Strong Resistance of Oligo(phosphorylcholine) Self-Assembled

via ATRP and Their Thermally-Induced Aggregation Behavior in Aqueous Media ..... Zheng Zhang , Min Zhang , Shengfu Chen , Thomas A. Horbett , Budd...
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Strong Resistance of Oligo(phosphorylcholine) Self-Assembled Monolayers to Protein Adsorption Shengfu Chen,† Lingyun Liu,‡ and Shaoyi Jiang*,†,‡ Department of Chemical Engineering, and Department of Bioengineering, UniVersity of Washington, Seattle, Washington 98195 ReceiVed October 23, 2005. In Final Form: January 8, 2006 In this work, we demonstrate the strong resistance of oligo(phosphorylcholine) (OPC) self-assembled monolayers (SAMs) to protein adsorption and cell adhesion. OPC SAMs were characterized using X-ray photoelectron spectroscopy (XPS), and protein adsorption was measured using a surface plasmon resonance (SPR) sensor. Results are compared with those of phosphorylcholine (PC) SAMs. Despite the existence of negative charge on OPC SAMs and the simple synthesis procedure of OPC thiols, OPC SAMs resist protein adsorption as effectively as or better than PC SAMs formed from highly purified PC thiols. The ease of their preparation and the effectiveness of their function make OPC SAMs an attractive alternative for creating nonfouling surfaces.

Introduction Noufouling materials draw much attention because of their ability to prevent nonspecific protein adsorption, which degrades the performance of surface-based diagnostic devices and may have an adverse effect on the healing process for implanted biomaterials.1,2 Poly(ethylene glycol) (PEG)3 and poly(2-methacryloyloxyethyl phosphorylcholine) (MPC)4,5 are two major synthetic nonfouling materials, although other nonfouling functional group candidates have been extensively explored.3,6,7 Whereas PEG exhibits an excellent nonfouling capability, it faces the problem of relatively low stability in the presence of oxygen and transition-metal ions found in most biochemically relevant solutions.6,8 Poly(MPC)-based materials are believed to be more biocompatible because of the abundant phosphorylcholine (PC) structures in the cell membranes. However, they often do not match the excellent protein-resistant properties of PEG.4,5 Recently, it was shown that poly(MPC)9,10 grafted onto surfaces by an atom-transfer radical polymerization (ATRP) method exhibits protein resistance that is as effective as PEG. Self-assembly is an excellent method for creating thin films with a high degree of structural control. Our recent experimental results11 show that PC self-assembled monolayers (SAMs) have very low protein adsorption when the N/P ratio is close to 1.0, thus exhibiting surface charge neutrality. Our simulation results11 show that pure PC SAMs have packing densities similar to those of membrane lipids and that PC headgroups prefer to have an antiparallel orientation for dipole minimization. Oligo- or poly(PC) containing a unique PC repeat unit and exhibiting excellent water * Corresponding author. E-mail: [email protected]. † Department of Chemical Engineering. ‡ Department of Bioengineering. (1) Ratner, B. D.; Bryant, C. J. Annu. ReV. Biomed. Eng. 2004, 6, 41. (2) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28. (3) Ostuni, E.; Chapman, R. G.; Holmlin, R. K.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605. (4) Ishihara, K.; Fukumoto, K.; Iwasaki, Y.; Nakabayashi, N. Biomaterials 1999, 20, 1545. (5) Ishihara, K.; Nomura, H.; Mihara, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi, N. J. Biomed. Mater. Res. 1998, 39, 323. (6) Luk, Y. Y.; Kato, M.; Mrksich, M. Langmuir 2000, 16, 9604. (7) Kitano, H.; Kawasaki, A.; Kawasaki, H.; Morokoshi, S. J. Colloid Interface Sci. 2005, 282, 340. (8) Shen, M. C.; Martinson, L.; Wagner, M. S.; Castner, D. G.; Ratner, B. D.; Horbett, T. A. J. Biomater. Sci., Polym. Ed. 2002, 13, 367. (9) Feng, W.; Brash, J.; Zhu, S. P. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 2931. (10) Feng, W.; Zhu, S. P.; Ishihara, K.; Brash, J. L. Langmuir 2005, 21, 5980. (11) Chen, S. F.; Zheng, J.; Li, L. Y.; Jiang, S. J. Am. Chem. Soc. 2005, 127, 14473.

solubility was previously synthesized for studies of polyelectrolyte properties.12 We expect that oligo(PC) (OPC) SAMs should be as effective as or better than PC SAMs at resisting protein adsorption. This phenomenon is similar to what was previously observed for oligo(ethylene glycol) [EGn or HS(CH2)11(OCH2CH2)nOH] SAMs.13 Although high protein adsorption was observed on EG1 SAMs, EG2 SAMs dramatically decrease protein adsorption, and EG4 SAMs prevent almost all protein adsorption. In this work, we synthesized OPC thiols, characterized OPC SAMs on Au(111) using X-ray photoelectron spectroscopy (XPS), and evaluated the adsorption of fibrinogen (Fg), bovine serum albumin (BSA), and lysozyme (Lyz) using a surface plasmon resonance (SPR) sensor. In addition, we also patterned OPC on a surface using a microcontact printing method to demonstrate its excellent nonfouling property in biologically relevant applications. Materials and Methods Chemicals. 1-Hexadecanethiol (C16 thiol), 11-mercaptoundecanol, iodine, triethylamine, tetrahydrofuran (THF, anhydrous, 99.9%), N,N-dimethylformamide (DMF), 1,4-dithio-DL-threitol(DTT), sodium bisulfite, and acetone were purchased from SigmaAldrich and used as received. 2-Chloro-1,3,2-dioxaphospholane2-oxide and N,N-dimethanolamine were purchased from Acros Organics. Human Fg, BSA, and Lyz were also purchased from SigmaAldrich. Synthesis of 11-Mercaptoundecyl-oligo-phosphorylcholine. The reaction route for the synthesis of 11-mercaptoundecyl-oligophosphorylcholine (OPC thiol) is presented in Scheme 1, which is similar to the synthesis route used in our PC thiol.11 A methanol solution (30 mL) containing 11-mercapto-1-undecanol (2.05 g) (1) was titrated with 1 M iodine methanol solution until the solution turned light yellow. The reaction was then quenched with sodium bisulfite. 11-Hydroxyundecyl disulfide (2) was precipitated from the methanol solution at 0 °C and recrystallized from ethanol with a yield of 1.85 g (90%). N,N-Dimethanolamine (2.08 mL) and triethylamine (5 mL) were dissolved in 15 mL of anhydrous THF. This solution was slowly added to a 2-chloro-1,3,2,-dioxaphospholane-2-oxide (2.1 mL) anhydrous THF (40 mL) solution at -18 °C. The mixture was allowed to warm to ambient temperature over 2 h. The reaction mixture was then cooled to 0 °C and filtered. The filtrate was concentrated in vacuo at room temperature without further purification. Product 3 is a colorless to light-yellow oil. 2 (0.075 g) was dissolved into 5 mL of DMF. 3 (0.1 mL) was then added to the solution in a N2-filled reactor once a day for 5 days (12) Nakaya, T.; Yasuzawa, M.; Imoto, M. Macromolecules 1989, 22, 3180. (13) Li, L. Y.; Chen, S. F.; Zheng, J.; Ratner, B. D.; Jiang, S. J. Phys. Chem. B 2005, 109, 2934.

10.1021/la052851w CCC: $33.50 © 2006 American Chemical Society Published on Web 02/09/2006

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Scheme 1. Synthesis Route of 11-Mercaptoundecyl-oligo-phosphorylcholine (OPC Thiol)

Figure 1. High-resolution spectra of the S 2p region from XPS for OPC SAMs prepared from a solution at pH 7.4. The peaks were fit using two S 2p doublets with a 2:1 area ratio and a splitting of 1.2 eV. Results show up to 88.1% sulfur species at 162 eV, indicating that a majority of the sulfur species are bound to the gold surface.

and the solution was continuously stirred at 55 °C. The reaction mixture was cooled to 0 °C and filtered to yield a white powder (4). The white powder (4) was dissolved in ethanol (3 mL), and DTT (0.4 g) was added to the solution. The pH of the solution was adjusted to 9 with concentrated NH3‚H2O. The solution was then stirred for half an hour. After the addition of 50 mL of acetone, the precipitate (5) was separated by centrifugation. 5 was dissolved in 3 mL of ethanol, precipitated in 50 mL of acetone, and again separated by centrifugation. This procedure was repeated three times. The final product obtained after drying in vacuo at room temperature is a white powder. SAM Preparation and Characterization by X-ray Photoelectron Spectroscopy. Gold-coated chips were washed with ethanol and cleaned in a UV cleaner for 15 min. They were dipped into a pH 7.4 PBS aqueous solution (10 mM sodium phosphate, 138 mM NaCl, and 2.7 mM KCl at pH 7.4) or a pH >10 basic aqueous solution of 0.2 mg/mL OPC thiol overnight. The chips covered with OPC SAMs were washed with Millipore water and then dried with filtered, compressed air. The procedure was repeated three times. OPC SAMs analyzed by XPS were stored in N2 and put in a vacuum chamber within 30 min of preparation. XPS measurements were conducted using a Surface Science Instruments X-Probe spectrometer (Mountain View, CA) equipped with a monochromatic Al K source (KE ) 1486.6 eV), a Hemispherical analyzer, and a multichannel detector. All XPS data were acquired at a nominal photoelectron takeoff angle of 55°. SSI data analysis software was used to calculate elemental compositions from peak areas. Protein Adsorption by a Surface Plasmon Resonance Sensor. A custom-built highly sensitive SPR biosensor was used.14 The chips were coated with an adhesion-promoting chromium layer (thickness ∼2 nm) and surface plasmon active gold layer (thickness 50 nm) by electron beam evaporation in vacuum. The chip modified with a sensing layer was attached to the base of the prism, and optical contact was established using a refractive index matching fluid (Cargille). A dual-channel Teflon flow cell containing two independent parallel flow channels with small chambers was used to contain a liquid sample during experiments. A peristaltic pump (Ismatec) was utilized to deliver the liquid sample to the two chambers of the flow cell. A flow rate of 0.05 mL/min was used throughout the experiments. (14) Chen, S. F.; Liu, L. Y.; Zhou, J.; Jiang, S. Langmuir 2003, 19, 2859.

All protein adsorption measurements were performed in physiological PBS solution (10 mM sodium phosphate, 138 mM NaCl, and 2.7 mM KCl at pH 7.4.). SPR was first stabilized with PBS solution for 20 min, protein solution flowed into the system at 1 mg/mL for 10 min, and then the SPR was flushed with PBS solution for 5 min. Microcontact Printing. The gold-coated cover slides were washed with ethanol and cleaned in a UV cleaner for 15 min. The PDMS stamp was cleaned with ethanol. A C16 thiol ethanol solution (200 µL, 8 mM) was dropped onto the cleaned PDMS stamp and remained for 30 s. The stamp was dried using flowing compressed air for 30 s. A cleaned gold-coated slide was placed on the top of the stamp and remained for 30 s. The gold-coated slide covered with the patterned C16 SAM was then soaked in a PBS solution containing OPC thiol overnight, followed by washing with Millipore water and drying with filtered compressed air. Cell Culture. Bovine aortic endothelial cells (BAECs) were isolated from bovine aortas and characterized as previously described.15 BAECs were maintained in continuous growth in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% nonesssential amino acids, and 2% penicillin-streptomycin solution at 37 °C in a humidified atmosphere containing 5% CO2 on TCPS flasks. To passage cells, BAECs were removed from the flask surfaces by washing twice with 5 mL of PBS, followed by incubation in 2 mL of trypsin/EDTA (0.05%/0.53 mM) for detachment. After cells had detached, cells were then resuspended in supplemented medium and replated onto TCPS flasks. Cells were passaged once a week and discarded after 15 passages. Cell culture medium and reagents were obtained from Gibco (Gaithersburg, MD). The substrates were transferred to a 24-well culture plate and washed with sterile PBS three times. Freshly confluent BAECs were incubated with 2 mL of trypsin/EDTA and then resuspended in PBS buffer. After centrifugation at 1000 rpm for 5 min, the supernatant was removed, and the cells were resuspended and diluted in DMEM medium supplemented with 2% fetal bovine serum at a final concentration of 100 000 cells/mL. A cell suspension (2 mL) was added to each well. The cells were then incubated with the samples for 1 to 2 days at 37 °C. Cell seeding density was determined with a hemocytometer. Phase-contrast images (10×) were captured using the Nikon TE200 inverted microscope.

Results and Discussion Characterization of OPC SAMs. OPC SAMs were characterized using XPS. The high-resolution spectra of the S 2p region from XPS (Figure 1) show that most of the sulfur species (15) Liaw, L.; Almeida, M.; Hart, C. E.; Schwartz, S. M.; Giachelli, C. M. Circ. Res. 1994, 74, 214-224.

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Figure 2. Illustration of three packing structures of OPC SAMs. Structure a should dominate, whereas structure b could exist. Structure c is unlikely due to a very strong dipole. The arrow indicates the direction of a dipole. Table 1. Elemental Compositions of OPC and PC SAMs on Au(111) Determined from XPS element SAMs OPC PC

C

O

Au

S

N

P

40.5 ( 1.5 48.7 ( 1.3

30.9 ( 2.1 20.5 ( 0.1

21.2 ( 0.7 23 ( 0.7

1.1 ( 0.1 1.8 ( 0.1

2.8 ( 0.5 3.1 ( 0.2

4.0 ( 0.5 2.8 ( 0.2

are bound to the surface (88.1%), with 11.9% unbound sulfur and without obvious oxidized sulfur species. The elemental compositions of OPC SAMs are shown in Table 1. For comparison, the elemental compositions of PC SAM are also listed in the Table. The similar Au compositions of OPC and PC SAMs indicate that the effective thickness of OPC and PC SAMs is similar. The number of PC repeat units can be estimated by comparing the (N + P)/S ratio of OPC SAMs with that of PC SAMs because of the similar attenuation of the S signal for both SAMs. Our results show that OPC SAMs from this work have two repeat PC units on average, although various repeat units may occur. Because of the preferential adsorption of shorter OPC chains, we expect that OPC thiols with more PC repeat units remain in the SAM-formation solution. Poly(PC) was previously synthesized in solution.12 In that study, it was shown that an average molecular weight of poly(PC) could reach 15 000 g/mol or ∼75 PC repeat units, as determined from gel permeation chromatography (GPC) with PEG as a reference. Our lowmolecular-weight OPC SAMs could be due to the lower reactivity of the disulfide structure (compound 2), the lower ratio of compound 3 to compound 2 used in this work, and the preferential adsorption of low-molecular-weight OPC thiols onto a surface. It has been shown in previous work11 that PC headgroups in PC SAMs lie nearly parallel to the surface and have an antiparallel orientation for dipole minimization. We speculate that OPC chains fold in such a way as to minimize their dipole within a chain and with other OPC chains as illustrated in Figure 2a. The distance between two OPC chains in this case should be greater than that between two PC chains. The lower chain density and higher chain height of OPC compared to those for PC chains in this model are consistent with XPS results (i.e., lower S/Au ratio for OPC and similar Au composition) shown in Table 1. Another possible structure is illustrated in Figure 2b, in which OPC chains lie nearly parallel to the surface and have an antiparallel orientation for dipole minimization similar to that of PC groups.11 The packing density of this structure is very low. Although this structure could exist, the structure illustrated in Figure 2a should dominate. The structure illustrated in Figure 2c is unlikely because all OPC chains pointing out of the surface will lead to a very strong dipole.

Protein Adsorption on OPC SAMs. For OPC SAMs, their N/P ratio is not as critical to achieve protein resistance as in the case of PC SAMs.11 For OPC SAMs, unequal N and P were often observed, and the N/P ratio could be as low as 0.6, indicating that these surfaces were negatively charged. However, the adsorption of proteins, including Fg, BSA, and Lyz on these OPC SAMs, was all very low or even lower than on PC SAMs formed from highly purified PC thiols. As observed previously, a net surface charge often leads to strong protein adsorption on PC SAMs. For example, Fg adsorption on PC SAMs is ∼18% and ∼3.5% of a Fg monolayer (ML) on CH3-terminated SAMs when the N/P ratio is ∼0.6 16 and ∼0.8711 for formation in acidic ethanol solution, respectively. The typical SPR wavelength shift for Fg adsorption on CH3-terminated SAMs is 16 nm13 (or 2.4 mg/m2). With the increasing number of PC repeat units, OPC SAMs exhibit excellent resistance to protein adsorption even though there is a net surface charge. It is shown in Figure 3 that the adsorbed amounts of Fg, BSA, and Lyz on OPC SAMs are 0.003, 0.014, and 0.01 mg/m2 from PBS buffer (0.15 M and pH 7.4) containing 1 mg/mL Fg, BSA, and Lyz, respectively, as compared to 0.03 mg/m2 for Fg and 0.01 mg/m2 for BSA on PC SAMs reported in our previous work.11 It should be pointed out that OPC SAMs resist the adsorption of positively charged Lyz even though there is a net negative surface charge on OPC SAMs. This indicates that increasing the number of PC repeat units will increase their capability to resist protein adsorption and suppress protein adsorption caused by the presence of a net charge. On the basis of these results, it can be seen that much stricter conditions in the synthesis and purification of PC thiols are required for PC SAMs whereas a much simpler procedure is needed to synthesize OPC thiols and to prepare OPC SAMs to achieve a similar protein-resistant capability. Thus, OPC SAMs are a much simpler and more effective alternative to PC SAMs for preventing protein adsorption. Cell Adhesion on Patterned OPC SAMs. Figure 4a shows a pattern of 50 µm × 50 µm on a gold-coated slide covered with OPC and hydrophobic C16 SAMs on alternative bands. Most of (16) Chung, Y. C.; Chiu, Y. H.; Wu, Y. W.; Tao, Y. T. Biomaterials 2005, 26, 2313.

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Figure 4b is a pattern of 50 µm × 50 µm on a gold-coated slide similar to Figure 4a except for replacing OPC with PC. To create a cell pattern, it is essential to have a coating that resists the nonspecific adsorption of proteins and cells.17 After 2 days of cell culture, cells remain in their respective bands in both cases and exhibit well-defined patterns. No obvious difference was observed in cell behavior between PC and OPC SAMs. Thus, both OPC and PC SAMs can strongly resist protein adsorption and cell adhesion.

Conclusions

Figure 3. Adsorption of 1 mg/mL Fg, BSA, and Lyz in PBS buffer (0.15 M and pH 7.4) on OPC SAMs from SPR. The wavelength shift of 1 nm in SPR is equivalent to 0.15 mg/m2 adsorbed proteins.18 Protein adsorption is defined as the wavelength shift before the Fg injection and after the buffer wash. The wavelength shift after the protein injection is mainly due to the change in the bulk refractive index.

In this work, OPC thiol was successfully synthesized. OPC SAMs were formed on Au(111) and characterized using XPS. Results show that most of the sulfur species are bound to the surface without obvious oxidized sulfur species. On the basis of the (N + P)/S ratio from XPS, it is estimated that OPC SAMs in this work contain two repeat PC units on average. Even though OPC SAMs have a N/P ratio of less than 1.0, thus creating a negative surface charge, OPC SAMs strongly resist protein adsorption and cell adhesion. In fact, SPR results show that OPC SAMs resist protein adsorption as effectively as or better than PC SAMs reported previously. This indicates that increasing the number of PC repeat units will improve their ability to resist protein adsorption as well as suppress protein adsorption caused by the presence of a net surface charge.These results show that much stricter conditions are required in the synthesis and purification of PC SAMs than for OPC SAMs to achieve protein resistance. Thus, OPC SAMs are a much simpler and more effective alternative to PC SAMs for preventing protein adsorption. Acknowledgment. This work is supported by the Office of Naval Research (N000140410409). The XPS experiments were performed at the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO) supported by NIBIB grant no. EB02027.

Figure 4. Optical images of BAECs on gold-coated slides covered with nonfouling and hydrophobic C16 SAMs on alternative bands after 2 days of cell culture. OPC (a) and PC (b) SAMs are used as nonfouling coatings. The width of each band (cell-adhesive or nonfouling) is 50 µm.

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the adhered BAECs were aligned along the patterned bands covered with C16 SAMs. These hydrophobic bands will adsorb cell-adhesive proteins when in contact with cell culture medium.

(17) Zhang, S. G.; Yan, L.; Altman, M.; Lassle, M.; Nugent, H.; Frankel, F.; Lauffenburger, D. A.; Whitesides, G. M.; Rich, A. Biomaterials 1999, 20, 1213. (18) Jung, L. S.; Campbell, C. T.; Chinowsky, T. M.; Mar, M.; Yee, S. S. Langmuir 1998, 14, 5636.