Force Measurements between Bacteria and Poly(ethylene glycol

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Force Measurements between Bacteria and Poly(ethylene glycol)-Coated Surfaces Anneta Razatos,†,‡ Yea-Ling Ong,§ Fabienne Boulay,† Donald L. Elbert,| Jeffrey A. Hubbell,| Mukul M. Sharma,†,§ and George Georgiou*,†,⊥ Department of Chemical Engineering, Department of Petroleum Engineering, and Institute for Molecular and Cell Biology, University of Texas, Austin, Texas 78712, and Department of Materials and Institute for Biomedical Engineering, ETH-Zurich and University of Zurich, Moussonstrasse 18, CH-8044 Zurich, Switzerland Received June 12, 2000. In Final Form: September 5, 2000 The atomic force microscope (AFM) was used to directly measure the forces of interaction between E. coli D21 bacteria and hydrophilic glass or hydrophobic N-octadecyltrichlorosilane (OTS)-treated glass substrates coated with the block copolymers, poly(ethylene glycol) (PEG)-lysine dendron or Pluronic F127 surfactant, respectively. Short-range repulsive interactions between bacterial cells and substrates coated with the block copolymers were detected by the AFM over distances of separation comparable to the extended length of the PEG polymer chains. In contrast, glass and OTS-treated glass devoid of PEG-lysine dendron or Pluronic F127 exerted long-range attractive forces on E. coli D21 bacteria. Thus, polymeric brush layers appear to not only block the long-range attractive forces of interaction between bacteria and substrates but also introduce repulsive steric effects.

Introduction Bacterial adhesion onto biomaterial surfaces results in the establishment of biofilm infections that are highly refractile to host defense mechanisms and antibiotic therapy.1-3 If left untreated, biofilm infections can lead to chronic inflammation, tissue necrosis, or even septicemia.4 Currently, the only consistently effective solution to biofilm infections is removal of the infected medical device.1,4 One approach to preventing biomaterial-associated infections is to modify the solid-liquid interface to block the first step in biofilm formation, bacterial adhesion.1,5,6 The initial interaction and adsorption of bacteria on an interface is mediated by the physicochemical properties of the bacterial cell and substrate surfaces.3,7,8 Frequently, poly(ethylene glycol) (PEG) is incorporated into biomaterial surfaces to increase the biocompatibility of the interface without altering the bulk properties of the biomaterial.5 PEG chains immobilized onto biomaterial surfaces extend from the interface into solution to form * To whom correspondence may be addressed. E-mail: gg@ che.utexas.edu. Phone: (512) 471-6975. Fax: (512) 471-7963. † Department of Chemical Engineering, University of Texas. ‡ Current address: Department of Chemical & Materials Engineering, Arizona State University, Tempe, AZ 85287. § Department of Petroleum Engineering, University of Texas. | Department of Materials and Institute for Biomedical Engineering, ETH-Zurich and University of Zurich. ⊥ Institute for Molecular and Cell Biology, University of Texas. (1) Gristina, A. G.; Giridhar, G.; Gabriel, B. L.; Naylor, P. T.; Myrvik, Q. N. Int. J. Artif. Organs 1993, 16, 755. (2) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Science 1999, 284, 1318. (3) Costerton, J. W.; Lewandowski, Z.; Caldwell, D. E.; Korber, D. R.; Lappin-Scott, H. M. Annu. Rev. Microbiol. 1995, 49, 711. (4) Stickler, D. J.; McLean, R. J. C. Cells Mater. 1995, 5, 167. (5) Han, D. K.; Park, K. D.; Kim, Y. H. J. Biomater. Sci., Polym. Ed. 1998, 9, 163. (6) Park, K. D.; Kim, Y. S.; Han, D. K.; Kim, Y. H.; Lee, E. H.; Suh, H.; Choi, K. S. Biomaterials 1998, 19, 851. (7) Ofek, I.; Doyle, R. J. Bacterial Adhesion to Cells and Tissues; Chapman Hall, Inc.: New York, 1994. (8) Christensen, G. D.; Baldassarri, L.; Simpson, W. A. Methods Enzymol. 1995, 253, 477. (9) Elbert, D. L.; Hubbell, J. A. J. Biomed. Mater. Res. 1998, 42, 55. (10) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; Gennes, P. G. d. J. Colloid Interface Sci. 1991, 142, 149.

a brush layer that reduces the adhesion of platelets, proteins, and cells.5,6,9-15 Poly(ethylene glycol) can be incorporated onto biomaterial surfaces via surface grafting, plasma polymerization, surface interpenetrating networks, or simple adsorption of PEG-containing block copolymers.12,16 Elbert et al.17 developed a PEG-lysine dendron consisting of a PEG chain (MW 20 000) coupled to a lysine dendron with 32 positively charged amino acid residues. The lysine dendron adsorbs onto anionic substrates via electrostatic interactions while the PEG chains extend from the interface forming brush layers.9,17 Physicochemical adsorption of PEG-lysine dendrons onto tissue culture polystyrene plates was found to reduce protein adsorption and prevent the attachment of eukaryotic cells to the surface. While the adsorption of PEG-lysine dendrons is reversible, the time scale of desorption is long such that on the experimental time scale, adsorption may be considered irreversible.17 Likewise, hydrophobic surfaces can be treated with Pluronic surfactants, PEG-containing ABA block copolymers consisting of a central hydrophobic segment of poly(propylene oxide) (PPO) flanked by two hydrophilic segments of PEG.14,18 Pluronics adsorb onto hydrophobic substrates via the hydrophobic PPO segment while the hydrophilic PEG segments extend into solution to form stable brush layers.14,18 In a study by Portole´s et al.,18 addition of Pluronic F127 was found to inhibit 9299% of Pseudomonas aeruginosa adhesion onto contact lenses. (11) Lee, J. H.; Kopecek, J.; Andrade, J. D. J. Biomed. Mater. Res. 1988, 23, 351. (12) Lee, J.; Matric, P. A.; Tan, J. S. J. Colloid Interface Sci. 1989, 131, 252. (13) Milner, S. T. Science 1991, 251, 905. (14) O’Connor, S. M.; Gehrke, S. H.; Retzinger, G. S. Langmuir 1999, 15, 2580. (15) Owens, N. F.; Gingell, D.; Rutter, P. R. J. Cell Sci. 1987, 87, 667. (16) Drumheller, P.; Hubbell, J. J. Biomed. Mater. Res. 1995, 29, 207. (17) Elbert, D. Polymeric steric stabilization of proteins, cells and tissues by adsorption of polycations and biologically inert polymers; The University of Texas at Austin: Austin, 1997. (18) Portole´s, M.; Refojo, M. F.; Leong, F. L. J. Biomed. Mater. Res. 1994, 28, 303.

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In earlier studies the ability of PEG-containing copolymers to impede bacterial adhesion was evaluated by the enumeration of cells adhering to surfaces. Until recently, no information on the nature of the forces of interaction between bacteria and substrates had been available, a fact that precluded a detailed understanding of the effects of polymeric brush layers on adhesion. Recently, we developed an atomic force microscope (AFM)-based bacterial adhesion assay capable of directly measuring the forces of interaction between bacteria and planar substrates as bacteria initially approach the interface.19-21 In this study, we use the AFM to demonstrate that surfaces coated with PEG-lysine dendron and Pluronic F127 impede bacterial adhesion by two complementary mechanisms: they block the long-range forces of interaction responsible for the attraction of bacteria to a surface, and they introduce shortrange, steric interactions that prevent direct contact between bacteria and the surface. Experimental Procedures Bacteria Strains and Growth Conditions. The Escherichia coli (E. coli) K-12 strain, D21, was obtained from the E. coli Genetic Stock Center (Department of Biology, Yale University, New Haven, CT). D21 was grown aerobically in Luria broth at 37 °C. AFM Measurements. The AFM-based bacterial adhesion assay developed to measure forces of interaction between bacteria and planar substrates has been described previously.19 Briefly, bacterial cells were harvested in mid-exponential phase and fixed with 2.5% v/v glutaraldehyde for 2 h at 4 °C. After being rinsed, a pellet of glutaraldehyde-treated cells was manually transferred onto standard AFM cantilevers with silicon nitride tips (Digital Instruments, Santa Barbara, CA). AFM cantilevers were precoated with 1% v/v polyethylenimine for 2-3 h prior to transfer of bacterial cells. Cantilevers coated with bacteria were treated with an additional drop of 2.5% v/v glutaraldehyde and incubated at 4 °C for 1-2 h. After incubation, tips were rinsed with distilled deionized water (ddH2O) and stored at 4 °C. Nanoscope III Contact Mode AFM (Digital Instruments, Santa Barbara, CA) was used throughout this work. All experiments were performed in a fluid cell filled with either 1 mM Tris buffer (tris(hydroxymethyl)aminomethane, Eastman Kodak Co., Rochester, NY; pH 7.4) or phosphate-buffered saline (PBS; pH 7.5). Force measurements were performed with a Z scan size of 300 nm at 1 Hz. Following the completion of every experiment, cantilevers were examined by scanning electron microscopy (JEOL, JSM-T220A, Tokyo, Japan) to confirm the presence of cells on the tip. Results are presented in terms of force (nanonewtons) versus distance of separation (nanometers) curves as described by Razatos et al.20 Each force curve represents the average of at least four separate experiments; the standard deviations calculated for each of the averaged force curve were less than 30% of the mean. Flow Cell Experiments. A parallel-plate flow cell apparatus (Water Technologies, Bozeman, MT) was also used to evaluate bacterial adhesion. The flow cell apparatus consists of a well (53 mm × 12 mm × 436 µm) covered by the planar substrate under investigation. Thin glass microscope cover slips (Corning, NY) were used as substrates for flow cell experiments. Once the flow cell was sealed with the glass substrate, it was filled with buffer and oriented substrate-side down. E. coli D21 bacteria were harvested in mid-exponential phase and resuspended in PBS to an OD600 of 2.5. Aliquots (0.5 mL) of the bacterial cell suspension were fluorescently stained using 1 µL of the LIVE/DEAD BacLight staining kit (Molecular Probes, Eugene, OR). Over 95% of the cells became fluorescent upon staining. A 0.5 mL portion of the (19) Razatos, A.; Ong, Y. L.; Sharma, M. M.; Georgiou, G. Proc. Natl. Acad. Sci. 1998, 95, 11059. (20) Razatos, A.; Ong, Y. L.; Sharma, M. M.; Georgiou, G. J. Biomater. Sci., Polym. Ed. 1998, 9, 1361. (21) Ong, Y. L.; Razatos, A.; Georgiou, G.; Sharma, M. M. Langmuir 1999, 15, 2719.

Letters cell suspension was injected into the flow cell, and the bacteria were allowed to settle onto the substrate in the absence of flow for 30 min. The flow cell was flushed for 5 min with PBS buffer at a volumetric flow rate of 9 × 10-8 m3/s. Bacteria adsorbed on the substrate were visualized using an inverted fluorescence microscope (Olympus IX70 Inverted Systems Microscope; Melville, NY) and photographed using an integrated camera (Olympus SC35; Melville, NY). Substrate Preparation. Corning glass cover slips were cleaned by soaking in 1 M HNO3 for 24 h, followed by rinsing with ddH2O and MeOH. Glass was rendered hydrophobic by coating with N-octadecyltrichlorosilane (OTS; United Chemical Technologies, Inc., Bristol, PA) in a 1% w/v OTS/toluene solution for 2 h followed by rubbing with detergent and rinsing with hot water. PEG-lysine dendron was synthesized and used to coat glass substrates as described by Elbert et al.17 For PEG-lysine dendron adsorption, glass was cleaned by soaking in 1% w/v NaOH at 130 °C for 1.5 h, rinsed with ddH2O, soaked in 1 M HCl at 130 °C for 1.5 h, and rinsed with ddH2O. The glass was then dried at 130 °C and stored in sterile ddH2O at 4 °C. The PEG-lysine dendron polymer was dissolved in sterile ddH2O to a final concentration of 5% w/v (pH 7). Subsequently, the PEG-lysine solution was deposited onto clean glass and allowed to adsorb for 3.5 h at room temperature. Glass coated with PEG-lysine was rinsed with ddH2O and air-dried for 1 h prior to AFM measurements. Pluronic F127 (BASF Corp., Mount Olive, NJ) consists of two PEG segments (MW 4500) flanking a central PPO segment (MW 3600). Pluronic F127 was dissolved in PBS buffer and filtersterilized through 0.22 µm syringe filters (Corning, NY) to a final concentration of 1% w/v. Pretreatment with Pluronic for AFM experiments consisted of incubating OTS-treated glass substrates or bacteria-coated cantilevers in 1% w/v Pluronic F127 solution for 15 min followed by air-drying. For the flow cell experiments, a suspension of 109 bacteria/mL was incubated with 1% w/v Pluronic F127 for 15 min. Subsequently, the cells were pelleted by centrifugation and resuspended in PBS buffer. Alternatively, the OTS-treated glass substrate in the flow cell was treated with 1% w/v Pluronic F127 solution for 15 min followed by flushing the flow cell with PBS for 5 min, prior to the introduction of the bacterial suspension. Contact Angle Measurements. Contact angles were measured by the sessile drop method using a Rame-Hart goniometer (Rame-Hart, Inc., Mountain Lakes, NJ). Probe liquids used to measure contact angles were ddH2O and dodecane (Aldrich Chemical Co., Milwaukee, WI). For most surfaces in this study, advancing contact angles were measured using water droplets in air. In the case of OTS-treated glass pretreated with Pluronics, advancing contact angles were measured using dodecane as the probe liquid while both the substrate and the dodecane droplet were submerged in PBS buffer.

Results and Discussion 17

Elbert et al. demonstrated that adsorption of PEGlysine dendron onto anionic tissue culture polystyrene inhibits protein adsorption and eukaryotic cell spreading. In this study, PEG-lysine dendron was adsorbed onto clean glass, which is anionic at neutral pH. Contact angles measured with water on glass and PEG-lysine coated glass were 14 ( 1° and 24 ( 3°, respectively. The AFM was used to measure the forces of interaction between PEG-coated substrates and E. coli D21, which expresses core lipopolysaccharide molecules but no O-antigen on its surface.22 In this study, E. coli D21 cells immobilized onto AFM cantilevers were attracted to clean, hydrophilic glass in 1 mM Tris buffer (Figure 1) consistent with previous AFM results.20 Adsorption of PEG-lysine dendron onto glass, however, not only eliminated the attractive forces between the E. coli D21 bacteria and hydrophilic glass but also gave rise to short-range, repulsive interactions (22) Boman, H. G.; Monner, D. A. J. Bacteriol. 1975, 121, 455.

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Figure 1. Force versus distance of separation curves between D21 and (2) clean glass in 1 mM Tris and (0) PEG-lysine dendron coated glass in 1 mM Tris.

Figure 2. Force versus distance of separation curves between D21 and (2) OTS-treated glass in PBS and (0) OTS-treated glass in PBS + 1 wt % Pluronic F127.

between the bacteria-coated AFM cantilevers and the PEG-lysine coated glass (Figure 1). The interaction of bacteria with a hydrophobic surface treated with a PEG-containing block copolymer was also examined. Hydrophilic glass was made hydrophobic by coating with octadecyltrichlorosilane (OTS). Contact angles measured with water were 14 ( 1° for untreated glass and 95 ( 1° for OTS-treated glass confirming that glass became hydrophobic following treatment with OTS. Pluronic F127 is known to adsorb onto hydrophobic substrates via its PPO block, resulting in a stable PEG brush layer at the interface.14,18 To characterize OTStreated glass that had been coated with Pluronic F127, contact angles were measured in PBS buffer using the apolar probe liquid dodecane. Contact angles measured with dodecane were 5 ( 1° for OTS-treated glass and 43 ( 2° for OTS-treated glass coated with Pluronic. E. coli D21-coated cantilevers were strongly attracted to OTS-treated glass in PBS buffer (Figure 2). Force measurements between E. coli D21 and OTS were repeated with the addition of 1% w/v Pluronic F127 dissolved in the PBS buffer in which force measurements were conducted. The presence of 1% w/v Pluronic F127 in the PBS buffer completely eliminated the adhesive interactions between E. coli D21 bacteria and OTS-treated glass (Figure 2). Moreover, instead of an attractive interaction, a repulsive force was measured between the bacteriacoated cantilevers and OTS-treated glass in the presence of Pluronic (Figure 2). To investigate whether Pluronic modified the bacterial cell or substrate surface, OTS-treated glass and E. coli D21 bacteria immobilized onto AFM cantilevers were incubated separately in a 1% w/v Pluronic F127 solution. Forces of interaction were measured between (i) OTSglass pretreated with Pluronic and clean E. coli D21-coated cantilevers and between (ii) E. coli D21-coated cantilevers pretreated with Pluronic F127 and clean OTS-treated glass. Pretreatment of either the OTS-treated glass

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substrates or bacteria-coated tips with Pluronic resulted in force curves identical to those obtained when Pluronic F127 was simply added to the PBS buffer filling the AFM fluid cell (data not shown). These results indicate that Pluronic surfactant can prevent bacterial adhesion by adsorbing onto either the hydrophobic substrate or the bacterial cell surface. The ability of Pluronic F127 to impede bacterial adhesion was also investigated using a parallel-plate flow cell apparatus. Figure 3A is a photograph of fluorescent E. coli D21 bacteria adhering to OTS-treated glass in PBS buffer. It is evident that the substrate in this case was completely covered with bacteria. However, when Pluronic F127 was added to the E. coli cell suspension, only a few bacterial cells adsorbed onto the OTS-treated glass (Figure 3B). Flow cell experiments were also conducted to verify that pretreatment of either the bacteria or the hydrophobic substrate with Pluronic F127 reduced bacterial adhesion. OTS-treated glass that had been pretreated with a 1% w/v Pluronic F127 solution in the flow cell prior to the injection of the bacterial suspension was resistant to bacterial adhesion (Figure 3C). Similarly, E. coli D21 bacteria pretreated with a 1% w/v Pluronic F127 solution and rinsed once with PBS buffer did not adhere to a great extent on OTS-treated glass (Figure 3D). Therefore, the presence of Pluronic F127 on the bacteria and/or the substrate significantly reduced the number of bacteria adhering to hydrophobic substrates. The AFM measured repulsive interactions at 650 Å for PEG-lysine dendron and 300 Å for Pluronics. PEG-lysine dendron repelled bacteria over a longer distance of separation consistent with its longer chain length of PEG in comparison to Pluronic. Scaling law analysis indicates a linear relationship between the thickness of a polymeric brush layer on a surface and the molecular weight of the polymer.23 Using the surface force apparatus and assuming that brush thickness approaches zero as molecular weight approaches zero, Claesson and Golander23 found the coefficient of the linear equation for brush thickness as a function of molecular weight to be 0.0263 for PEG (assuming that PEG is present only on one surface). By use of this linear coefficient, the thickness of the brush layer of PEG-lysine dendron (MW of PEG 20 000) should be 526 Å and that of Pluronic F127 (MW of PEG 4500) should be 118 Å. However, the distances of separation over which repulsion became significant measured by the AFM were 124 and 182 Å greater than the brush layer thickness calculated for PEG-lysine dendron and Pluronics, respectively. This discrepancy between the measured distance of separation and the calculated brush layer thickness indicates that other forces may be acting in addition to steric repulsion by the polymeric brush layer. Additional AFM studies using buffer solutions with higher salinity than the 1 mM Tris and PBS buffers revealed that electrostatic interactions were not involved in the short-range repulsive interactions observed between bacteria and the PEG-coated substrates (data not shown). One possible explanation for the longer distance of separation over which repulsion was measured by the AFM is steric exclusion effects between the PEG-brush layers and the linear lipopolysaccharide molecules coating the E. coli D21 bacterial cell surface. In summary, the AFM was used to directly measure the forces of interaction between bacteria and hydrophilic or hydrophobic substrates treated with PEG-lysine dendron or Pluronic F127, respectively. Contact angle measurements revealed that coating glass with PEG(23) Claesson, P.; Golander, C. J. Colloid Interface Sci. 1986.

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Figure 3. Photographs of D21 fluorescent bacteria adhering to OTS-treated glass substrate in the flow cell: (A) D21 adhering to OTS-treated glass in PBS; (B) D21 adhering to OTS-treated glass with 1 wt % Pluronic F127 dissolved in PBS; (C) D21 adhering to OTS-glass that had been pretreated with 1 wt % Pluronic F127; (D) D21 that had been pretreated with 1 wt % Pluronic F127 adhering to OTS-glass.

lysine dendron rendered the glass slightly more hydrophobic. However, despite this small increase in hydrophobicity, the presence of PEG-lysine dendron not only blocked adhesive forces of interaction but actually gave rise to repulsive steric interactions between E. coli bacteria and glass. Moreover, Pluronic F127 effectively blocked the strong attractive forces of interaction between E. coli D21 bacteria and OTS-treated glass in part by disrupting hydrophobic interactions known to play a significant role in bacterial adhesion.21,24 Addition of Pluronic F127, however, not only eliminated the strong, long-range attraction between bacteria and hydrophobic OTS-treated glass but also gave rise to short-range repulsive interactions. This repulsive force is due to a combination of entropic and enthalpic factors, collectively referred to as steric stabilization in colloid science. (24) Klotz, S. A. Role of hydrophobic interactions in microbial adhesion to plastics used in medical devices; Doyle, R. J., Rosenberg, M., Eds.; ASM: Washington, DC, 1990; p 107.

It is important to note that under physiological conditions, biomaterials become coated with organic as well as inorganic molecules to form conditioning films, which chemically alter the interface and hence influence bacterial adhesion.4 The effect of polymeric brush layers on conditioning films and subsequent bacterial interactions is outside the scope of this work but will be considered in future studies. In conclusion, this study presents direct, quantitative evidence that reversible adsorption of PEG-containing block copolymers at an interface reduces long-range attractive forces between the substrate and bacterial cells while introducing short-range repulsive interactions. Coating substrates with nontoxic, PEG-containing polymers such as PEG-lysine dendron and Pluronic F127 represents a simple and effective method of modifying solid-liquid interfaces to create a physical barrier to microbial adhesion. Moreover, spontaneous adsorption of these polymers at solid-liquid interfaces eliminates the need for any covalent modification of biomaterial surfaces. LA000818Y