Colloids and Surfaces B: Biointerfaces 117 (2014) 185–192

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Biomimetic PEG-catecholates for stabile antifouling coatings on metal surfaces: Applications on TiO2 and stainless steel Faiza Khalil a , Elisa Franzmann a , Julian Ramcke a , Olga Dakischew b , Katrin S. Lips b , Alexander Reinhardt c , Peter Heisig c , Wolfgang Maison a,∗ a

University of Hamburg, Department of Chemistry, Pharmaceutical and Medicinal Chemistry, Bundesstrasse 45, 20146 Hamburg, Germany Justus-Liebig-University Giessen, Laboratory of Experimental Trauma Surgery, Schubertstr. 81, 35392 Giessen, Germany c University of Hamburg, Department of Chemistry, Institute of Biochemistry, Pharmaceutical Biology and Microbiology, Bundesstrasse 45, 20146 Hamburg, Germany b

a r t i c l e

i n f o

Article history: Received 4 December 2013 Received in revised form 4 February 2014 Accepted 12 February 2014 Available online 20 February 2014 Keywords: Biofouling Catecholates Multivalency Surface engineering Antifouling surfaces

a b s t r a c t Trimeric catecholates have been designed for the stable immobilization of effector molecules on metal surfaces. The design of these catecholates followed a biomimetic approach and was inspired by natural multivalent metal binders, such as mussel adhesion proteins (MAPs) and siderophores. Three catecholates have been conjugated to central scaffolds based on adamantyl or trisalkylmethyl core structures. The resulting triscatecholates have been immobilized on TiO2 and stainless steel. In a proof of concept study we have demonstrated the high stability of the resulting nanolayers at neutral and slightly acidic pH. Furthermore, polyethylene glycol (PEG) conjugates of our triscatecholates have been synthesized and were immobilized on TiO2 and stainless steel. The PEG coated surfaces showed excellent antifouling properties upon exposure to human blood and bacteria as demonstrated by fluorescence microscopy, ellipsometry and a bacterial assay with Staphylococcus epidermidis. In addition, our PEG-triscatecholates showed no cytotoxicity against bone-marrow stem cells on TiO2 . © 2014 Elsevier B.V. All rights reserved.

1. Introduction Functional materials are important tools in industry and academia. However, several applications in marine technology [1], hygiene technology [2], biosensors, drug delivery systems [3,4], and medical implants [5] are seriously hampered by the rapid adhesion of biomolecules, microorganisms or macroorganisms to almost any material surface [6]. In a clinical context, the formation of microbial biofilms is a serious problem in implant medicine and clinical hygiene often leading to infections [7–10]. The first step of biofilm formation is the rapid adhesion of biomolecules such as proteins to almost any material brought into biological media. A common approach to prevent this undesirable nonspecific protein adsorption is the formation of molecular monolayers [11] by chemisorption of polymer-functionalized anchor molecules (Fig. 1A) [12–17]. Suitable polymers for this strategy include polyethylene glycol (PEG) [13], polyglycerol (PG) [16,17], poly(2methyl-2-oxazoline) (PMOXA) [18], poly-saccharides [19,20], peptidomimetics [21], and zwitterionic polymers [22,23], which

∗ Corresponding author. Tel.: +49 40 42838 3477; fax: +49 40 42838 3497. E-mail address: [email protected] (W. Maison). http://dx.doi.org/10.1016/j.colsurfb.2014.02.022 0927-7765/© 2014 Elsevier B.V. All rights reserved.

confer biopassive properties to surfaces [24]. To attach these polymers to the surface, bifunctional anchor molecules may be used, containing a tailored functional group for surface immobilization such as a thiol for noble metal surfaces [23,25–27], silanols for glass surfaces [28], and phosphonates for various metal surfaces [29–31]. Recently, bifunctional catechols, inspired by mussel-adhesive proteins (MAPs) [32–37] and bacterial siderophores [38–40], have received considerable interest as anchor groups for pharmaceutically relevant metal surfaces on titanium and stainless steel [41,42]. They have good binding affinities to various surfaces and bifunctional derivatives such as dopamine or DOPA (L-3,4dihydroxyphenylalanine) are readily available natural products (Fig. 1B) [43–49]. Conjugates of catecholates and PEG have antifouling properties on metal oxides [34,50–59]. In water, binding of simple catecholates (such as dopamine) to metal surfaces is reversible at neutral and acidic pH and continuous leaching of grafted material is the consequence. Multivalent catecholates such as oligo-DOPA, in contrast, show increased binding affinities to metal surfaces and are therefore attractive anchors for antifouling coatings [60]. We and others have recently reported non-peptidic trimeric catecholates based on adamantyl, trisalkylmethyl or silyl core structures [49,61]. Examples include the Cbz-protected derivatives

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Fig. 1. (A) Schematic drawing of a bifunctional anchor molecule and its chemisorption on a surface resulting in a molecular monolayer for the prevention of biofouling and (B) typical functionalized catechol derivatives which have been used as bifunctional anchors for surface immobilization.

Fig. 2. Cbz-dopamine 1 (as a monomeric catecholate) and trimeric catecholates 2 and 3 are bifunctional anchors for TiO2 and stainless steel.

2 and 3 (Fig. 2). These triscatecholates have been conjugated to effector molecules such as dyes and bacterial autoinducers and may be grafted on metal surfaces by simple dip-and-rinse protocols [62]. In the present work, we compare the binding stability of our triscatecholates with monomeric catecholates such as Cbz-dopamine 1 at physiologically relevant pH and describe antifouling applications of PEG-triscatecholates on clinically relevant materials such as TiO2 and stainless steel. 2. Experimental 2.1. Materials TiO2 nanoparticles have been obtained from Degussa (TiO2 , P25). These nanoparticles are specified to have an average particle size of 21 nm and a purity of 99.5%. The specific surface area was determined by the Brunauer–Emmet–Teller method using the Thermoscientific Surfer. The particles were dried in vacuo at a temperature of 40 ◦ C for 2 h prior to measurement. The specific surface area was found to be 51.27 m2 /g. Homogenous TiO2 films (∼100 nm adlayer thickness, see supporting information) on silicon wafers or glass slides have been prepared as previously described [49]. The resulting surfaces were cleaned twice with water and methanol and dried in a compressed air stream before immobilization of catecholates. Stainless steel plates (type X5CrNi18-10, no 1.4301, 2 cm diameter) were obtained from HOPPE AG. The plates were treated with piranha solution (H2 SO4 :H2 O2 , 7:3) for 2 min, then rinsed with water and methanol and were dried in a stream of compressed air before immobilization of catecholates.

2.2. Syntheses The following starting materials were prepared according to literature procedures: 1 [49]; 2 [49]; 3 [49]; 4 [63]; 8 [64] and 11 [65]. 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 9.86): 0.1 m MOPS/0.6 m NaCl/0.6 m K2 SO4 . The pH of the buffer was adjusted to 9.86 by addition of 1 m NaOH. Synthesis of triester 5: Pentynoic acid (1.10 g, 11.6 mmol) dissolved in anhydrous CH2 Cl2 (20 mL). N-(3was Dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC·HCl) (1.80 g, 11.6 mmol) and N-hydroxysuccinimide (NHSOH) (1.30 g, 11.6 mmol) were added and the solution was stirred at room temperature (rt) for 12 h. Amine 4 (3.70 g, 8.9 mmol) and Et3 N (1.60 mL, 11.6 mmol) were added and the solution was stirred at rt for 48 h. The solvent was removed in vacuo and the residue was dissolved in CH2 Cl2 , washed with 1 M HCl (3 × 30 mL), saturated NaHCO3 (3 × 30 mL) solution, H2 O (3 × 20 mL) and brine. The combined organic layers were dried (Na2 SO4 ), filtered and the solvent was evaporated in vacuo to give the triester 5 (4.60 g, 9.3 mmol, 80%) as a colorless solid. mp.: 125 ◦ C; Rf = 0.34 (petrol ether/EtOAc; 2:1); molybdophosphoric acid staining; 1 H NMR (400 MHz, CDCl3 ): ı (ppm) = 6.00 (s, 1H), 2.49 (dt, 2H, 4 J = 2.6 Hz, 3 J = 7.0 Hz), 2.32 (t, 2H, 3 J = 7.0 Hz), 2.24 (t, 6H, 3 J = 7.3 Hz), 2.00 (t, 1H, 4 J = 2.6 Hz), 1.97 (t, 6H, 3 J = 7.3 Hz), 1.43 (s, 27H); 13 C NMR (100 MHz [CD3 OD]): ı = 173.5, 170.4, 83.3, 80.8, 69.6, 57.8, 36.3, 30.0, 29.9, 28.2, 15.2; IR (KBr):  [cm−1 ] = 3365, 2979, 1720, 1679, 1534, 1154; HRMS (ESI): calcd for C27 H45 NNaO7 [M−Na]+ 518.3088; found 518.3066; elemental anal. calcd. for C27 H45 NO7 : C, 65.43%; H, 9.15%; N, 2.83%. Found: C, 65.47%; H, 9.15%; N, 2.89%.

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Synthesis of triscatecholate 6: A solution of the triester 5 (0.85 g, 1.73 mmol) in trifluoroacetic acid (TFA)/CH2 Cl2 (25 mL/75 mL) was stirred at rt for 3 h. The reaction mixture was concentrated in vacuo and coevaporated three times with CH2 Cl2 to give the corresponding triacid (0.50 g, 1.73 mmol, quant.). 1 H NMR (400 MHz, [CD3 OD]): ı = 2.44 (dt, 2H, 4 J = 2.5 Hz, 3 J = 7.0 Hz), 2.38 (t, 2H, 3 J = 7.0 Hz), 2.30 (t, 6H, 3 J = 7.3 Hz), 2.25 (t, 1H, 4 J = 2.5 Hz), 2.03 (t, 6H, 3 J = 7.3 Hz); 13 C NMR (100 MHz, [CD3 OD]): ı = 177.1, 173.6, 83.8, 70.4, 58.7, 36.6, 30.4, 29.2, 15.8; HRMS (ESI): calcd for C15 H21 NNaO7 [M−Na+ ] 350.1210; found 350.1213; elemental anal. calcd. for C15 H21 NO7 : C, 55.04%; H, 6.47%; N, 4.28%. Found: C, 54.78%; H, 6.50%; N, 4.27%. The triacid (0.10 g, 0.30 mmol) and Et3 N (0.16 mL, 1.17 mmol) were dissolved in anhydrous dimethylformamide (DMF) (10 mL) and cooled to 0 ◦ C. EDC·HCl (0.18 g, 1.17 mmol), 1-hydroxybenzotriazole (HOBt) (0.15 g, 1.17 mmol) and dopamine hydrochloride (0.17 g, 1.17 mmol) were added and the resulting solution was stirred at rt for 72 h. The reaction mixture was diluted with EtOAc (20 mL) and washed three times with 1 m HCl (3 × 30 mL), H2 O (3 × 20 mL) and brine. The combined organic layers were dried (Na2 SO4 ), filtered and the solvent was evaporated in vacuo. This crude product was suspended in Et2 O (100 mL) and stirred for 2 h. Filtration of the solid and drying in vacuo gave triscatecholate 6 (0.04 g, 0.06 mmol, 23%). 1 H NMR (400 MHz, [DMSO-d6 ]): ı = 8.77 (s, 3H), 8.64 (s, 3H), 7.80 (br, 3H), 7.26 (s, 1H), 6.62 (d, 3H, 3 J = 7.5 Hz), 6.56 (s, 3H), 6.41 (d, 3H, 3 J = 7.5 Hz), 3.16–3.11 (m, 6H), 2.73 (t, 1H, 4 J = 2.4 Hz), 2.50–2.47 (m, 6H), 2.34–2.32 (m, 2H), 2.29–2.27 (m, 2H), 2.00–1.96 (m, 6H), 1.81–1.77 ppm (m, 6H); 13 C NMR (100 MHz [DMSO-d6 ]): ı = 172.0, 169.8, 145.0, 143.5, 130.3, 119.2, 115.9, 115.5, 83.9, 71.3, 56.9, 40.7, 34.9, 34.7, 30.1, 29.6, 14.5 ppm. HRMS (ESI): m/z: calcd for C39 H48 N4 NaO10 : 755.3263 [M + Na]+ , found: 755.3257. Synthesis of triscatecholate 9: Compound 8 (250 mg, 0.62 mmol) was dissolved in dimethylsulfoxide (DMSO) (20 mL). Et3 N (0.35 mL, 2.50 mmol) and succinimidyl-4-pentiolate (181 mg, 0.93 mmol) were added and stirred for 12 h at rt. The reaction mixture was concentrated in vacuo and the residue was dissolved in EtOAc (20 mL). The resulting solution was washed with saturated aqueous KHSO4 (20 mL) solution. The organic layer was concentrated in vacuo and the residue was dissolved in aqueous 1 M NaOH (20 mL). The resulting solution was washed with EtOAc (10 mL) and concentrated again to give the intermediate tricarboxylate. This was dissolved in DMF (60 mL) and the solution was cooled to 0 ◦ C (ice bath). Et3 N (0.90 mL, 8.0 mmol) was added and the solution was stirred for 5 min. EDC HCl (405 mg, 2.05 mmol) and HOBt (277 mg, 2.05 mmol) were added and the solution was stirred for 5 min. Dopamine hydrochloride (389 mg, 2.05 mmol) was added and the solution was stirred for 72 h at rt. The reaction mixture was concentrated in vacuo and the residue was dissolved in EtOAc (30 mL) and 1 M HCl (5 mL) and washed with saturated, aqueous KHSO4 solution (10 mL) three times. The organic layer was dried (Na2 SO4 ), filtered and the solvent was evaporated in vacuo. Freeze drying from MeCN/H2 O gave a colorless solid, which was suspended in Et2 O (100 mL) and stirred for 30 min at 30 ◦ C. The resulting solid was collected by filtration and the procedure was repeated to give triscatecholate 9 (282 mg, 0.331 mmol, 53%, slightly impurified with residual HOBt) after drying in vacuo; 1 H NMR (400 MHz, CD3 OD): ı = 6.65 (d, 3H, 3 J = 8.0 Hz), 6.61–6.60 (m, 3H), 6.50 (d, 3H, 3 J = 8.0 Hz), 3.32–3.27 (m, 6H), 2.61–2.58 (m, 6H), 2.39–2.35 (m, 2H), 2.29–2.23 (m, 2H), 2.11–2.07 (m, 7H), 1.59–1.57 (m, 6H), 1.43–1.39 (m, 6H), 1.01–1.04 (m, 6H); 13 C NMR (100 MHz, CD3 OD): ı = 176.8, 173.3, 146.3, 144.8, 132.0, 121.1, 116.9, 116.4, 79.3, 67.2, 46.3, 45.6, 42.3, 40.2, 36.8, 36.1, 35.8, 31.2, 19.7, 15.8 ppm; HRMS (ESI): calcd. for C48 H59 N4 O10 [M−H]− = 851.4237 found = 851.4232. Synthesis of PEG-triscatecholates 7 and 10: To a solution of alkyne 6 or 9 (1 equiv.) and PEG (5 kDa)-azide (1 equiv.) in H2 O/tBuOH (1:1), sodium ascorbate (0.1 equiv.) and CuSO4

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(0.1 equiv.) were added and the reaction mixture was stirred for 12 h at rt. The reaction mixture was filtered over celite and the solvent was removed in vacuo to give the PEG-triscatecholates 7 and 10 in quantitative yield. The PEG derivatives were characterized by MALDI-MS. PEG-triscatecholate 7: MALDI-ToF-MS m/z (%): 5790.8 (100) [MH]+ ; IR (KBr):  [cm−1 ] = 3422 (m), 2887 (s), 1466 (s), 1342 (s), 1113 (s). PEG-triscatecholate 10: MALDI-ToF-MS m/z (%): 5912.8 (86) [MH]+ ; IR (KBr):  [cm−1 ] = 3258 (m), 2880 (s), 1731 (w), 1650 (m), 1527 (m), 1466 (s), 1359–1342 (s), 1280–1241 (s), 1091 (s), 962 (s) and 841–802 (s). Synthesis of PEG-triacid 12: Aminotrimethylester (HCl–salt) 11 (100 mg, 0.22 mmol) was dissolved in 100 mL CH2 Cl2 and treated with EDC·HCl (85.8 mg, 0.45 mmol), DMAP (6.00 mg, 0.05 mmol) and PEG-COOH (1.00 g, 0.20 mmol). The resulting solution was stirred for 72 h at rt and washed with 2 M aqueous HCl (50 mL), two times with aqueous KHSO4 (50 mL each) and water (50 mL). The organic layer was dried over Na2 SO4 , filtered and the solvent was evaporated in vacuo. The remaining solid was purified by flash chromatography on silica (CH2 Cl2 /MeOH, 9:1, v/v) to give the intermediate PEG-trimethylester (724 mg, 133 mmol, 67%) as a colorless solid. 1 H NMR (300 MHz, CDCl3 ): ı = 6.45 (s, 1H, NH); 3.75 (s, 2H, 10-H); 3.62–3.49 (m, n-H, 11-H, 12H); 3.29 (s, 3H, 13-H); 2.18 (t, 6H, 3 J = 8.4 Hz, 6-H); 1.56 (s, 6H, 2-H); 1.45 (t, 6H, 3 J = 8.4 Hz, 5-H); 1.08 (d, 3H, 2 J = 12.0 Hz, 4aH); 0.97 (d, 3H, 2 J = 12.0 Hz, 4b-H); 13 C NMR (75 MHz, CDCl3 ): ı = 174.2 (C-7); 168.7 (C-9); 70.4 (C-11, C-12); 59.9 (C-13); 58.9 (C-10); 53.1 (C-1); 51.5 (C-8); 44.9 (C-3); 44.4 (C-2); 37.3 (C-4); 34.7 (C-5); 27.9 (C-6); MS-ESI: m/z [M + 4H + Na]5+ = 1035.58 (for C237 H467 NNaO114 5+ ); IR: /cm−1 = 3266; 2881; 1727; 1466; 1341; 1279; 1100; 1059; 960; 841; mp: 47 ◦ C; Rf : 0.20 (CH2 Cl2 /MeOH, 9:1, v/v). PEG-trimethylester (190 mg, 34.9 ␮mol) was dissolved in THF (50 mL), treated with KOTMS (potassium trimethylsilanolate, 38.5 mg, 300 ␮mol) and stirred for 17 h at rt. The solvent was evaporated in vacuo and the residue was dissolved in 1 M aqueous HCl and extracted three times with CH2 Cl2 (75 mL each). The combined organics were dried over Na2 SO4 , filtered and the solvent was evaporated to a volume of 1 mL. The solution was treated with Et2 O (3 mL) and crystallized at 4 ◦ C. The title compound 12 was obtained by filtration as a colorless solid (160 mg, 29.6 ␮mol, 85%). 1 H NMR (300 MHz, CDCl3 ): ı = 3.85 (s, 2H, 9-H); 3.71–3.56 (m, n-H, 10-H, 11-H); 3.36 (s, 3H, 12-H); 2.26 (t, 6H, 3 J = 8.2 Hz, 6-H); 1.64 (s, 6H, 2-H); 1.53 (t, 6H, 3 J = 8.2 Hz, 5-H); 1.15 (d, 3H, 2 J = 12.1 Hz, 4a-H); 1.06 (d, 3H, 2 J = 12.1 Hz, 4b-H); MS-ESI: m/z [M−2H]2− = 2707.52 (for C248 H483 NO121 2− ). Synthesis of PEG-triscatecholate 13: Tricarboxylic acid 12 (160 mg, 29.6 ␮mol) was dissolved in 10 mL DMF and DIEA (N,Ndiisopropylethylamine, 8 ␮L, 47.5 ␮mol) were added at 0 ◦ C. The solution was treated with EDC·HCl (18.8 mg, 98.0 ␮mol) and HOBt (13.2 mg, 98.0 ␮mol) in DMF (5 mL). The resulting solution was stirred for 30 min at 0 ◦ C and dopamine hydrochloride (18.6 mg, 98.0 ␮mol) in DMF (5 mL) were added. The mixture was stirred for 48 h at rt and the solvent was removed in vacuo. The resulting residue was dissolved in CH2 Cl2 (150 mL) and washed three times with 1 M aqueous HCl (25 mL each), three times with aqueous KHSO4 (25 mL each) and water (25 mL). The organic layer was dried over Na2 SO4 , filtered and the solvent was evaporated in vacuo. The title compound was obtained as a colorless solid (166 mg, 28.5 ␮mol, 96%). MS-ESI: m/z [MNa−7H]6− = 964.54 (for C270 H501 NNaO123 6− ).

2.3. Immobilization on TiO2 nanoparticles TiO2 nanoparticles and the appropriate catecholate (5 equiv. by weight) were treated with aqueous MOPS buffer (pH = 9.86) with

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sonication. After 12 h, the solid was separated by centrifugation, washed with H2 O and MeOH and was finally dried in vacuo. 2.4. Stability measurement The following solutions were prepared with phosphate buffered saline (PBS): pH = 7.75, 7.0, 6.75, 6.50 and 6.01. 10 mg of coated TiO2 nanoparticles (see Section 2.3) were suspended in these buffer solutions and stirred for 2 h at rt. The suspensions were then centrifuged at 1200 rpm for 7 min. The solid was separated and washed with H2 O and MeOH and was finally dried in vacuo. Loading of the particles with catecholate was followed by FTIR spectroscopy and integration of the carbonyl stretching band. Samples of 4 mg were analyzed as KBr pellets (KBr:sample, 200:4). The sum of 200 scans is shown (see supporting information). 2.5. Immobilization on TiO2 surfaces The appropriate PEG-catecholate (5 mg) was dissolved in MeOH (1 mL) and MOPS buffer (10 mL, pH 9.86). TiO2 surfaces were dipped into the solution of catecholate in buffer and left for 12 h. The plates were then rinsed with H2 O and MeOH. 2.6. Fouling assay with human blood The PEG coated surfaces were stirred in aqueous (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer for 2 h before treatment with fresh human blood. After 2 h, incubation with blood at 37 ◦ C, the surfaces were rinsed with H2 O and phosphate buffered saline (PBS buffer). The surfaces were then stained with acridine orange solution (0.1% in EtOH) for 10 min at rt in the dark, washed with H2 O and PBS buffer and dried with a stream of compressed air. 2.7. Cell culture and cell imaging In accordance with local ethical approval, bone-derived stromal cells were harvested from human reaming debris of 2 adult male patients at the age of 64 and 75 years that went through a routine trauma surgical procedure of femoral fracture. Both patients did not suffer from secondary diseases or additional injuries. Debris were removed from reamer head, placed into F12K medium containing 20% fetal calf serum (FCS, PAA, Austria), 100 U/mL penicillin and 0.05 ␮mol/mL streptomycin in a humidified 95% air, 5% CO2 atmosphere. Outgrowing bone-derived stromal cells were collected and seeded (20,000/cm2 ) for testing the materials. Media was changed twice a week. After 1 week media was replaced by osteogenic differentiation media that contained only 10% FCS but additional 0.1 ␮M dexamethasone, 0.05 mM ascorbic acid-2 phosphate, 10 mM glycerolphosphate (Sigma, Taufkirchen, Germany) and penicillin/streptomycin. The cells were cultured for one month under these conditions. Life cell images were performed twice a week using an inverted microscope (Leica, Wetzlar, Germany). 2.8. Comparative bacterial adhesion test The strain Staphylococcus epidermidis DSM20044 was grown at 37 ◦ C upon shaking with 130 rpm in LB medium (10 g/L tryptone, 5 g/L yeast-extract and 10 g/L NaCl adjusted to pH 7.0). Cells were harvested during early exponential growth phase (optical density at 550 nm [OD550] 0.2) via centrifuging (5 min, 5000 × g) and suspended in sterile PBS to obtain a cell density of 4.5 × 10exp7 cfu/mL. Three coated and three uncoated stainless steel surfaces (27.3 mm diameter) were sterilized for 5 min in 70% ethanol and placed in a 6-well plate (each well 9.6 mm2 ). Surfaces were covered with 3 mL

of the bacterial suspension and incubated for 1 h at 37 ◦ C. The substrates were rinsed three times with each 200 mL sterile PBS buffer. To keep the shear forces constant for each measurement, the stainless steel substrates were mounted and the PBS buffer was rinsed over the surface with a funnel from a fixed height. The slides were then slightly pressed upside down onto contact plates (LB medium containing 15 g/L agar) and removed after 30 s. Transferred cells were incubated overnight at 37 ◦ C. 2.9. Characterization Thin layer chromatography (TLC) was performed on silica gel aluminum sheets. Reagents used for developing plates include molybdatophosphoric acid stain (5 g molybdatophosphoric acid, 2.5 g cerium sulfate tetrahydrate, 25 mL sulfuric acid and 225 mL water), potassium permanganate (0.5% in 1 N NaOH, w/v) and detection by UV light was used when applicable. Flash column chromatography was performed on silica gel (60–200 ␮m). 1 H chemical shifts are referenced to residual non-deuterated solvent (CDCl3 , ıH = 7.26 ppm; DMSO-d6 , ıH = 2.50 ppm; CD3 OD, ıH = 3.31 ppm). 13 C chemical shifts are referenced to the solvent signal (CDCl , 3 ıC = 77.16 ppm; DMSO-d6 , ıC = 39.50 ppm; CD3 OD, ıH = 49.00 ppm). NMR spectra were recorded on 300 (75) or 400 (100) MHz instruments. ESI mass spectra were recorded on a TOF instrument operated in positive or negative mode (Bruker MicrOTOF Q). Samples were dissolved in MeOH or H2 O/MeCN-mixtures and directly injected via syringe. If indicated with abs, solvents were purified by standard techniques prior to application [66]. Ellipsometry measurements were carried out with a SE 400adv. Fluorescence images were taken using a Leica fluorescent microscope Leica CTR6000. The surface polarity was characterized by water (HPLC grade) contact angle measurement (DAS 10-MK 2). The water droplet (volume = 4.0 ␮L) permeation process was recorded using speed optimum video measuring technology (1000 picture sequence) equipped with the computer program (DSA = drop shape analyze). The measurements of the advancing contact angle were performed at 23 ◦ C and 60% relative air humidity by the sessile drop method. All samples were analyzed 3–4 times and the average was taken. 3. Results and discussion A central motivation for the design of trimeric catecholates was their high potential to form stable monolayers on metal surfaces. To verify this hypothesis, a comparative study of simple monomeric catecholates and our trimeric analogs was designed. 3.1. Stability of catecholate monolayers on TiO2 For a comparative binding study we selected three catecholate derivatives 1–3 (Fig. 2) as structurally simple model anchors and TiO2 nanoparticles as carrier material [19]. This model system allows a quantitative evaluation of the catecholate loading on TiO2 by IR-spectroscopy. Our aim was to compare the stability of molecular monolayers on TiO2 assembled from monomeric catecholate 1 and two trimeric catecholates 2 and 3 in buffered solutions of neutral or slightly acidic pH values from 7.8 to 6.0. We focused on TiO2 as solid support because it is a preferred material for implants and thus of high clinical relevance in terms of antifouling applications. Catecholates 1–3 were immobilized on commercial TiO2 nanoparticles according to a known protocol (12 h in MOPS-buffer at pH 9.86) [49]. It is well known that some derivatives of catecholamine tend to oxidize under alkaline conditions [67]. If formed with our acylated catecholates, the resulting quinone derivatives and oligomerization products would affect the following binding studies to nanoparticles and flat surfaces. We have therefore checked the stability of our catecholates in aqueous solution at

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Fig. 4. Comparison of adlayer thickness on TiO2 surfaces as measured by ellipsometry. TiO2 probes were treated with MOPS buffer, HEPES buffer and blood for 1 h and 12 h. The gray columns indicate adlayer thickness measured after each treatment (mean value of three probes) on TiO2 surfaces which were coated with compound 10. The black columns indicate adlayer thickness measured after each treatment (mean value of three probes) on uncoated TiO2 surfaces.

Fig. 3. Loading of catecholate derivatives 1, 2 and 3 immobilized on TiO2 nanoparticles after treatment with PBS buffer at different pH. The particles were incubated at the indicated pH for 2 h or 24 h. After centrifugation, the catecholate loading was measured by IR-spectroscopy of 4 mg samples and is given relative to the initial loading measured after immobilization of the appropriate catecholate on TiO2 nanoparticles in MOPS buffer. Columns are mean values of three independent measurements.

adamantyl scaffold 8, which has been previously used as a versatile 3 + 1-scaffold [65,74,75]. As a second, copper-free route to a PEG-conjugate the synthesis of PEG-triscatecholate 13 via the tricarboxylic acid 12 is shown.

pH 10 for 24 h by ESI-MS and found no oxidized or oligomerized catecholate derivatives. The loading of triscatecholates 1–3 on TiO2 nanoparticles was followed with IR-spectroscopy (see supporting information). For the stability test, the coated TiO2 nanoparticles were incubated with aqueous PBS buffer at selected pH values (pH = 7.8, 7.0, 6.8, 6.5 and 6.0). After 2 h, the suspensions were centrifuged and the separated particles were washed and freeze-dried. The loading with catecholate was measured by IR-analysis of defined nanoparticle aliquots before and after PBS treatment. In Fig. 3, the loading of TiO2 nanoparticles with catecholates 1–3 is given relative to 100% catecholate loading after immobilization in MOPS buffer. Binding of catecholates to Ti is known to be reversible at neutral and acidic pH [68,69]. The monomeric Cbz-protected dopamine derivative 1 therefore leaches from the nanoparticles and after 2 h of incubation in PBS buffer at pH 6, about 40% of the initial catecholate loading is lost from the nanoparticles. In contrast, both trimeric catecholates 2 and 3 form more stable coatings on TiO2 nanoparticles. Even at pH 6, no loss of catecholates 2 or 3 was detected after 2 h (Fig. 3) and also after 24 h at pH 6, no loss of catecholates 2 and 3 from the surfaces was detected, confirming the remarkable stability of these coatings in aqueous media.

PEG-triscatecholates 7 and 10 were immobilized on TiO2 and stainless steel (both materials of high clinical relevance). The immobilization was performed following a dip-and-rinse protocol in MOPS-buffer under cloud-point-conditions as described in the literature [49,53]. The immobilization of triscatecholates 7 and 10 on TiO2 films was followed with standard surface analyses techniques such as ellipsometry and contact angle measurement. The results were in agreement with the formation of molecular monolayers upon immobilization of catecholates 1–3 confirming findings of other groups with N-acylated derivatives of dopamine [29,31,35,45,47,48,50]. After immobilization of triscatecholate 10 on TiO2 , an adlayer thickness of 2.1 nm was observed by ellipsometry as depicted in Fig. 4 (left gray column), which is in good agreement with reported data on similar PEG layers on TiO2 [51]. The resulting PEG-coated TiO2 -plates were subsequently evaluated with respect to their antifouling properties. TiO2 surfaces resistant to biofilm formation are particularly valuable for the design of dental implants preventing microbial infections (periimplantitis) as a common clinical problem [76]. To evaluate the properties of our PEG-triscatecholates for this application, we focused first on the antifouling efficiency of the coatings in human blood and then on the effect of the coated material on the growth of bone cells. The latter factor is important for a good integration of implants into living bone, which one might expect to be impaired by the presence of antifouling coatings in close vicinity to the bone. For the first assay, TiO2 -plates were coated with PEGtriscatecholate 10 on one half of the plate by the standard dip-and-rinse protocol. The resulting plates (50% PEG-coated) were immersed in 1 mL 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution for 2 h and subsequently incubated in fresh human blood for 1 h and 12 h at 37 ◦ C. The TiO2 plates were extensively rinsed with PBS buffer and water. After drying in a stream of compressed air, the adlayer thickness on the plates was analyzed by ellipsometry. As depicted in Fig. 4, the adlayer thickness on TiO2 films upon exposure to human blood is strongly

3.2. Synthesis of PEG-triscatecholates PEG-triscatecholate 7 was synthesized according to Scheme 1 in four steps from trisalkylmethyl scaffold 4, which is readily available in gram quantities [63]. The free amine in 4 was acylated with pentynoic acid via NHS-ester coupling to give the alkyne 5. Acidic cleavage of the tert-butyl ester and subsequent conjugation to three equivalents of dopamine with EDC/HOBt gave triscatecholate 6. A following copper catalyzed [3 + 2]-cycloaddition of the alkyne moiety with commercial PEG-N3 (5 kDa) gave the PEG-triscatecholate 7 in quantitative yield [70–73]. The corresponding adamantyl derivative 10 was prepared in a similar sequence starting from

3.3. Evaluation of antifouling surfaces

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Fig. 5. Biological evaluation of TiO2 slides, 50% coated with PEG-triscatecholate 10: antifouling assay with human blood and incubation with bone marrow stem cells. Image (A) image of transparent TiO2 films on a glass slide after incubation of bone marrow stem cells for 30 days and (B) fluorescence image of a TiO2 slide after incubation with human blood (2 h) and acridine staining.

dependent on surface coating. On non-coated TiO2 (black columns), a regular increase of adlayer thickness with incubation time was observed leading to a value of 3 nm after 12 h incubation time, most likely reflecting the adhesion of biomolecules to the surface. In contrast, the adlayer thickness of TiO2 coated with PEG-triscatecholate 10 (gray columns) remained almost constant around 2 nm upon treatment with human blood even after 12 h of incubation. The results from the ellipsometry measurements cannot rule out, that the constant adlayer of ∼2 nm seen for the PEG-coated surfaces is just a consequence of a replacement of PEG-triscatecholate 10 with biomolecules amounting by chance to the same adlayer. In a second experiment, we have thus evaluated the presence of biomolecules on the TiO2 plates by fluorescence staining. For this assay, the TiO2 plates (on one half coated with PEG-triscatecholate 10) were again incubated with blood for 2 h, washed with PBS buffer and then stained with acridine orange for 10 min at room temperature. After washing with water and PBS buffer, the plates were analyzed by fluorescence microscopy. A resulting image is shown in Fig. 5(B) and shows a high level of non-specific adsorption of

biomaterial to the uncoated TiO2 surface, whereas the PEG-coated area was almost free of biomaterial. In a second assay, TiO2 -plates were incubated with bone marrow stem cells for four weeks. In this case, the analysis of the surfaces was done by standard light microscopy and therefore transparent TiO2 -films on glass slides were used. Again 50% of the slides were coated with PEG-triscatecholates 7 and 10. After incubation with the stem cells, a clear dividing line was observed between the coated and the uncoated half of the slide as depicted in image (A) in Fig. 5. As expected, the PEG-coated area on the left of the slide is not covered with cells, whereas the uncoated right half is densely populated by growing cells. Notably, no toxicity of the coating (no cell-repelling effect at the coatings boundary) was observed. It is thus unlikely that the coatings would have a negative impact on osseointegration of neighboring uncoated TiO2 -areas in implants. Stainless steel is an important material for clinical hygiene, because a number of instruments and mountings are composed of this material. In a third assay, we have thus evaluated the antifouling properties of PEG-triscatecholate 10 on small slides of stainless

Fig. 6. Antifouling assay on steel slides. Top line: incubation of steel slide (2 cm diameter) coated with PEG-triscatecholate 10 with Staphylococcus epidermidis, followed by washing and plating onto agar; bottom line: incubation of uncoated steel slide (2 cm diameter) with S. epidermidis, followed by washing and plating onto agar. The area of bacterial growth is enlarged on the right.

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Scheme 1. Synthesis of PEG-triscatecholates 7, 10 and 13. EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt, hydroxybenzotriazole; TFA, trifluoroacetic acid; DMSO, dimethylsulfoxide; NHS, N-hydroxysuccinimide; DMAP, 4-(dimethylamino)-pyridine.

steel (2 cm in diameter) by incubation with S. epidermidis (skin colonizing bacteria). In a comparative experiment, uncoated slides and slides coated with PEG-triscatecholate 10 were incubated with bacteria, washed and then plated onto a petri dish containing agar. After additional incubation overnight, images were taken. A clear antifouling effect of the PEG-triscatecholate 10 is again observed on these images (Fig. 6), with almost no bacteria colonizing on the PEG-coated steel slide. The same effect was observed with PEG-triscatecholate 13 on stainless steel plates (see supporting information). In contrast to 10, compound 13 was prepared using a copper-free route. Since both compounds showed identical antifouling properties, the observed effect is most likely not a consequence of possible copper traces from click-functionalization of 10.

Acknowledgments We acknowledge analytical support from Dipl.-Chem. Alexander Rein and helpful discussions with Prof. Dr. Bernd Smarsly, Dipl.-Biol. Christian Zimmermann and Prof. Dr. Silvia Rudloff. TiO2 slides were kindly provided by Dipl.-Chem. Michael Schröder and Dipl.-Chem. Christoph Weidmann. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. colsurfb.2014.02.022. References

4. Conclusions We have described the synthesis of novel non-peptidic trimeric catecholates based on central 3 + 1-scaffolds either derived from adamantane or a trisalkylmethyl core. These trimeric catecholates have been used as anchor groups for immobilization of effector molecules on TiO2 and stainless steel. The resulting surface coatings are remarkably stable in aqueous media even under slightly acidic pH. The obtained trimeric catecholates have been conjugated to PEG for antifouling applications. In three independent assays, a high antifouling capacity has been found for TiO2 and stainless steel coated with our PEG-triscatecholate 10. Trimeric catecholates such as 7, 10 and 13 may thus find applications in implant engineering and for antifouling coatings of instruments and mountings relevant for clinical hygiene.

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