Bacteria-Repulsive Polyglycerol Surfaces by Grafting Polymerization

Article pubs.acs.org/Langmuir

Bacteria-Repulsive Polyglycerol Surfaces by Grafting Polymerization onto Aminopropylated Surfaces Theresa Weber, Yasmin Gies, and Andreas Terfort* Institute of Inorganic and Analytical Chemistry, University of Frankfurt, 60438 Frankfurt, Germany ABSTRACT: The formation of hydrogels on surfaces is a frequently used strategy to render these surfaces biorepulsive. Hyperbranched polyglycerol layers are a promising alternative to the frequently used polyethyleneglycol layers. Here, we present a strategy to covalently graft polyglycerol layers onto surfaces by first depositing an aminopropylsiloxane layer, which then acts as initiator layer for the ring-opening polymerization of 2-(hydroxymethyl)oxirane (glycidol). For silicon surfaces, the resulting polyglycerol layers start being biorepulsive for E. coli at a thickness of 2 nm and reach their highest bacterial repulsion (98%) at thicknesses of 7 nm or larger. This deposition strategy promises general applicability because the formation of aminopropylsiloxane layers has already been described for many materials.

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INTRODUCTION A well-known problem in industry and medicine is the settlement and growth of microorganisms, like bacteria, on surfaces. This process, called biofouling, has a huge impact on the functionality and persistence of materials in contact with water, such as machines in paper production, food industry, cooling towers, membrane technology, or deep water sensors.1 In medicine, bacteria growing on biomedical implants such as catheters or prostheses often lead to life-threatening infections.2,3 While for some medical applications biocides are a suitable measure to suppress biofouling processes, for others less invasive strategies are more appropriate regarding the evolution of antibiotic resistance of bacteria and side effects for patients. One of these strategies is the direct modifying of surfaces to provide them with biorepulsive properties to prevent or at least minimize the adhesion of proteins and bacteria and thus the formation of plaques or biofilms. In recent years, many strategies to prevent the interaction between surfaces and cells have been developed.4 Since the early eighties, surface-bound polyethyleneglycol (PEG, −(CH2CH2O)n−) is known to suppress or diminish protein and bacterial adhesion by the formation of hydrogels.5,6 Those studies were extended by the development of self-assembled monolayers (SAMs) on gold with oligoethyleneglycol (OEG) or oligoethyleneglycol monomethylether headgroups and thiolate anchoring groups.7,8 Other SAM systems containing hydrogel-forming headgroups such as sulfobetaines,9−11 oligopropylenesulfoxides,12 phosphorylcholines,13,14 and a series of compounds15 based on carbohydrates such as mannitol16 and galactose17 were found to be protein-repulsive but none of these was as effective as the OEG-based monolayers. Although monolayers based on OEG building blocks have been studied extensively and are still widely used for biochemical applications, they are prone to oxidation and subsequent degradation.18−22 This limits their use for the © 2012 American Chemical Society

modification of medicinal surfaces such as implants and catheters. It has been predicted that, at comparable coverage, branched molecules prevent the adsorption of proteins more efficiently than linear molecules do.23 Therefore, branched polymers with nonfouling properties attracted a lot of interest for biomedical applications.24−26 Such polymers can be connected to the surface directly or via suitable linkers. In contrast to OEG-SAMs these ultrathin polymer films often are chemically and mechanically stable. One of the polymers becoming very popular in the last years is polyglycerol (PG), which has structural similarities to PEG (Figure 1). Although the material can be formed as a linear polymer, its hyperbranched form is used more commonly.27 Basically two strategies can be envisioned for the deposition of PG-based layers onto surfaces. The first strategy resembles the one used for the formation of PEG-terminated SAMs,

Figure 1. Structure of hyperbranched polyglycerol (PG). The part highlighted in red demonstrates the structural similarity to oligoethyleneglycol. The formation of these polymers is typically initiated by the reaction of a nucleophile (Nuc) with glycidol. Received: September 2, 2012 Revised: October 15, 2012 Published: October 16, 2012 15916

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Figure 2. Outline of the presented process: After deposition of an APTMS monolayer, the surface exposes nucleophilic amino groups, which can inititiate the grafting of hyperbranched PG by ring-opening polymerization of glycidol.

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where preformed molecules containing both the biorepulsive part and the surface-reactive linkers become deposited to the surfaces. This strategy has been introduced by Haag et al.28 and since been used by several groups.29−31 Its big advantage is the potentially high degree of control over the molecular structure of the materials to be deposited. In the second approach, the PG units are directly formed at the respective surfaces, for example by grafting polymerization of 2-(hydroxymethyl)oxirane (glycidol). The promise of this approach is the formation of covalent bonds having a higher stability than the bonds used in most of the SAM systems. This approach was pursued by Huck et al., who deprotonated the native oxide surfaces of silicon to use the oxide groups for the initiation of the ring-opening polymerization of glycidol.32 It has already been demonstrated that PG layers obtained with both strategies inhibit protein adsorption as much as PEG systems do33−35 and that high biocompatibility makes PG monolayers more attractive for biomedical applications.36 An important difference to the PEG materials is the very good thermal and radiative stability of PG dendrimers28,39 making them favorable for real-world applications. Although experiments have been done previously to show the biorepulsive effect of PG coatings on proteins, there has been − to the best of our knowledge − only one study on the effect on eukaryotic cells,27 and none to take a closer look into the adhesion of bacteria, the major cause of biofouling. Because not many surfaces can be activated as easily as the silica surface by simple deprotonation,32 we wanted to extend the grafting system by using amino-terminated surfaces as initiators for the formation of covalently attached PG layers. For this, we wanted to use siloxane chemistry, which reportedly has been used for the modification of many kinds of materials (metals, glass, oxides, graphene oxide).40−43 When (3aminopropyl)trimethoxysilane (APTMS) is used for the surface modification, it could be expected that the exposed amino groups would not need any further treatment or reagents to act as polymerization initiators, providing an experimentally facile access to PG covered, biorepulsive surfaces (Figure 2). To check this property, we used bacteria (E. coli), which represent a real-world challenge.

EXPERIMENTAL SECTION

Reagents. All reagents were purchased from Acros or Aldrich and used as received unless stated otherwise. Millipore grade water and freshly distilled ethanol and acetone were used to rinse the samples after different preparation steps. APTMS was destilled before use and stored under nitrogen. Toluene was dried over sodium/benzophenone and destilled under nitrogen. PBS buffer contained 10 mM PO43−, 138 mM NaCl, and 3 mM KCl at pH 7.4 (25 °C). E. coli K12 (JM101) were obtained from New England BioLabs Inc. and grown in CASO broth (Roth, Germany). To visualize the bacteria after adsorption, they were stained with a 1 μg/mL solution of 4′,6-diamidino-2phenylindole (DAPI) in water. Surface Cleaning. Silicon substrates (4 cm2) were cleaved from silicon wafers with ⟨100⟩ orientation, cleaned with freshly prepared piranha solution (7:3 v/v mixture of H2SO4 and H2O2. Caution, this material reacts violently with organic materials!) for one hour, rinsed thoroughly with Millipore water, and dried in a flow of nitrogen. Film Formation. After the cleaning process, the thickness of the oxide layer on the substrates was determined by ellipsometry. For the formation of the aminopropylsiloxane layers, different concentrations of APTMS in dried toluene (1%, 10%, 30%), different coating times (2 h, 17 h, 67 h) and different coating temperatures (6 °C, 26 °C, 80 °C) were tested to find the optimal conditions for monolayer growth. After the coating process, the substrates were cleaned with ethanol thoroughly and dried in a flow of nitrogen. The monolayers were characterized using ellipsometry and contact angle goniometry. The samples were then placed into a PTFE screw cap tube and completely covered with a 10% solution of glycidol in N-methyl-2pyrrolidon (NMP). The tube was closed and heated to the desired temperature without stirring. After the growth of the PG layers, all samples were rinsed with acetone and cleaned by sonication in acetone for five minutes. Then the samples were kept in fresh acetone for another 30 min to remove any remaining physisorbed PG, and finally dried in a nitrogen flow. Ellipsometry. Measurements were carried out using a Sentech SE400 ellipsometer with a 632.8 nm laser at an incidence angle of 70° using a multilayer model (ambient-film-substrate) on the basis of the Sentech analysis software. A minimum of three different spots was measured on at least three samples. For the silicon substrate, parameters of n = 3.858 and k = 0.018 were used while for the oxide layer n = 1.45 and k = 0 was assumed. For the organic layers (siloxane and PG films) n = 1.45 and k = 0 were used as input in the respective multilayer simulations, and their thickness was calculated by subtracting the thickness of the previous layer(s) from the total adlayer thickness determined in the respective step. 15917

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Contact Angle Goniometry. The water contact angles were measured using the sessile-drop method. The reported values are the average of at least three samples with three different measurements taken at different locations of the surface of each sample. IRRAS. Infrared absorption measurements were recorded using Nicolet 6700 FTIR spectrometer (thermo) purged with dry and CO2 free air and equipped with a nitrogen-cooled MCT detector. Surface studies were performed with a Smart SAGA setup utilizing p-polarized light at an angle of incidence of 80° relative to the surface normal; 1024 scans were recorded at a resolution of 4 cm−1 recording from 600 to 3500 cm−1 at room temperature. An untreated piece of the same silicon wafer was used as reference. Bacteria Adsorption. For the adsorption experiments, a suspension of E. coli K12 (JM101) in CASO broth with an optical density of 0.5, corresponding to about 6 × 108 bacteria/cm3 and the beginning of the exponential growth phase, was used. For each modified sample one cleaned, unmodified piece of silicon wafer was tested as reference. All samples were heated to 80 °C for 20 min to sterilize the surfaces. Adsorption tests were carried out at 37 °C using 200 mL of the bacteria suspension. The samples were placed in a PTFE carrier, completely covered with the bacteria suspension and shaken with 100 rpm for two hours. Each sample was then dipped into PBS solution, shaken with 100 rpm in a 45 mL Falcon tube filled with PBS solution, and again dipped into fresh PBS solution afterward. After staining with DAPI, the number of bacteria was determined using an Olympus BX51 microscope equipped with a metal halide lamp with a 365 nm band filter for excitation and a 450 nm edge filter for detection.

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Figure 3. Formation of APTMS SAMs on silicon surfaces at different concentrations, temperatures, and coating times (top, two hours deposition time; bottom, 67 h).

RESULTS AND DISCUSSION Preparation of APTMS SAMs. Although several reports have been published on the deposition of aminopropylsiloxane monolayers44,45 we were not able to reproducibly obtain monolayers with constant thicknesses and wetting behavior. We therefore decided to redetermine the optimum conditions for the deposition of such layers onto silicon surfaces with native oxide layers. For this, three parameters were varied: (1) the concentration of the precursor, APTMS, (2) the deposition time, and (3) the deposition temperature. Because most of the previous studies used toluene, this solvent was used as well. The main difference was that the toluene was dried over sodium/benzophenone before use to have a reproducible water content (below 40 ppm). The resulting siloxane layers were characterized by ellipsometry (Figure 3) and water contact angle goniometry (Table 1). In these measurements, it was not only important to obtain the expected values for the layer thickness (0.7 nm46) and the contact angles (44−54°47), but also to obtain reproducible results, as visualized by the error bars. From the results given in Figure 3 and Table 1, we deduced that the most reproducible aminopropylation procedure involves the treatment of the silica surface with a 10% solution of APTMS in toluene for 67 h at 26 °C. Using this protocol, a monolayer was deposited onto the native oxide layer of a highly doped silicon wafer (resistivity 0.01−0.02 Ωcm) and characterized by infrared reflection absorption spectroscopy (IRRAS). The respective spectrum is depicted in Figure 4. While it should be kept in mind that the selection rules on silicon are very different compared to the ones, for example on metallic substrates,48 all of the expected features of a surface covered by aminopropylsiloxanes became visible: (a) The amino groups produce the signals of the δ−N− H vibrations at around 1550 cm−1, whereas (b) the signals at 2950 cm−1 stem from the alkylchain, and (c) the broad Si−O bands at 960 to 1200 cm−1 are indicative for the siloxane binding chemistry.

Table 1. Water Contact Angles of the APTMS Layers on Silicon at Different Coating Conditions coating time

APTMSconcentration

6 °C (adv./ rec.)

26 °C (adv./ rec.)

80 °C (adv./ rec.)

2h

1%

33° ± 2°/ 26° ± 3° 33° ± 3°/ 28° ± 3° 31° ± 3°/ 14° ± 3° 29° ± 4°/ 11° ± 3° 30° ± 3°/ 11° ± 2° 26° ± 3°/ 10° ± 3°

32° ± 3°/ 22° ± 3° 31° ± 3°/ 19° ± 5° 31° ± 3°/ 22° ± 4° 27° ± 4°/ 9° ± 3° 27° ± 9°/ 11° ± 5° 26° ± 2°/ 7° ± 3° 28° ± 3°/ 25° ± 3° 29° ± 2°/ 25° ± 3° 26° ± 4°/ 23° ± 3°

33° ± 5°/ 20° ± 3° 31° ± 2°/ 18° ± 2° 31° ± 3°/ 20° ± 2° 23° ± 2°/ 7° ± 2° 23° ± 3°/ 9° ± 1° 25° ± 2°/ 9° ± 2° 23° ± 2°/ 22° ± 3° 33° ± 3°/ 28° ± 3°

10% 30% 17 h

1% 10% 30%

67 h

1% 10% 30%

30° ± 3°/ 25° ± 4°

Grafting of Hyperbranched PG onto Aminopropylated Surfaces. For the grafting of polyglycerol layers, neat glycidol32 or glycidol solutions in inert solvents have been used.35 Because the polymerization proceeds by an ionic mechanism, we chose the polar N-methyl-2-pyrrolidone (NMP) to promote the reaction. In this study, for all grafting reactions a 10% w/w solution of glycidol in NMP was used, but the reaction temperatures and times were varied to optimize the layer deposition. As can be seen in Table 2, at 20 °C as well as 80 °C the surface reaction proceeds quite slowly, so that even after 50 h only very thin layers could be obtained. To aid the reaction by stabilizing the intermediate alkoxide species, we repeated the 80 °C reaction in the presence of 0.5% of 15918

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Figure 4. IRRAS spectrum of an APTMS-coated silicon surface. See text for details.

Table 2. Thickness and Water Contact Angles of PG Layers Grafted onto Aminopropylated Silicon Surfaces under Different Conditions, in One Case (Entry 5), an Auxiliary Base, Diisopropylethylamine, Was Added deposition conditions 1 2 3 4 5 6 7

20 °C, 20 h 20 °C, 50 h 80 °C, 20 h 80 °C, 50 h 80 °C, 21 h, 1% DIPEA 140 °C, 17 h 140 °C, 68 h

layer thickness/ nm 0.3 1.3 1.0 1.0 0.1 2.8 9.6

± ± ± ± ± ± ±

0.1 0.8 0.2 0.4 0.4 0.6 0.9

CA (adv.)

CA (rec.)

± ± ± ± ± ± ±

24 ± 4° 17 ± 5° 11 ± 2° 16 ± 7° 5 ± 2° 16 ± 4° 8 ± 3°

46 34 39 30 33 30 20

3° 2° 3° 7° 1° 3° 2°

Figure 5. Polyglycerol layer thickness on an APTMS monolayer as a function of deposition time (top), relation between contact angle and thickness of PG layer (bottom).

therefore used E.coli, a very common microorganism associated with hygiene, to test the biorepulsion of the grafted PG layers. Even on the thinnest PG layer (2.3 nm, obtained after 8 h), the bacterial adhesion was suppressed by more than 80% (Figure 6). With increasing thickness, the bacteria repulsion also

diisopropylethylamine (DIPEA). For hitherto unknown reasons, this did not lead to an improvement of the grafting process but to a hampering because the layer thicknesses after 21 h were significantly lower than for the base-free case. Thick PG adlayers could only be obtained when the reaction temperature was raised to 140 °C and the reaction time was prolonged (Table 2, entries 6 and 7). With a suitable protocol for the formation of the PG layer at hand, we wished to determine the correlation between layer thickness and biorepulsivity. To obtain PG layers of different thickness, the deposition time was varied between 8 and 128 h. As can be seen in Figure 5, the growth of the PG layer seems to approach a maximum, because the thickness gain between 64 and 128 h is only about 20%. It might be possible that the proximity of the interface-bound amino groups is still needed for the polymerization to occur, so that with increasing distance the reaction becomes slower. This hypothesis nevertheless does not go in line with the previous observation that the addition of external catalyst (DIPEA) is obviously not helping the polymerization reaction, making further studies necessary in the future. An important observation is that with increasing PG layer thickness the water contact angle decreases. It is known that strongly hydrophilic surfaces typically inhibit the adsorption of proteins and bacteria, in particular, when the hydrophilicity is caused by the formation of a hydrogel.49 Bacterial Adhesion. While often protein adsorption is used to test surfaces for their biorepulsion, typical real-world scenarios involve living bacteria settling onto surfaces. We

Figure 6. E. coli K12 adhering to bare silicon (left, reference) and to silicon coated with 0.7 nm APTMS and 2.3 nm PG (middle) and 11.6 nm PG (right).

increased, leveling off at about 98% at a thickness of 7.8 nm (obtained after 32 h of grafting). Increasing the deposition time, and thus the layer thickness, further did not result in improved biorepulsivity (Figure 7).

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CONCLUSIONS In conclusion, we could demonstrate that PG layers, which resist the adhesion of bacteria, can be covalently grafted to surfaces using a two-step procedure. In the first step, the deposition of amino-terminated monolayers basically activates the surface, so that in the second step the ring-opening polymerization of glycidol to the PG layer can occur. We found that the thickness of the PG layer can be varied up to 12 nm by 15919

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(6) Desai, N. P.; Hubbel, J. A. Solution technique to incorporate polyethylene oxide and water-soluble polymers into surfaces of polymeric biomaterials. Biomaterials 1991, 12, 144−153. (7) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. Formation of self-assembled monolayers by chemisorption of derivatives of oligo(ethylene glycol) of structure HS(CH2)11(OCH2CH2)mOH on gold. J. Am. Chem. Soc. 1991, 113, 12−20. (8) Prime, K. L.; Whitesides, G. M. Adsorption of proteins onto surfaces containing end-attached oligo(ethylene oxide): a model system using self-assembled monolayers. J. Am. Chem. Soc. 1993, 115, 10714−10721. (9) West, S. L.; Salvage, J. P.; Lobb, E. J.; Armes, S. P.; Billingham, N. C.; Lewis, A. L.; Hanlon, G. W.; Lloyd, A. W. The biocompatibility of crosslinkable copolymer coatings containing sulfobetaines and phosphobetaines. Biomaterials 2004, 25, 1195−1204. (10) Zhang, Z.; Finlay, J. A.; Wang, L.; Gao, Y.; Callow, J. A.; Callow, M. E.; Jiang, S. Polysulfobetaine-grafted surfaces as environmentally benign ultralow fouling marine coatings. Langmuir 2009, 25, 13516− 13521. (11) 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, 10799−10704. (12) Deng, L.; Mrksich, M.; Whitesides, G. M. Self-assembled monolayers of alkanethiolates presenting tri(propylene sulfoxide) groups resist the adsorption of protein. J. Am. Chem. Soc. 1996, 118, 5136−5137. (13) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Protein resistant surfaces: Comparison of acrylate graft polymers bearing oligo-ethylene oxide and phosphorylcholine side chains. Biointerphases 2006, 1, 50− 60. (14) 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. (15) Metzke, M.; Bai, J. Z.; Guan, Z. J. A novel carbohydrate-derived side-chain polyether with excellent protein resistance. J. Am. Chem. Soc. 2003, 125, 7760−7761. (16) Luk, 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. (17) Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. Surface Plasmon Resonance Imaging Studies of Protein-Carbohydrate Interactions. J. Am. Chem. Soc. 2003, 125, 6140−6148. (18) Crouzet, C.; Decker, C.; Marchal, J. Caractérisation de réactions primaires de dégradation oxydante au cours de l’autoxydation des poly(oxyéthylène)s à 25°C: étude en solution aqueuse avec amorcage par radiolyse du solvant. Macromol. Chem. 1976, 177, 145−157. (19) Hamburger, R.; Azaz, E.; Donbrow, M. Autoxidation of polyoxyethylenic non-ionic surfactants and of polyethylene glycols. Pharm. Acta Helv. 1975, 50, 10−17. (20) Gerhardt, W.; Martens, C. Zur oxydation von polyethylenoxiden und polyethylenoxidethern; die bildung von acetaldehyd bei der oxidation von diethylenglycol mit sauerstoff. Z. Chem. 1985, 25, 143. (21) Talarico, T.; Swank, A.; Privalle, C. Autoxidation of pyridoxalated hemoglobin polyoxyethylene conjugate. Biochem. Biophys. Res. Commun. 1998, 250, 354−358. (22) Sharma, S.; Johnson, R. W.; Desai, A. Evaluation of the stability of nonfouling ultrathin poly(ethylene glycol) films for silicon-based microdevices. Langmuir 2004, 20, 348−356. (23) Irvine, D. J.; Mayers, A. M.; Griffith-Cima, L. Self-consistens field analysis of grafted star polymers. Macromolecules 1996, 29, 6037− 6043. (24) Cho, W. K.; Kong, B.; Choi, I. S. Highly efficient non-biofouling coating of zwitterionic polymers: Poly((3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)ammonium hydroxide). Langmuir 2007, 23, 5678−5682. (25) Obermeier, B.; Wurm, F.; Mangold, C.; Frey, H. Multifunctional poly(ethylene glycol)s. Angew. Chem., Int. Ed. 2011, 50, 7988−7997.

Figure 7. Adhesion of E. coli K12 on the grafted PG layers. The first value shows the number of bacteria adhering to untreated silicon.

the deposition time and also found that the bacterial repulsion already reaches its optimum (of 98% adhesion suppression) at layer thicknesses of about 7 nm. As a proof of concept, we restricted ourselves to the use of silicon surfaces covered by a native oxide layer, for which the deposition of the aminopropylsiloxane monolayer was optimized. Upon variation of deposition temperature, concentration of the precursor (APTMS), and deposition time, we found that monolayers of high quality can be obtained from a 10% APTMS solution in toluene at 26 °C after 67 h. Because many surfaces reportedly can be covered with such aminopropylsiloxane monolayers, this approach should be applicable to all kinds of materials, making it quite general. For the future, we plan to use technically relevant materials, such as glass, aluminum, and stainless steel, to provide these materials with antifouling properties in particular regarding the real-world challenge of bacterial adhesion.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank New England BioLabs Inc. for the donation of the E. coli K12 (JM101) strain. REFERENCES

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