR prism using refractive index matching liquid (Cargille, Cedar Grove, NJ). A four-channel flow cell with a microfluidic gasket was then mounted on the sensor chip. A baseline signal was established by flowing PBS buffer over the chip for 10 min. For the adsorption test, 100% human blood serum or 100% human blood plasma was flowed through different channels for 10 min. PBS was run over the surface again for 10 min to remove the unbound protein molecules to establish the postadsorptive baseline. Flow rate was 0.05

mL/min for all SPR experiments. The protein adsorption amount was finally quantified based on the wavelength shift between the preadsorptive and postadsorptive baselines. A 1 nm SPR wavelength shift at 750 nm corresponds to protein adsorption of 15 ng/cm2 for our sensor.6 Cell Adhesion. BAECs were maintained in continuous growth on tissue culture polystyrene flasks at 37 °C and 5% CO2, in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% nonessential amino acids, and 2% penicillin streptomycin. Cells were passaged once a week and used only before passage 15. To evaluate cell adhesion on pAspAA- or pGluAA-grafted surfaces, cells were detached from the flask surfaces by treating with trypsin/ ethylenediaminetetraacetic acid (0.05%/0.53 mM), washed with PBS once, and resuspended in culture medium to a concentration of 105 cells/mL. The gold-coated glass substrates, grafted with or without polymers, were washed with sterile PBS three times and transferred to individual wells of a 6-well plate. 2 mL of cell suspension was then added in each well and incubated with the substrates for 1 week at 37 °C, with culture medium refreshed on the fourth day. Phase-contrast images (10×) were obtained with an EVOS xl core inverted microscope (Advanced Microscopy Group, Bothell, WA).

■

RESULTS AND DISCUSSION

To synthesize the monomers N4-(2-methacrylamidoethyl)asparagine (AspAA) and N5-(2-methacrylamidoethyl)glutamine (GluAA), CDI was employed as the condensation agent to carry the amidation between the amine of AEMAA·HCl and the side chain acid of Asp or Glu, via a one-pot procedure (Scheme 2). Since aspartic acid and glutamic acid have two carboxylic and one amino groups, the α-acid and α-amine of the amino acids were both protected, leaving alone the side chain acid available for reaction. Zwitterionic monomers were later regenerated by hydrolysis of the protective groups in an acidic 12622

dx.doi.org/10.1021/la502789v | Langmuir 2014, 30, 12619−12626

Langmuir

Article

The water contact angle (WCA) of pAspAA and pGluAA grafted surfaces was measured under different UV-irradiation time. As shown in Table 1, WCAs of the polymer-grafted gold chips were significantly smaller compared to that of the photoiniferter SAM (80°), suggesting successful surface grafting and hydrophilic nature of polymers. The WCA decreased dramatically with the increased UV-irradiation time up to 4 h. When the irradiation time was prolonged to 6 h, the contact angle changed only slightly compared to that at 4 h, which is consistent with the ellipsometry results. The strongest surface hydration of the polymer-grafted surfaces at 4 or 6 h can be attributed to the increased film thickness with stronger polymer−water interactions. Surface morphology of pGluAA-grafted surfaces was characterized by AFM (Figure 3). With the increasing UV

environment (TFA) after the amide formation. The structures of two vinyl monomers were confirmed by NMR (Figure 1). To prepare the polymer surfaces, DTCA first formed selfassembled monolayers on gold chips to work as the surface photoiniferter. Next, the polymer brushes were grafted from gold surfaces by SI-PIMP of the AspAA or GluAA monomer. While atom transfer radical polymerization (ATRP) has been widely employed to obtain surface-tethered polymer brushes with high density and finely tuned thickness, there are certain limitations, mostly related to the catalyst used in the reaction. One major problem we encountered was that the amino acidderived monomers in our work can complex strongly with the transition metal catalyst; therefore, ATRP was difficult to proceed. Alternatively, we employed the surface-initiated polymerization based on a dithiocarbamate photoiniferter chemistry to overcome those limitations. Some advantages of SI-PIMP include simple experimental procedure, no use of toxic catalyst, and easy control of film thickness by varying UVirradiation time.23 As film thickness is a key factor affecting the antifouling properties of polymer brushes, we first established the relationship between UV-irradiation time and film thickness (Figure 2). Film thickness of pGluAA and pAspAA was shown

Figure 3. Tapping mode AFM images of the photoiniferter modified gold chip (a) and pGluAA-grafted surfaces with UV irradiation for 1 (b), 2 (c), and 4 h (d). Figure 2. Film thickness dependence of pAspAA and pGluAA on the polymerization time, measured by ellipsometry. Each measurement was repeated three times to obtain the average and standard deviation.

irradiation time from 1 to 4 h, the root-mean-squared (rms) surface roughness value of pGluAA brushes changed from 1.1 to 0.6 nm when the polymer film thickness increased from ∼8 to ∼12 nm and the water contact angle decreased from 56.7° to 42°. Such a difference of rms is minimal and should not be critical enough to affect contact angle values.24 Under the UV irradiation time of 4 h, the pGluAA brushes formed uniform, flat and smooth surface with rms of only 0.6 nm. They were dense enough to cover the surface, which positively supported our protein adsorption results (described later in Figure 4). AFM results for pAspAA-grafted surfaces are similar to that of pGluAA-grafted surfaces. We can thus attribute the increased surface hydration with the increasing UV irradiation time mainly to the effect of film thickness.

to increase linearly with the irradiation time initially up to 4 h, with thickness reaching 11−12 nm for both polymers. The nonlinearity of the plot occurred when the irradiation time was prolonged to 6 h, and the thickness did not change significantly beyond 4 h, which is likely due to chain−chain termination reactions.21Although molecular weight of polymers is important, the unavailability of a photoiniferter soluble in the aqueous reaction solution and possible interference of the solution polymerization with the surface polymerization prevented us from determining molecular weight of the polymer brushes.

Table 1. Water Contact Angle of PAspAA and PGluAA Grafted Surfaces contact angle (deg) polymerization time

1h

2h

3h

4h

6h

pAspAA pGluAA

60.4 ± 0.9 56.7 ± 5.8

54.2 ± 1.5 49.6 ± 1.9

45.3 ± 2.9 45 ± 3.3

40 ± 2.8 42 ± 3.3

41.7 ± 2.3 42.7 ± 2.4

12623

dx.doi.org/10.1021/la502789v | Langmuir 2014, 30, 12619−12626

Langmuir

Article

Figure 5. SPR curves of minimal nonspecific adsorption from 100% human blood plasma and 100% human blood serum onto the pAspAA-grafted surfaces.

As discussed above, protein adsorption on the pAspAA- or pGluAA-grafted surfaces generally decreases with the increasing irradiation time, which can be correlated with the increased hydration ability of the polymer-modified surfaces as the polymer film thickness increases. For the thinner pAspAA and pGluAA films under irradiation time less than 4 h, shorter polymer chains do not form enough density of zwitterionic groups, leading to insufficient surface hydration and limiting the resistance to protein adsorption. Although the adsorption on pAspAA with 6 h UV appears slightly higher than that with 4 h UV, they do not show statistically significant difference (p > 0.05). It is true that for some polymer films (e.g., poly(sulfobetaine methacrylate) (pSBMA), pSerMA),18,25 there is an optimal film thickness associated with minimal protein adsorption because when the polymer chains get too long, strong intra- and intermolecular interactions weaken the polymer−water interaction (i.e., less surface hydration) and thus increase protein adsorption. But that should not be the situation here since ellipsometry results show similar pAspAA film thickness at 4 and 6 h polymerization. Protein adsorption results show pAspAA has less minimal adsorption than pGluAA, which can be related to the hydrophilicity of two polymers. Compared to AspAA, GluAA has one more methylene group between amide and zwitterionic amine−carboxyl terminal pair. PAspAA is therefore more hydrophilic, which results in less protein adsorption. It is reported in our previous work that the serine-derived poly(serine methacrylate) (pSerMA) is an excellent antifouling polymer. For pSerMA-grafted surfaces, the lowest nonspecific adsorption from 100% serum and 100% plasma was 9.2 ± 1.3 and 12.9 ± 2.1 ng/cm2 at optimal polymer film thickness of 37 nm (Table S1 in the Supporting Information).18 Compared to pSerMA, pAspAA and pGluAA exhibited even better antifouling ability, with lower serum and plasma adsorption at much thinner thickness (11−12 nm). To derive O-methacryloyl-L-serine (SerMA), serine was conjugated with a methacryloyl group through ester bond, while AspAA and GluAA were synthesized by forming an amide bond. Amide has both hydrogen acceptor and donor and therefore is capable of forming strong hydration layer by interacting with water via hydrogen bond. Amide belongs to the hydrophilic antifouling family.26 The contribution of amide group to antifouling performance has been proved in several

Figure 4. Nonspecific adsorption from 100% human blood serum and 100% human blood plasma onto the (a) pAspAA or (b) pGluAA grafted gold surfaces with different polymer film thicknesses. Each measurement was repeated six times to obtain the average and standard deviation.

To evaluate the nonspecific protein adsorption levels of the pAspAA- and pGluAA-grafted surfaces, 100% human blood serum and plasma were used. Figure 4 shows that protein adsorption from serum and plasma is a function of the UVirradiation time. There were significant amounts of protein adsorption on both polymer brush modified surfaces with 1 h UV irradiation; adsorption from serum was about 49.1 and 65 ng/cm2 for pAspAA and pGluAA, respectively; adsorption from plasma was 146.4 and 102 ng/cm2 for pAspAA and pGluAA, respectively. When the irradiation time was increased to 3 h, protein adsorption dramatically decreased for both polymer brushes. The minimal protein adsorption occurred at 4 or 6 h UV, which was 0.75 and 1.88 ng/cm2 from serum and 5.18 and 10.15 ng/cm2 from plasma, for pAspAA and pGluAA, respectively. As an example, Figure 5 provides the raw SPR curves of minimal nonspecific adsorption from undiluted human blood serum and plasma on the pAspAA-grafted surface. It is also observed from SPR that the polymer surfaces with 1 h UV irradiation showed faster protein adsorption and slower desorption than the surfaces with 4 h UV (Figure S2), further proving that the thinner films of polymer brushes were not strong enough to resist nonspecific protein adsorption. 12624

dx.doi.org/10.1021/la502789v | Langmuir 2014, 30, 12619−12626

Langmuir

Article

previous works. For example, polyacrylamide brushes grafted on gold strongly resisted protein, cell, and bacterial attachment.5 Poly(carboxybetaine acrylamide) suppressed more protein adsorption from complex medium than poly(carboxybetaine methacrylate) at similar thickness.27,28 Because AspAA and GluAA structures we design here have zwitterionic structure and two amide groups on each monomer, the structures can bind water molecules via both electrostatic interaction through zwitterions and hydrogen bonding through hydrophilic groups at the same time. The excellent antifouling property of the polymer-grafted surfaces we introduce here is consistent with the expectation from their structures. A previous molecular dynamic (MD) simulation work by Zhao et al. has shown that a zwitterionic amide molecule, sulfobetaine acrylamide, has higher water−solute radial distribution function and longest water residence time, compared to three other antifouling molecules including sulfobetaine methacrylate (zwitterionic ester), N-hydroxyethylacrylamide (nonzwitterionic amide), and 2-hydroxyethyl acrylate (nonzwitterionic ester).29 Their results correlated very well with our experimental work. The pAspAA and pGluAA grafted surfaces reported in this work are also better in serum resistance than the surface grafted with pSBMA, a widely used antifouling polymer in recent years, which has minimal serum adsorption of 6.1 ng/cm2 at a 62 nm polymer film thickness.25 We further evaluated the stability and antifouling performance of the aged pAspAA and pGluAA surfaces (4 h UV irradiation). After storage in PBS for 7 days, thickness of the pAspAA and pGluAA films was still about 12 nm, similar to that of the fresh samples. The nonspecific adsorption from 100% serum was 2.8 ± 2.9 and 4.7 ± 7.2 ng/cm2 for pAspAA and pGluAA, respectively; the adsorption from 100% plasma was 13.2 ± 10.6 and 7.5 ± 9.1 ng/cm2 for pAspAA and pGluAA, respectively. Compared to the fresh pGluAA surface (Table S1 in the Supporting Information), protein adsorption on the aged pGluAA did not change much. Though the protein adsorption level for pAspAA after storage appeared higher than that of the freshly prepared chip, the data did not show significant difference (p > 0.05). The results indicate that both pAspAA and pGluAA are stable and can maintain their antifouling properties after 7 days in PBS. Figure 6 shows there was no cell attaching onto pAspAA- or pGluAA-grafted gold chips. The endothelial cells were cultured on bare gold or polymer grafted surfaces with FBSsupplemented medium for 1 week. On bare gold, cells attached onto it quickly and developed into a confluent layer. The distinct difference among different surfaces suggests that the pAspAA- and pGluAA-grafted surfaces are not only resistant to protein adsorption strongly but also effective in suppressing cell adhesion. The naturally derived structure from amino acid renders AspAA and GluAA biomimetic. In the meantime, the existence of multiple functional groups (−NH2 and −COOH) in the structure provides the possibility to conveniently conjugate with multiple biomolecules for further biomedical applications. Also, the hydrophobic Boc-AspAA and Boc-GluAA render even broader applications, for example, for the synthesis of amphiphilic block copolymers.30 With these unique properties along with the excellent antifouling performance, pAspAA and pGluAA demonstrate them as promising candidates for drug delivery, biomedical implants, and biosensors.

Figure 6. Microscopic images of BAECs adhered on (a) pAspAA grafted, (b) pGluAA grafted, and (c) bare gold surfaces after 1 week culture. The polymer brushes were prepared with 4 h UV irradiation.

■

CONCLUSIONS AspAA and GluAA monomers derived from aspartic acid and glutamic acid were successfully polymerized from gold chips via SI-PIMP. The polymer-grafted surfaces can resist protein adsorption from 100% serum and plasma effectively, with the minimal adsorption realized at optimal polymer film thickness as low as 11 nm. Furthermore, the polymer-modified surfaces can greatly resist endothelial cell adhesion. Zwitterionic pAspAA and pGluAA can therefore be regarded as promising alternatives for other antifouling materials to be applied in biomedical and industrial field. 12625

dx.doi.org/10.1021/la502789v | Langmuir 2014, 30, 12619−12626

Langmuir

■

Article

Charged Gold Nanoparticles with Essentially Zero Serum Protein Adsorption in Undiluted Fetal Bovine Serum. J. Am. Chem. Soc. 2013, 135, 7799−7802. (13) Lin, P.; Ding, L.; Lin, C.-W.; Gu, F. Nonfouling Property of Zwitterionic Cysteine Surface. Langmuir 2014, 30, 6497−507. (14) Li, P.; Cai, X.; Wang, D.; Chen, S.; Yuan, J.; Li, L.; Shen, J. Hemocompatibility and Anti-biofouling Property Improvement of Poly(ethylene terephthalate) via Self-Polymerization of Dopamine and Covalent Graft of Zwitterionic Cysteine. Colloids Surf., B 2013, 110, 327−32. (15) Alswieleh, A. M.; Cheng, N.; Canton, I.; Ustbas, B.; Xue, X.; Ladmiral, V.; Xia, S. J.; Ducker, R. E.; El Zubir, O.; Cartron, M. L.; Hunter, C. N.; Leggett, G. J.; Armes, S. P. Zwitterionic Poly(amino acid methacrylate) Brushes. J. Am. Chem. Soc. 2014, 136, 9404−9413. (16) Shiraishi, K.; Ohnishi, T.; Sugiyama, K. Preparation of Poly(methyl methacrylate) Microspheres Modified with Amino Acid Moieties. Macromol. Chem. Phys. 1998, 199, 2023−2028. (17) Chen, S.; Cao, Z.; Jiang, S. Ultra-Low Fouling Peptide Surfaces Derived from Natural Amino Acids. Biomaterials 2009, 30, 5892− 5896. (18) Liu, Q.; Singh, A.; Liu, L. Amino Acid-Based Zwitterionic Poly(serine methacrylate) as an Antifouling Material. Biomacromolecules 2012, 14, 226−231. (19) Liu, Q. S.; Li, W. C.; Singh, A.; Cheng, G.; Liu, L. Y. Two Amino Acid-Based Superlow Fouling Polymers: Poly(lysine methacrylamide) and Poly(ornithine methacrylamide). Acta Biomater. 2014, 10, 2956−2964. (20) Chan, G. Y.; Jhingran, A. G.; Kambouris, P. A.; Looney, M. G.; Solomon, D. H. Approaches to the Controlled Formation of Network Polymers: 1. Synthesis and Evaluation of Monomers with Vinyl Differentiation. Polymer 1998, 39, 5781−5787. (21) Krause, J. E.; Brault, N. D.; Li, Y.; Xue, H.; Zhou, Y.; Jiang, S. Photoiniferter-Mediated Polymerization of Zwitterionic Carboxybetaine Monomers for Low-Fouling and Functionalizable Surface Coatings. Macromolecules 2011, 44, 9213−9220. (22) Benetti, E. M.; Zapotoczny, S.; Vancso, G. J. Tunable Thermoresponsive Polymeric Platforms on Gold by “Photoiniferter”Based Surface Grafting. Adv. Mater. 2007, 19, 268−271. (23) Rahane, S. B.; Kilbey, S. M.; Metters, A. T. Kinetics of SurfaceInitiated Photoiniferter-Mediated Photopolymerization. Macromolecules 2005, 38, 8202−8210. (24) Cheng, N.; Brown, A. A.; Azzaroni, O.; Huck, W. T. ThicknessDependent Properties of Polyzwitterionic Brushes. Macromolecules 2008, 41, 6317−6321. (25) Yang, W.; Chen, S.; Gang, C.; Vaisocherova, H.; Xue, H.; Li, W.; Zhang, J.; Jiang, S. Film Thickness Dependence of Protein Adsorption from Blood Serum and Plasma onto Poly(sulfobetaine)-Grafted Surfaces. Langmuir 2008, 24, 9211−9214. (26) Liu, L. Y.; Li, W. C.; Liu, Q. S. Recent Development of Antifouling Polymers: Structure, Evaluation, and Biomedical Applications in Nano/Micro-structures. WIREs Nanomed. Nanobiotechnol. 2014, DOI: 10.1002/wnan.1278. (27) Ladd, J.; Zhang, Z.; Chen, S.; Hower, J. C.; Jiang, S. Zwitterionic Polymers Exhibiting High Resistance to Nonspecific Protein Adsorption from Human Serum and Plasma. Biomacromolecules 2008, 9, 1357−1361. (28) Yang, W.; Xue, H.; Li, W.; Zhang, J.; Jiang, S. Pursuing “Zero” Protein Adsorption of Poly(carboxybetaine) from Undiluted Blood Serum and Plasma. Langmuir 2009, 25, 11911−11916. (29) Zhao, C.; Zhao, J.; Li, X.; Wu, J.; Chen, S.; Chen, Q.; Wang, Q.; Gong, X.; Li, L.; Zheng, J. Probing Structure-Antifouling Activity Relationships of Polyacrylamides and Polyacrylates. Biomaterials 2013, 34, 4714−4724. (30) Cao, Z. Q.; Yu, Q. M.; Xue, H.; Cheng, G.; Jiang, S. Y. Nanoparticles for Drug Delivery Prepared from Amphiphilic PLGA Zwitterionic Block Copolymers with Sharp Contrast in Polarity between Two Blocks. Angew. Chem., Int. Ed. 2010, 49, 3771−3776.

ASSOCIATED CONTENT

S Supporting Information *

Table S1 shows the tested film thickness range of five amino acid based polymer brushes, their optimal thickness when protein adsorption was minimal, and the minimal protein adsorption values from undiluted human blood serum and plasma; Figure S1 shows the 1H NMR spectrum of the photoiniferter DTCA; Figure S2 shows the SPR curves of adsorption from 100% human blood serum onto the pAspAA surfaces prepared under 1 and 4 h UV irradiation. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Ph (330) 972-6187; Fax (330) 9725856 (L.L.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We greatly appreciate help from Dr. Shaoyi Jiang at the University of Washington for providing SPR chips, Dr. Jie Zheng and Mr. Rundong Hu for the help with AFM studies, and Dr. Stephen Z. D. Cheng for the use of ellipsometer. The work is supported by the University of Akron Faculty Research Summer Fellowship.

■

REFERENCES

(1) Ratner, B. D.; Bryant, S. J. Biomaterials: Where We Have Been and Where We Are Going. Annu. Rev. Biomed. Eng. 2004, 6, 41−75. (2) Zhang, L.; Xue, H.; Cao, Z.; Keefe, A.; Wang, J.; Jiang, S. Multifunctional and Degradable Zwitterionic Nanogels for Targeted Delivery, Enhanced MR Imaging, Reduction-Sensitive Drug Release, and Renal Clearance. Biomaterials 2011, 32, 4604−4608. (3) Rosenhahn, A.; Schilp, S.; Kreuzer, H. J.; Grunze, M. The Role of “Inert” Surface Chemistry in Marine Biofouling Prevention. Phys. Chem. Chem. Phys. 2010, 12, 4275−4286. (4) Luk, Y.-Y.; Kato, M.; Mrksich, M. Self-Assembled Monolayers of Alkanethiolates Presenting Mannitol Groups Are Inert to Protein Adsorption and Cell Attachment. Langmuir 2000, 16, 9604−9608. (5) Liu, Q.; Singh, A.; Lalani, R.; Liu, L. Ultralow Fouling Polyacrylamide on Gold Surfaces via Surface-Initiated Atom Transfer Radical Polymerization. Biomacromolecules 2012, 13, 1086−1092. (6) Lin, S. H.; Zhang, B.; Skoumal, M. J.; Ramunno, B.; Li, X. P.; Wesdemiotis, C.; Liu, L. Y.; Jia, L. Antifouling Poly(b-peptoid)s. Biomacromolecules 2011, 12, 2573−2582. (7) McArthur, S. L.; McLean, K. M.; St. Kingshott, P.; John, H. A.; Chatelier, R. C.; Griesser, H. J. Effect of Polysaccharide Structure on Protein Adsorption. Colloids Surf., B 2000, 17, 37−48. (8) Chen, S.; Zheng, J.; Li, L.; Jiang, S. Strong Resistance of Phosphorylcholine Self-Assembled Monolayers to Protein Adsorption: Insights into Nonfouling Properties of Zwitterionic Materials. J. Am. Chem. Soc. 2005, 127, 14473−14478. (9) Zhang, Z.; Chen, S.; Chang, Y.; Jiang, S. Surface Grafted Sulfobetaine Polymers via Atom Transfer Radical Polymerization as Superlow Fouling Coatings. J. Phys. Chem. B 2006, 110, 10799−10804. (10) Zhang, Z.; Chao, T.; Chen, S.; Jiang, S. Superlow Fouling Sulfobetaine and Carboxybetaine Polymers on Glass Slides. Langmuir 2006, 22, 10072−10077. (11) He, Y.; Hower, J.; Chen, S.; Bernards, M. T.; Chang, Y.; Jiang, S. Molecular Simulation Studies of Protein Interactions with Zwitterionic Phosphorylcholine Self-Assembled Monolayers in the Presence of Water. Langmuir 2008, 24, 10358−10364. (12) Murthy, A. K.; Stover, R. J.; Hardin, W. G.; Schramm, R.; Nie, G. D.; Gourisankar, S.; Truskett, T. M.; Sokolov, K. V.; Johnston, K. P. 12626

dx.doi.org/10.1021/la502789v | Langmuir 2014, 30, 12619−12626