Bioconjugate Chem. 2006, 17, 21−28
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Generic Bioaffinity Silicone Surfaces Hong Chen,†,‡,§ Michael A. Brook,*.‡ Heather D. Sheardown,*,† Yang Chen,‡ and Bettina Klenkler† Department of Chemical Engineering and Department of Chemistry, McMaster University, 1280 Main Street W., Hamilton ON Canada, L8S 4M1. Received June 18, 2005; Revised Manuscript Received October 17, 2005
Synthetic polymer surfaces require surface modification to improve biocompatibility. A generic route to biocompatible silicone elastomers is described involving high yield surface functionalization of standard silicones with hydrosilanes, hydrosilylation using asymmetric, allyl-, NSC-terminated PEO of narrow molecular weight, and covalent modification in one step with amine-containing biological molecules including oligopeptides (YIGSR, RGDS), proteins (EGF, albumin, fibrinogen, mucin), and glycosaminoglycans (heparin). Efficient, high-density binding (e.g., 0.2 EGF molecules/nm2) was demonstrated using radiolabeling studies. The resulting surfaces were demonstrated to be biocompatible by further reaction with biomolecules, for example, thrombosis suppression on surfaces modified by heparin + ATIII, and the formation of confluent corneal epithelial cell layers on EGF, RGDS, or YIGSR surfaces.
INTRODUCTION When synthetic biomaterials are implanted, they are met with a complex and aggressive biological system that ultimately passivates the material or creates a fibrotic capsule, essentially walling the material off from the system. Various synthetic strategies have made impressive inroads to the problems of preparing compatible biomaterials (1). One promising approach exploits the plasma polymerization of hydrophilic monomers such as alkylamines or tetraglyme onto an existing polymer surface (2-4). However, the most general and powerful methods (5) involve the formation of layers of hydrophilic polymers on the surface, of which oligo- (6-8) and poly(ethylene glycol) (9-12) are exemplary. The polymers either bloom from polymer blends to an aqueous interface or are covalently grafted onto an activated polymer surface (13, 14). While promising, it is clear that more biocompatible surfaces can be produced when constituents of the local biology are harnessed to “bioactivate” or “biopassivate” the surface (15), either alone or in combination with hydrophilic polymers. Such approaches include modification with amino acids, cell adhesion peptides, proteins, and glycosaminoglycans. These materials have traditionally been tethered at multiple sites, reducing the mobility of the linking chain. The specific spacing of the tethered biomolecules from the polymer interface is not normally controllable. Silicone polymers offer many advantages as biocompatible supports, including their very high oxygen transmissibility and the ease with which a variety of different substrates can be conformally coated using several chemically different crosslinking processes. Silicones suffer, however, from an extremely high surface hydrophobicity to which biomolecules readily adhere (16, 17) generally resulting, in the case of proteins, in the subsequent mediation of biological reactions (15, 18). Herein we describe the development of a flexible, narrow molecular weight range, asymmetric linker that provides a facile route to convert hydrophobic silicones into activated ester-terminated, * To whom correspondence should be addressed. E-mail: mabrook@ mcmaster.ca,
[email protected]. † Department of Chemical Engineering. ‡ Department of Chemistry. § Current address: Wuhan University of Technology, Institute of Materials Science and Engineering, 133 Luoshird, 430070 Wuhan, PR China.
PEO-modified surfaces. These surfaces react effectively with alcohols and particularly amines and thus serve as key intermediates in the preparation of saccharide-, amino acid-, peptide-, and nucleotide-modified surfaces. We report on the preparation of high density films on silicone of biomolecules including the cell adhesion peptides, RGDS and YIGSR, proteins (EGF, lysozyme, albumin, fibrinogen, and mucin), and the glycosaminoglycan heparin, to which ATIII was complexed. The resulting surfaces are thus tailored to be selectively repellent or adherent to biomolecules and, as a result, biocompatible in a variety of environments.
EXPERIMENTAL PROCEDURES Reagents. Poly(ethylene glycol) monoallyl ether (average MW 500) was obtained as a gift from JuTian Chemical Co. (Nanjing, China). It was dried by azeotropic distillation with toluene before use. N,N′-Disuccinimidyl carbonate, o-xylene (97%, anhydrous), triethylamine (99%), acetonitrile (99%, anhydrous), Karstedt’s Pt catalyst (2-3 wt % in xylene, [(Pt)2(H2CdCH-SiMe2OSiMe2CHdCH2)3]), 2-mercaptoethanol, and CF3SO3H were purchased from Aldrich Chemical Co. Sylgard 184 (a platinum cured silicone rubber H2CdCH-silicone + HSi-silicone f silicone-CH2CH2Si-silicone) and DC1107 (MeHSiO)n were purchased from Dow Corning (Midland, MI). Human serum albumin (HSA), Tyr-Ile-Gly-Ser-Arg (YIGSR), Arg-Gly-Asp-Ser (RGDS), and Sephadex G-25 columns were obtained from Sigma. Epidermal growth factor (EGF) was obtained from Research Diagnostics Incorporated. Fibrinogen was obtained from Enyzme Research Laboratories. Toluene was dried by refluxing over Na prior to distillation, and MeOH was dried by refluxing over Mg and was distilled before use. Materials Characterization. 1H and 13C NMR spectra were recorded at 30 °C on a Bruker AC-200 spectrometer (at 200 and 50.3 MHz for 1H and 13C, respectively). Attenuated total reflection Fourier transform IR spectroscopy (ATR-FTIR) measurements were carried out on a Bruker VECTOR 22 Fourier transform infrared spectrometer (Bruker Instruments, Billerica, MA) equipped with a Harrick ATR accessory MUP with GeS crystal; 200 scans were collected for each sample. Electrospray mass spectra (ESI-MS) were recorded on a Micromass Quattro Ultima LC, triple quadruple MS. Water Contact Angle. Advancing and receding sessile drop contact angles were measured on PEO-grafted surfaces using
10.1021/bc050174b CCC: $33.50 © 2006 American Chemical Society Published on Web 12/23/2005
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Scheme 1: Synthesis of 3 and NMR Assignments of 2
Rame´ Hart NRL C.A. goniometer (Mountain Lakes, NJ). Milli-Q water (18 MΩ/cm) was used with a drop volume of approximately 0.02 mL. Results are presented as an average of 18 measurements or more on at least three different surfaces. Contact angles were also measured using the captive bubble method, where an air bubble was injected from a syringe onto an inverted sample surface immersed into Milli-Q water. Results are presented as the average of at least 10 measurements on three different surfaces. X-ray photoelectron spectroscopy (XPS) was performed at Surface Interface Ontario, University of Toronto, using a Leybold Max 200 X-ray photoelectron spectrometer with a MgK-R nonmonochromatic X-ray source. Preparation of N-Succinimidyl Carbonate PEG-Grafted PDMS Surfaces. Synthesis of R-Allyl-ω-N-succinimidyl Carbonate-Poly(ethylene glycol), 2. To a solution of poly(ethylene glycol) monoallyl ether (2.0 g, 4.0 mmol) and triethylamine (1.62 g, 16 mmol) in CH3CN (10 mL) was added N,N′disuccinimidyl carbonate (4.1 g, 16 mmol). The mixture was allowed to stir at room temperature over 10 h under N2. After removal of the solvent in vacuo, the residue was dissolved in dry toluene (25 mL), and the solution was cooled to 0 °C. A pale brown precipitate was filtered off. The toluene was removed under reduced pressure. This procedure was repeated three times. The resultant compound 2 was a yellow oil (1.2 g, 60% yield). IR (neat): 1739 (NCdO), 1788 (OCdO). 1H NMR (200.2 MHz, CDCl3, Scheme 1, see also Suppporting Information): δ 2.78 (s, 4H, OdCCH2CH2CdO), 3.57 (bs, 40H, PEO’s OCH2), 3.72 (bs, 2H, OCH2CH2OCdO), 3.95 (d, 2H, J ) 5.6 Hz, CH2d CHCH2O), 4.39 (m, 2H, OCH2CH2OCdO), 5.20 (m, 2H, CH2d CHCH2O), 5.82 (m, 1H, CH2dCHCH2O) ppm. 13C NMR (50.3 MHz, CDCl3): δ 25.2 (OdCCH2CH2CdO), 68.1 (OCH2CH2OCdO), 69.2 (OdCOCH2CH2O), 70.4 (PEG’s OCH2), 72.0 (CH2dCHCH2O), 117.0 (CH2dCHCH2O), 134.5 (CH2d CHCH2O), 151.4 (OCdOO), 168.5 (NCdOCH2) ppm. MS (ESI): m/z ) 745.6 (M + NH4+, n ) 12 100). Elastomer Preparation. Silicone elastomers were prepared according to the procedure provided by Dow Corning. Sylgard 184 PDMS prepolymer and catalyst was mixed thoroughly with its cross-linker in a 10:1 ratio (w/w) in a plate mold and degassed
under vacuum. Films were allowed to cure at room temperature for 48 h. After being cured, the silicone elastomer films were punched into disks, approximately 5 mm in diameter and 0.5 mm thick. The disks were washed with hexane and then dried under vacuum for further use. Si-H Surface Functionalization 1. For Si-H functionalization of the surface, 20 silicone elastomer disks were immersed in a mixture of DC1107 (3 mL) and methanol (5 mL). To this was added F3CSO3H (0.02 mL, 0.26 mmol). After being stirred at room temperature for 30 min, the functionalized surfaces were rinsed with methanol and hexane and dried under vacuum (for surface characterization, see below). Addition of PEG Derivative, 3. Si-H modified silicone surfaces 1 were incubated in a solution of 2-methoxyethyl ether solvent and 2 (80:20 wt %:wt %, 3 mL). Pt-catalyst (platinumdivinyltetramethyldisiloxane complex, 1 drop) was added, and the mixture was stirred for 15 h at room temperature. Following modification, the PEG-modified surfaces 3 were washed thoroughly with dry acetone and dried under vacuum. Characterization of NSC and Modified Surfaces. ATRFTIR. As described above, N,N′-disuccinimidyl carbonate was used to activate the hydroxy-terminal of R-allyl-ω-poly(ethylene glycol). The desired compound 2 was characterized by 1H NMR, with the resonance of the CH2CH2 on the NHS (2.78 ppm) being particularly diagnostic. Two types of CdO groups were observed on the NSC-activated termini (O-C(O)-O and imide) by 13C NMR (168.8 and 151.7 ppm, respectively). Assignments of the FT-IR spectrum of the NSC-activated PEO are shown in Figure 1. The band at 1739 cm-1, representing the CdO stretch of the NHS group, can be further used to diagnose the succinimidyl carbonate PEG grafting process. H-Si-functionalized silicone surfaces 1 were obtained by acid-catalyzed equilibration of a silicone elastomer in the presence of (MeHSiO)n as noted above. The ATR-FTIR spectra of the resulting surfaces exhibited a band at 2166 cm-1 due to the Si-H stretch. Succinimidyl carbonate PEO 2 was grafted onto the silicone rubber surfaces via a hydrosilylation reaction with the H-Si groups. In the ATR-FTIR spectrum of the succinimidyl carbonate PEG-grafted surfaces 3, the band at 2166 cm-1 due to H-Si
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Figure 1. FT-IR spectra of (a) PDMS, (b) Si-H-modified PDMS, (c) succinimidyl carbonate PEO-modified PDMS surfaces 3, (d) RGDmodified 4, and (e) YIGSR-modified 5 PDMS surfaces, respectively. Table 1. XPS Surface Analysis of Unmodified PDMS, Succinimidyl Carbonate PEO-Modified 3, RGDS-PEO-Modified 4, and YIGSR-PEO-Modified 5 PDMS Surfaces elemental at. %
control PDMS
NSC-PEO 3
RGDS-PEO 4
YIGSR-PEO 5
C1s N1s O1s Si2p
46 0 26.5 27.4
52.2 0.9 26.3 20.6
54.6 1.9 25.2 18.4
55.4 2.7 26.5 15.5
was no longer visible following the reaction. There were two CdO stretches at 1741 and 1789 cm-1, respectively, which were assigned to the CdO groups at the succinimidyl carbonate termini, and the O-C(O)-O linkage, which was also present in the starting material. The PEO CH2 scissoring band at 1454 cm-1, the antisymmetric stretch mode of the CH2-O-CH2 chain at 1351 cm-1, and the symmetric stretch mode of the CH2-O-CH2 chain at 1258 cm-1 indicated the presence of PEO chains on the resulting surface. XPS. NSC-PEO binding to the surface 3 was further confirmed by the presence of an N1s signal in the XPS survey scan due to the amine groups in the NSC-PEO polymer (Table 1, see Supporting Information). The C1s high-resolution spectrum shows a distinct peak at 286.4 eV which corresponds to the C-C-O bond in PEO repeat unit. Conjugation of Various Molecules to the NSC-Modified Surface. Peptide Conjugation. The covalent conjugation of peptides to the functionalized surfaces was carried out in a phosphate-buffered saline (PBS) buffer solution (pH 7.5). The N-succinimidyl carbonate PEG-grafted surfaces 3 were immersed in PBS buffer containing the peptide RGDS or YIGSR, (10 µg/mL) for 12 h to give 4 or 5, respectively. Washing Protocol. All samples were rinsed three times with PBS for 10 min, for a total of 30 min, and the surfaces were dried under vacuum. For radioactive samples, residual adherent buffer was removed by wicking onto filter paper and transferred to clean tubes, and the surface radioactivity was determined by
counting using a gamma counter. Radioactivity counts were converted to surface protein concentrations. One milliliter of a 2% sodium dodecyl sulfate (SDS) solution was then added to each tube and left at room temperature for 4 h and overnight at 4 °C. After three PBS rinses, the surfaces were transferred to clean tubes and radioactivity was measured to determine the levels of the protein remaining after the SDS treatment, which indicated that the growth factor was covalently immobilized to the surface (Table 2). Characterization. The IR spectra of modified surfaces 3-5, respectively, are presented in Figure 1 (see also Supporting Information). Distinct bands at 1652 and 1656 cm-1 due to amides were observed on both the RGDS- and YIGSR-modified surfaces: the CdO stretch mode at 1741 cm-1, due to the NHS group, disappeared in both cases following modification. These spectral changes indicated the coupling of the succinimidyl carbonate PEG to the peptides. Peptide immobilization was further demonstrated by an increase in the XPS N1s signal to 1.9 and 2.7% for RGDS and YIGSR, respectively, due to the amine groups in the peptides and a decrease in the Si2p signal (Table 1, see Supporting Information). Note that the starting succinimidyl carbonate PEO-modified PDMS 3 showed a very weak nitrogen peak due to the presence of a single nitrogen in NHS. The nitrogen intensity, postmodification, was much higher. EGF Conjugation 6. EGF was first labeled with 125I (ICN Pharmaceuticals, Irvine CA) using the iodogen method. The N-succinimidyl carbonate PEG-grafted surface 3 was immersed in a PBS buffer (pH 7.4) containing radiolabeled EGF (10 µg/ mL) for 2 and 24 h and rinsed three times with PBS for 10 min each (Table 2). Human Serum Albumin Conjugation 7. Human serum albumin was labeled with 125I (ICN Pharmaceuticals, Irvine CA) using the ICl method. The labeled protein was passed through an AG 1-X4 column (Bio-Rad Laboratories, Hercules, CA) to remove any free iodide. For measurement of nonspecific adsorption of protein from buffer and covalent coupling of albumin to the surfaces, a mixture of labeled and unlabeled protein (1:20) at a total concentration of 1 mg/mL was prepared. NSC-PEO-modified surfaces 3 were incubated with albumin for 2 h at room temperature, rinsed three times with PBS for 10 min, (250 µL per rinse per disk, 30 min total). The modification of surfaces by albumin, via adsorption or covalent grafting, is shown in Figure 3B. Figure 3D also shows the results of fibrinogen adsorption on the albumin pretreated surfaces. Fibrinogen Adsorption 11. Fibrinogen was labeled with 131I (ICN Pharmaceuticals, Irvine CA) using the ICl method. The labeled protein was passed through an AG 1-X4 column (BioRad Laboratories, Hercules, CA) to remove any free iodide. The untreated control (PDMS elastomer) surface, 125I-albuminpretreated control surface, and 125I-albumin-pretreated NSCPEG-modified surfaces 7 were incubated in PBS solution containing the radiolabeled fibrinogen at a concentration of 1
Table 2. Efficiency of Protein Binding to 3 onto control surfaces
YIGSR EGF albumin fibrinogen lysozyme heparin a
onto 3
before washing with SDS ng/cm2
after washing with SDS ng/cm2
before washing with SDS ng/cm2
after washing with SDS ng/cm2 (surface)
n/a 116 220 580 200
n/a 26 50 200a 40
19 190 180
n/a (5) 180 (6) 170 (7) 45 (11) 402 (9) 680 (10)
460
Adsorption on to albumin-coated control surface; i.e., 220 ng/cm2 is displaced by 200 ng/cm2 fibrinogen.
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Figure 2. Water contact angle of control, RGDS 4-, YIGSR 5-, and heparin 10-modified surfaces.
mg/mL for 2 h. The fibrinogen amounts on various surfaces were determined radioactively as described above (Figure 3D). Conjugation of Mucin 8. NSC surfaces 3 were incubated in 5 mg/mL solution of mucin from bovine (submaxillary glands, Type I-S, Sigma) in PBS buffer (pH ) 8.0) for 6 h. Surfaces were subsequently rinsed three times with fresh PBS. Conjugation of Lysozyme 9, 12. Lysozyme adsorption and conjugation to various surfaces was carried out in a phosphatebuffered saline (PBS, pH 7.4). Lysozyme was labeled with 125I (ICN Pharmaceuticals, Irvine CA) using the ICl method. The N-succinimidyl carbonate PEG-grafted surface 3, PEG350grafted surface, mucin-modified surface 8, and control surface, respectively, were immersed in a PBS buffer (pH 7.4) containing (unlabeled:radiolabeled ) 9:1) lysozyme (1 mg/mL) for 3 h and rinsed three times with PBS (Table 2).
Chen et al.
Heparin Conjugation 10. NSC surfaces 3 were incubated in 10 mg/mL solution of heparin (Sigma Aldrich) in PBS buffer (pH ) 8.0) for 6 h. Surfaces were subsequently rinsed three times with fresh PBS. The density of heparin on the NSC surface 10 was 0.68 µg/cm2, as shown by a toludine blue assay (see next section). For the assay, a series of heparin standard solutions with concentrations varying from 0 to 20 µg/mL were prepared by diluting a stock solution. The stock solution was obtained by dissolving 10 mg heparin in an aqueous 0.2 wt % NaCl solution. Toluidine blue (Sigma-Aldrich Canada, 50 mg) was dissolved in HCl (1 mL, 0.01 N solution), in which 0.2 wt % NaCl had been previously added and dissolved. The 50 mg/mL toluidine blue solution was diluted with deionized water to give a 0.005 mg/mL (0.0005%) solution. The solution (1.0 mL) was added to a 5 mL tube, followed by 0.1 mL of the above heparin 10 mg/mL standard solution. The mixed solution was vortexed for 30 s. n-Hexane (Aldrich-Sigma Canada, 1 mL) was added, and the solution was vigorously mixed for 30 s and then allowed to separate into two phases over 5 min. The heparin-toluidine blue complex was extracted into the upper transparent organic layer. After the organic layer was removed, the absorbance of the aqueous layer at 631 nm was measured on a Beckman DU640UV/VIS spectrophotometer. A linear standard calibration curve was obtained by plotting absorbance at 631 nm versus concentration of heparin in the aqueous NaCl solution. The amount of heparin immobilized on the polymer surfaces was determined using this calibration curve. Prior to testing, polymer samples were incubated in 0.05 M Tris-buffered saline (TBS) with pH 7.4 at room-temperature overnight to hydrate the surfaces. For each experiment, 0.1 mL of 0.2% NaCl solution and 1.0 mL of 0.005 mg/mL (0.0005%) toluidine blue solution were mixed in a 5 mL polypropylene test tube. The heparin-modified (polymer) surfaces 10 (0.77 cm2
Figure 3. A: Binding EGF to the surfaces: PDMS ) control, PDMS-PEO-NSC f 6. B: Covalent and physical association of albumin on PDMS and PDMS-PEO-NSC surfaces f 7, including the effect of SDS washing. C: Adsorption of lysozyme with PDMS (control), PDMSPEO-NSC f 9, PDMS-PEO-MeO and PDMS-PEO-mucin f 12 surfaces before and after SDS exposure. D: Adsorption of fibrinogen (Fg) onto albumin-coated control or onto 7 giving 11.
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Figure 4. A: Ability of thrombin to process N-p-tosyl-gly-pro-arg p-nitroanilide under various conditions. B: Thrombin inactivation by AT bound to heparin surface 10 and versus AT directly bound to 3.
Figure 5. Human corneal epithelial cells (HCEC) grown on A: control PDMS, B: PDMS-PEO-NSC 3, C: RGDS 4, D: YIGSR 5, and E: EGF 6-modified surfaces (5 days).
area) were immersed in the solution, which was vortexed for 30 s. Then, 1 mL n-hexane was added and shaken well. The mixture was allowed to phase-separate for 5 min after removal of the surfaces. As above, the upper organic layer was removed and the absorbance of the aqueous layer at 631 nm was investigated on a Beckman DU640 UV/vis spectrophotometer. The density of total heparin immobilized on the surfaces was calculated from the above calibration curve. For each surface, the heparin density was expressed by mass per unit surface area (µg/cm2). Thromboresistant Properties. Platelin was obtained from Organon Teknila Corp., Durham, NC (No. 35501). TBS/Ca2+/ Platelin (0.1 M CaCl2 with a 1:10 dilution of platelin) buffer solution was made by dissolving CaCl2 (1.11 g) and four standard vials of Platelin in 10 mL of Milli-Q water (10 mL). The volume was then brought to 100 mL with TBS (0.05 M, pH ) 7.4). Thrombin substrate N-p-tosyl-gly-pro-arg p-nitroanilide (Sigma-Aldrich) (5 mg) was dissolved in TBS (10 mL) to give a solution with a final concentration of 0.5 mg/mL. To passivate the walls of the 96-well microtitration plate, the wells were exposed to human serum albumin in TBS (10 mg/ mL) overnight at 4 °C. The albumin solution was then withdrawn from the wells, and the wells were aspirated and washed three times with fresh TBS (0.3 mL/well/time) before adding the unmodified and heparin-modified silicone surfaces. The heparin-modified surface 10 was incubated in antithrombin III in TBS buffer solution (0.25 mg/mL) for 30 min before testing. The disks were placed vertically in the wells, and 10% diluted pooled human citrated plasma (200 µL) was added to the wells. After the plate was warmed to 37 °C, TBS/Ca2+/platelin buffer solution (20 µL) and thrombin substrate (30 µL of 0.5 mg/mL) were added. The release of p-nitroaniline by thrombin was measured as a function of time by recording the optical density at 405 nm and 37 °C using a UV-vis plate reader (Figure 4).
Cell Culture on Peptide-Modified-Surfaces. Surfaces (∼5 mm disks) 4 or 5 as well as controls were washed three times with PBS supplemented with antibiotics (penicillin, streptomycin, and gentamycin) and subsequently stored overnight at 4 °C in Keratinocyte Serum Free Medium (KSFM, Invitrogen, Grand Island, NY) medium containing antibiotics. Under sterile conditions, the surfaces were transferred to a 24-well plate and plated with human corneal epithelial cells (HCECs, 104 cells per well) in KSFM supplemented with penicillin, streptomycin, gentamycin, and EGF. Surfaces 6 were cultured under identical conditions except the cells were grown in medium only; no exogenous EGF or antibiotics were added. The cells were cultured at 37 °C in a 5% CO2 atmosphere. Samples were imaged regularly. All images were taken at 100× magnification (Figure 5). Epithelial cell morphology was confirmed by AE5 staining.
RESULTS Unlike most polymers, silicones can be readily formed and degraded under thermodynamic control (19). Thus, treatment of monomers and/or polymers with endcapping molecules in the presence of acid or base allows the preparation of homo- or copolymers of various molecular weights. By carefully controlling the swelling conditions using relatively poor solvents for silicone, such as methanol, it was possible to preferentially introduce Si-H functional groups at the surfaces of a variety of pre-cured silicone elastomer by a redistribution polymerization with triflic acid (20), giving 1, as shown by the characteristic strong IR absorption at 2166 cm-1 (Scheme 1, Figure 1, see also Supporting Information). The Si-H group underwent efficient hydrosilylation with a series of olefins, including allylPEO (13, 14) and, more importantly, allyl-PEO-NSC 2, prepared by the reaction of allyl-PEO-OH with bis-N-hydroxysuccinimidyl carbonate to give a high density, reactive NSC
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Scheme 2: Generic Route to Biomolecule Attachment to Silicone Surfaces
surface 3. Surface properties (ATR-IR, Figure 1, Table 1, XPS, see also Suporting Information) were determined using traditional methods. It should be noted that, unlike model systems prepared on flat gold films (21), these functional surfaces can be prepared from any dimethylsilicone elastomer, and on objects with both simple and complex shapes. Surface 3 was modified, in phosphate-buffered saline (PBS) solutions (pH ) 8.0), with a series of peptides and proteins, respectively, including the cell adhesion peptides Arg-Gly-AspSer 4 (RGDS)and Tyr-Ile-Gly-Ser-Arg 5 (YIGSR), epidermal growth factor 6 (EGF), human serum albumin 7 (HSA), mucin 8, lysozyme 9, and the glycosaminoglycan heparin 10. Selected modified surfaces were further exposed to additional proteins. Thus, the HSA surface 7 was modified with fibrinogen 11, a mucin-modified surface 8 was treated with lysozyme 12, and heparin surface 10 was exposed to antithrombin III 13 (Scheme 2). The biologically modified silicones were characterized by IR, XPS, and contact angle (Figure 1, Figure 2, Table 1, see also Supporting Information). The total quantities of the surface linked and adsorbed peptides or proteins were determined by radioactivity assays before and after exhaustively washing the modified surface with sodium dodecyl sulfate (SDS) (Table 2). This method also provided a minimum estimate of the total graft density of the surface. For example, after 24 h of reaction of surface 3 with EGF, the resulting surface 6 exhibited a surface concentration of 190 ng/cm2 (ca. 0.2 EGF molecules/nm2): after washing with SDS, the control surface showed only 26 ng/cm2 while the EGF-g-PEO surface was essentially unchanged (Figure 3A). The smaller molecule YIGSR was found at a surface concentration of 19 ng/cm2, also about 0.2 molecules/nm2. Larger molecules, such as lysozyme (MW ca. 14400), also efficiently grafted, 402 ng/cm2 (0.15 molecules/nm2), to the surface to give 9 after extensive washing with SDS. Similarly, heparin bound to the surface 10 with a graft density of 0.68 µg/cm2 (see below). The silicone surfaces are not molecularly flat, and thus it is not possible make direct comparison of EO surface packing on these silicones with model systems, such as planar selfassembled monolayer-modified gold surfaces (21). The crosssectional area of an anhydrous EO oligomer when coiled in a helix is on the order of 0.2 nm2. Thus, an ideal PEO brush, rarely achieved with the exception of self-assembled monolayers on noble metal surfaces (9), could pack about 20-25 anhydrous EO chains/nm2 (22). Water-swollen PEO chains occupy significantly more space (23). We did not directly assess the packing density of PEO on these surfaces. . However, as noted
above, approximately 0.2 molecules/nm2 of peptides and EGF, and 0.15 molecules/nm2 of the larger lysozyme, were bound to the surface, surface densities which approach a monolayer (the diameter of lysozyme is approximately 3.0 nm (24)). These molecules cast a larger shadow on the surface than a PEO helix, and therefore efficiency of surface binding is more dependent on the size of the biomolecules to be tethered than on the PEO density: multiple attachments to the surface are anticipated. We note that the ability to tether biomolecules in high density in active form to rough, instead of smooth model, surfaces is a major advantage of the method: any silicone surface can be functionalized. Albumin, the most abundant protein in blood, can be used to passivate implanted synthetic surfaces (25). Less protein was initially found on the NSC-modified surface 7 than on the control (0.22 vs 0.18 µg/cm2, Figure 3B). However, the control surface was mostly washed free of the 125I-labeled protein with SDS (0.05 µg/cm2 remained), while more than 90%, 0.17 µg/ cm2, remained on the functionalized surface. These data are consistent with initial, patchy, protein physisorption that changed to chemisorption 7 before the protein could migrate across the NSC surface to form a coherent monolayer. That is, the albumin binds on contact, leaving a noncoherent film and accessible interstitial areas. Attempts to form a coherent albumin film prior to covalent linkage, by controlling the rate of surface binding, have not so far been successful. The ability to displace the albumin by 131I-labeled fibrinogen, from control and 7 surfaces, respectively, was examined. Fibrinogen adsorbed effectively to the control surface (0.58 µg/ cm2). However, much less fibrinogen was able to contact and bind to the albumin adsorbed surface (0.20 µg/cm2), and even less was found on the albumin-passivated NSC surface 11, Figure 3D (0.045 µg/cm2). This is consistent with a control surface in which albumin can be “nudged aside” by fibrinogen, but a covalently linked surface 7 in which only a few interstitial spaces are sufficiently large to accommodate the large fibrinogen molecule (340 kD) giving 11. Lysozyme, one of the proteins responsible for ophthalmic disinfection, was exposed to a variety of modified silicones. Significantly more lysozyme was adsorbed to the pregrafted mucin 8 (post SDS, 423 ng/cm2) and NSC surfaces 3 (post SDS, 402 ng/cm2) than to the control silicone (post SDS, 42 ng/cm2) or simple methoxy PEO-modified (post SDS, 0.3 ng/cm2) silicone surfaces (Figure 3C) (13, 26, 27). The natural surface with which lysozyme interacts in the eye is mucin (28). Thus, this modified polymer may prove useful as a model system to examine surface fouling by lysozyme in ophthalmic applications.
Generic Bioaffinity Silicone Surfaces
Heparin, a highly sulfonated, anionic polysaccharide that is a well-known antithrombotic agent was analogously grafted to the surface 3 in high density (0.68 µg/cm2) giving 10. The surface was subsequently exposed to thrombin, via the interaction of CaCl2 with plasma, in the presence of the chromogenic substrate N-p-tosyl-gly pro-arg p-nitroanilide. Release of the p-nitroaniline hydrolysis product was followed over 3 h (Figure 4A). Although nitroaniline was formed in the presence of 10, it did so at a significantly lower rate and with a significantly longer half-life than observed with plasma alone, or in the presence of the control silicone surface or the NSC-PEOmodified surface 3. The heparinized surface was demonstrably less thrombogenic than the other surfaces examined and was comparable to those previously described (29). Also of interest was the observation that antithrombin III (ATIII) covalently linked to the heparin surface 10, giving 13, was far more efficient at inhibiting thrombin than ATIII directly bound to the surface (after reaction with 3, Figure 4B). The most important criterion for any biomedical surface is the degree to which it is accepted by the local biological environment. To test the hypothesis that cell-compatible surfaces could be prepared using this method, human corneal epithelial cells (HCEC) were cultured on NSC surfaces modified with the cell adhesion peptides RGDS and YIGSR, 4 and 5, respectively, under serum-free conditions as well as on surfaces modified with EGF cultured under serum-free and exogenous EGF-free conditions. In this case, unlike other studies in which various proteins including EGF and a bovine pituitary extract are added back to the medium, potential exogenous stimulatory effects of adsorbed and soluble proteins in the medium were eliminated. As shown in Figure 5C,D, cells readily adhere to, spread, and mitose on the peptide-modified surfaces 4 and 5, respectively, to give confluent monolayers in less than 96 h. Significantly lower levels of confluence were observed on both the control and on the PEO-modified silicone surfaces (13), respectively (Figure 5A,B; see also Supporting Information). The lack of cell adhesion on the PEO-modified surfaces in particular is not surprising given that PEO has been widely described as cell and protein repellent. In fact, under conditions where there were no exogenous factors present in the medium, the surfaces were completely cell-free after 5 days of culture. Confluent epithelial cell monolayers could be formed on the EGF-modified surfaces 8 as rapidly in as rapidly as 5 days, demonstrating that the tethered EGF was active and able to stimulate the production of extracellular matrix proteins (Figure 5E, see also Supporting Information).
DISCUSSION The NSC group, related to NHS esters, has become one of the ‘workhorse’ functional groups for the attachment of biological molecules to various supports because it is mild and selective for amines over alcohols, and it reacts with both groups faster than with water (30). PEO is a polymer widely exploited by the biomaterials community because of the water-swollen layer it forms that is known, particularly in in vitro assays, to passivate surfaces from adsorption by various biomolecules (8). We chose to combine these two elements in the development of our generic surfaces 3. In this regard, we have borrowed from a key finding of Castner, who described the use of polyfunctional silanes, including NHS groups, for the preparation of well-defined gold surfaces on to which protein absorption could be studied (21). The preparation of NHS surfaces as self-assembled monolayers on gold surfaces are excellent model systems. However, these surfaces are not readily adaptable to complex devices themselves or to coatings on devices composed of other polymers. There are important distinctions between this work and previous reports. First, an advantage of this method is that any
Bioconjugate Chem., Vol. 17, No. 1, 2006 27
silicone elastomer will suffice for the biological surface modification described. Thus, one can exploit the ability of silicone materials to form complex shapes, for example, in various molding applications, or their ability to form conformal films on a variety of substrates. Second, a network is not formed on the surface. Instead, a series of monofunctional PEO chains, of approximately the same length (dependent on the original PEO polydispersity), are grafted onto a silicone. That is, the NSC functional PEO chains act as a brush to which biological molecules can covalently adhere. The concentration of these groups is enhanced by the absence of water in the brush-forming process, such that PEO swelling is reduced (9). These monofunctional species provide a more predictable distribution of functional groups along the surface than can a network system and, of course, the functional groups are only arrayed across the surface XY plane. The generic utility of these modified silicone surfaces 3 to create biologically modified surfaces is amply demonstrated by the wide variety of biomolecules that can be readily grafted to them, in high density (ca.0.1-0.2 molecules/nm2), to give 6-10, and the maintenance of the bioreactivity of the tethered molecules after modification (11-13), which can make the surface compatible and/or render it usefully bioactive. A simple two-step synthetic process, hydrosilylation of NSCPEO onto a Si-H functional silicone and reaction of the NSC group with a biomolecule, permits generic modification of silicone substrates or conformal silicone coatings. The density of groups can be varied as can the nature of groups to facilitate rejection or attraction of available biomolecules. Proteins including enzymes, peptides, or polysaccharides are readily attached in stable form to give biocompatible, PEO-stabilized surfaces.
ACKNOWLEDGMENT We acknowledge with gratitude the financial support by the Natural Sciences and Engineering Research Council of Canada. Supporting Information Available: XPS data for surfaces 3, 4, and 5; IR data for 1-5. This material is available free of charge via the Internet at http://pubs.acs.org.
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