Biomacromolecules 2003, 4, 1072-1079
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Maleic Anhydride CopolymerssA Versatile Platform for Molecular Biosurface Engineering Tilo Pompe, Stefan Zschoche, Nicole Herold, Katrin Salchert, Marie-Francoise Gouzy, Claudia Sperling, and Carsten Werner* Institute of Polymer Research Dresden and The Max Bergmann Center of Biomaterials Dresden, Hohe Str. 6, 01069 Dresden, Germany Received March 12, 2003; Revised Manuscript Received April 8, 2003
A platform of thin polymer coatings was introduced for the functional modulation of immobilized bioactive molecules at solid/liquid interfaces. The approach is based on covalently attached alternating maleic acid anhydride copolymers with a variety of comonomers and extended through conversion of the anhydride moieties by hydrolysis, reaction with functional amines, and other conversions of the anhydride moieties. We demonstrate that these options permit control of the physicochemical constraints for bioactive molecules immobilized at interfaces to influence important performance characteristics of biofunctionalized materials for medical devices and molecular diagnostics. Examples concern the impact of the substrate-anchorage of fibronectin on the formation of cell-matrix adhesions, the orientation of endothelial cells according to lateral anti-adhesive micropatterns using grafted poly(ethylene oxide), and the spacer-dependent activity of immobilized synthetic thrombin inhibitors. Introduction Advanced biomaterials have to reflect the rapidly growing knowledge about the molecular regulation of biointerfacial processes occurring in living matter. This task provokes strategies for the controlled immobilization of biomolecules onto solid materials. Examples include the interfacial binding of the extracellular matrix,1 growth factors,2-4 and coagulation inhibitors.5 Current limits of biomimetic surface engineering often have to be attributed to the insufficient control of the interfacial binding, orientation, and the degree of structural integrity of immobilized bioactive molecules.6-8 Beyond that, multimolecular assemblies affect several key phenomena, e.g., the formation of focal adhesions within the membranes of attached cells or the activation of the kinin cascade system of the blood plasma at solid surfaces9-12 and have to be addressed by surface engineering concepts accordingly. To meet those demands, platform technologies of surface modification are required. Coatings of synthetic polymers are most promising for that purpose because they provide high degrees of chemical and physical versatility.1-6,13-20 Making use of this advantage, we suggest herein a polymer coating strategy to switch the chemical reactivity of solid substrates toward biopolymers and to precisely adjust their physicochemical characteristics: Alternating maleic acid anhydride copolymers21 were deposited as thin films determining a variety of surface parameters through the choice of the comonomer and via conversions of the reactive anhydride moieties. Although earlier works20,22,23 already made use of the high reactivity of maleic anhydrides for * To whom correspondence should be addressed. Phone: +49 351 4658531. Fax: +49 351 4658533. E-mail:
[email protected].
several surface modification concepts, our study utilized maleic anhydride copolymers to combine the gradual variation of physicochemical surface characteristics with specific biomolecular functionalities and lateral constraints independent of the underlying substrate. Materials and Methods Copolymers and Preparation of Thin Films. Models of the molecular structures of the copolymers were calculated using software from Accelrys Inc. (San Diego, CA) with implementation of UNIVERSAL1.02 force field, and graphical displays were printed from Cerius molecular modeling system (Accelrys Inc.). Poly(octadecene-alt-maleic anhydride) MW ) 30 00050 000 (Polysciences Inc., Warrington, PA) was reprecipitated from tetrahydrofuran (Fluka, Deisenhofen, Germany) in hexane (Fluka), intensively washed with hexane for removing olefin impurities, and followed by annealing at 120 °C for 20 h. Poly(propene-alt-maleic anhydride) MW ) 39 000, poly(styrene-alt-maleic anhydride) MW ) 300 000 (both are special products of Leuna-Werke AG, Germany), and poly(ethylene-alt-maleic anhydride) MW ) 125 000 (Aldrich, Munich, Germany) were pretreated in a similar way. For thin film preparation, poly(octadecene-alt-maleic anhydride), poly(styrene-alt-maleic anhydride), and poly(propene-alt-maleic anhydride) were dissolved in THF at concentrations of 0.08%, 0.15%, and 0.1%, respectively, and poly(ethylene-alt-maleic anhydride) in acetone (Acros Organics, Geel, Belgium) was dissolved at a concentration of 0.15%. For preparation of stable thin films, the copolymers were covalently bound to SiO2 surfaces (silicon wafer or glass
10.1021/bm034071c CCC: $25.00 © 2003 American Chemical Society Published on Web 05/02/2003
Maleic Anhydride Copolymers
coverslips). Copolymer solutions were spin-coated (RC5, Suess Microtec, Garching, Germany) or solution-cast on surfaces which had been freshly oxidized in a mixture of aqueous solutions of ammonia (Acros Organics) and hydrogen peroxide (Merck, Darmstadt, Germany) and thereafter surface-modified with 3-aminopropyl-dimethylethoxy-silane (ABCR, Karlsruhe, Germany). Stable covalent binding of the polymer films was achieved by annealing at 120 °C for generation of imide bonds with the amino-silane on the SiO2 substrate. The copolymer films were thoroughly characterized in dry state by ellipsometry (VASE 44M, Woolam, Lincoln, NE; experimentally determined refractive index: 1.50), infrared spectroscopy in attenuated total internal reflection (FS66, Bruker, Ettlingen, Germany), and static advancing contact angle measurements of sessile water droplets (diameter: approximately 2 mm; G40, Kruess, Hamburg, Germany). RMS roughness data were determined from scanning force microscopy in TappingMode (Bioscope, Digital Instruments, Darmstadt, Germany; scan size: 10 µm) according to the equation RMS ) {∑(zi)2/N}1/2. Isoelectric points of the copolymer films were determined with an inhouse built streaming potential/streaming current device (Microslit Electrokinetic Setup24) in aqueous KCl solutions (1mM) of varied pH. For determination of the surface anhydride moieties the copolymer films were immersed in 50 mM aqueous solution of methionine amide hydrochloride (Bachem Biochemica, Heidelberg, Germany) for 1 h and subsequently analyzed by X-ray photoelectron spectroscopy (Escalab II, Visons, Durham, UK) as previously described.22 Elastomeric poly(dimethyl siloxane) (Sylgard 184, Dow Corning, Midland, MI) structures molded from silicon master structures in the micrometer range25 were surface modified to enable the preparation of copolymer surfaces with a wide range of topographical shapes. For that purpose, the PDMSstructures were solution-cast with the copolymer after creating amino functionalities by ammonia plasma treatment.26,27 The ammonia plasma treatment was carried out in a computer controlled MicroSys apparatus (Roth&Rau, Hohenstein-Ernstthal, Germany) at a pressure of 7 × 10-3mbar and microwave power of 600 W for 40 s. Chemical Conversion of Copolymer Films. Hydrolysis of the anhydride moieties of the copolymer film into the diacid form was achieved by autoclaving (120 °C, saturated water vapor, 20 min, autoclave: 2540 EL, Systec, Wettenberg, Germany). Anhydride groups were regenerated upon annealing of the films at 120 °C at dry atmosphere for 1 h. Amino functionalization of the film surface was obtained by dipping the copolymer films in 0.1 M aqueous solution of 1,4-butanediamine (Fluka) for 4 min with subsequent steps of rinsing in deionized water, 0.01 N HCl solution, and deionized water again, and annealing at 120 °C. Poly(ethylene oxide) (PEO) spacer functions for protein immobilization were introduced on the amine modified films by reaction with a mixture of 50 mM N-ethyl-N′-(3dimethylaminopropyl)carbodiimide hydrochloride (Merck) (EDC), 25 mM N-hydroxysulfosuccinimide sodium salt (Fluka) (sulfo-NHS), and 50 mM poly(ethylene oxide) diacid (MW ) 600, Fluka) in 1/15 M phosphate buffer at pH 7.4 for 4 h.
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Immersion of the films in a 10 mM aqueous solution of R-methoxy poly(ethylene oxide) ω-propyl-2-amine Jeffamine M-1000 (Huntsman, Houston, TX) resulted in a poly(ethylene oxide) modified surface. Hydrazide groups were introduced by reaction with 0.1 N aqueous hydrazine (Sigma, Munich, Germany) solution for 1 h with subsequent rinsing in deionized water and annealing at 120 °C. Perester functionalities were created from the anhydrides by reaction with 10% solution of butyl-hydroperoxide (tertbutyl hydroperoxide purum, 80% in di-tert-butylperoxide, Fluka) in pyridine (puriss., Fluka) for 66 h at room temperature and subsequent rinsing in acetone. Radical polymerizations with 6% aqueous solution of acrylic acid (purum, Fluka) with 0.2 mol-% of N,N′-methylene diacrylamide (>98%, MERCK-Schuchardt) at 80 °C for 2.5 h were preformed to give thick hydrogel films on top of the copolymer films.28 Surface modifications were characterized by infrared spectroscopy in attenuated total internal reflection, ellipsometry, or reactions with specific biopolymers. Similar surface modification steps were furthermore performed in a locally restricted fashion to create chemically patterned surfaces. Micro-contact printing25 of R-methoxy poly(ethylene oxide) ω-propyl-2-amine Jeffamine M-1000 was used to create copolymer surfaces with patterned poly(ethylene) oxide moieties for a patterned protein immobilization. Therefore, elastomeric poly(dimethyl siloxane) (Sylgard 184) structures molded from silicon master structures in the micrometer range were treated in oxygen plasma to create hydrophilic surfaces, then inked with a 2 mM solution of the PEO-amine in ethanol, dried by nitrogen stream, and placed on the copolymer surface by its own weight for 30 s. After that, the stamped patterns were annealed at 120 °C to create stable imide bonds. Immobilization of Bioactive Molecules. Human plasma fibronectin (Roche, Basel, Switzerland) adsorption was performed with solutions of 50 µg/mL in phosphate buffered saline pH 7.4 (PBS, Sigma) for 1 h at room temperature. For fluorescence measurements, fibronectin was rhodamineconjugated with a TRITC protein labeling kit (Molecular Probes, Eugene, OR). Rhodamine-conjugated bovine serum albumin was purchased from Biotrend (Cologne, Germany). Synthetic benzamidine derivatives to be utilized as surfacebound thrombin inhibitors were prepared as described elsewhere.29 Immobilization of the benzamidine derivatives (BZA) was accomplished by, first, direct modification of POMA films and, second, using a PEO spacer linked to POMA. For the direct immobilization reaction, the copolymer films were immersed with a 10mM solution of BZA in 0.1 M borate buffer (pH ) 8) overnight and subsequently rinsed with deionized water. For the immobilization of BZA via a PEO spacer (n ) 11), the carboxy groups of the PEO spacers were activated with EDC/sulfo-NHS (50 mM/25 mM, 2h) and subsequently incubated with a 10mM solution of BZA in 0.1M borate buffer (pH ) 8) overnight and, thereafter, washed with deionized water. Three-dimensional silicone structures coated with rhodamine-conjugated albumin and a stripe pattern of
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Figure 1. Molecular structures of the repeating units of different maleic anhydride copolymers. The anhydride group is brought out by the dark oxygen atoms. (A) POMA, (B) PSMA, (C) PPMA, and (D) PEMA.
rhodamine conjugated fibronectin were imaged with a fluorescence confocal laser scanning microscope (TCS SP, Leica, Bensheim, Germany) with a 40× oil immersion objective. Cell Culture Experiments. Human endothelial cells from the umbilical cord vein were collected according to the procedure suggested by Weis et al.30 and grown to confluence in endothelial cell growth medium ECGM (Promocell, Heidelberg, Germany) containing 2% fetal calf serum. After the first passage, the cells were seeded on the prepared substrates. Cells on patterned substrates were imaged with an optical microscope (IX50, Olympus Optical, Hamburg, Germany) with a 10× phase contrast objective. To visualize fibronectin rearrangement on homogeneous fibronectin coated substrates, cells were imaged by fluorescence confocal laser scanning microscopy with a 40× oil immersion objective. In Vitro Hemocompatibility Assays. 5 mL of freshly drawn human blood was incubated for 3 h with the polymer films at 37 °C avoiding the sedimentation of blood cells by rotation of the incubation chambers. Anticoagulation was achieved with heparin (2 U/mL). After incubation, the blood was analyzed with respect to changes in cell numbers and activation markers for coagulation (thrombin-antithrombin complex), complement activation (complement fragment C5a), and thrombogenicity (platelet factor 4, PF4). ELISA assays were used to quantify the latter substances. Results Characterization of the Copolymer Thin Films. Molecular model structures of the characteristic repeating units of poly(octadecene-alt-maleic anhydride) (POMA), poly(propene-alt-maleic anhydride) (PPMA), poly(styrene-altmaleic anhydride) (PSMA), and poly(ethylene-alt-maleic anhydride) (PEMA) were generated to visualize the decreasing ratio of the size of the apolar comonomer side chain and the polar anhydride group in the different copolymers in the order PEMA and PPMA < PSMA < POMA (Figure 1). Further, in the case of POMA, a tendency of the extended alkyl chains to self-assemble can be anticipated. For PSMA, the chain mobility is substantially reduced due to the bulky phenyl group. Thin films of the maleic acid anhydride copolymers were prepared by spin-coating, solution casting, or adsorption on top of amine-bearing surfaces. Covalent attachment of the
Figure 2. 3-D confocal fluorescence microscopy image of a silicone rubber structures coated with PPMA after ammonia plasma treatment and subsequently immobilized with rhodamine-conjugated albumin to visualize the homogeneous surface modification achieved with microtextured surfaces. Noncovalent immobilized albumin was cleaned off by a subsequent washing step. The images show the rhodamine labeling of the immobilized albumin. (A) Covalent attachment of albumin. (B) Negative control with greatly diminished albumin signal due to immobilization on a surface with hydrolyzed anhydride moieties (microscope settings as in A).
copolymers, realized through spontaneous reaction of the anhydride functions with the amines of the substrate, efficiently prevented delamination, reordering, and dewetting of the films during further modification and application. Amino functionalization of the substrates was achieved on SiO2 surfaces (glass coverslips or silicon wafer) by silanization with 3-aminopropyl-dimethylethoxy-silane. Polymer substrates were amino-functionalized by low-pressure plasma treatment in ammonia atmospheres26,27 or oxygen plasma modification with subsequent amino-silane functionalization. Note that this also allows for the functionalization of rough and micro-textured surfaces as shown by the homogeneous coating of microstructures made from silicone rubber (Figure 2). Alternative options for immobilization of maleic anhydride copolymer layers on polymer substrates comprise crosslinking via the perester-type of the copolymers (macroinitiators) and low pressure plasma treatment of preadsorbed copolymer films. Both approaches are currently studied in detail by the authors but do not concern the scope of the work presented herein. Thicknesses between 3 and 6 nm were determined by ellipsometry for the covalently attached layers in 3 measurements on 5 different samples for each copolymer surface (see Table 1). A low surface roughness of the films (RMS 0.3 nm) was measured by scanning force microscopy on smooth substrates (silicon wafer). Typical values are shown in Table 1. Compared to the roughness of a silicon wafer of 0.15 nm, an increase can be noted; however, the films represent very smooth surfaces. Concentrations of accessible anhydride moieties of about 1 × 1014 cm-2 were estimated from XPS data via determination of the sulfur and nitrogen content of the surface after reaction with methionine amide (Table 1). The nitrogen content of the copolymer films was compared with a SiO2 surface bearing an aminopropyl-silane monolayer of known amine density (3 × 1013 cm-2). Limitations of this method
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Maleic Anhydride Copolymers Table 1. Summary of Characteristic Properties of the Compared Copolymer Films
thickness (( 0.5 nm) RMS roughness anhydride surface density water contact angle (( 3deg) isoelectric point (1 mM KCl)
POMA
PSMA
PPMA
PEMA
3.5 nm 0.32 nm 6 × 1013 cm-2 100° 3.2
5.8 nm 0.31 nm 8 × 1013 cm-2 75° 2.0
3.5 nm 0.34 nm 1 × 1014 cm-2 52° 1.9
4.5 nm 0.8 nm
concern the possibly incomplete conversion of the anhydride groups and the fact that the information depth of the XPS measurement comprises the whole polymer layer. Therefore, the results probably do not reflect the anhydride density of the uppermost surface of the film but the frequency of anhydrides accessible for reaction with amines from aqueous solutions (which is the value of interest for this study). Anhydride densities of about 1 × 1014 cm-2 were obtained (Table 1). The number of the estimated anhydride surface concentrations for POMA, PSMA, and PPMA agrees with the expectation of higher values for smaller comonomers (Table 1). For comparison, the calculated molecular structure (Figure 1) of the anhydride unit was used to give an upper limit for the detectable groups at maximum packing density which was determined to about 3 × 1014 cm-2. Differences in the hydrophobicity of the copolymers have to be attributed to the comonomers.31 Means of the advancing water contact angles of two measurements on three different samples for each copolymer surface show differences in the hydrophobicity of the films related to the size of the hydrocarbon structure in the comonomer (see Table 1). No remarkable difference in the water contact angle was observed between the copolymers containing propene and ethylene comonomers, for both of them the size of the comonomer is comparable to the size of the anhydride group. Electrosurface phenomena on similar copolymer films were studied in detail by electrokinetic measurements. The results are reported in a companion paper.32 The low isoelectric points (Table 1) of PPMA and PSMA indicate that the first dissociation step is facilitated because of a hydrogen bridge of the formed anion to the undissociated second carboxylic acid. For POMA, the less acidic net characteristics were attributed to the larger comonomer generating additional surface sites for unsymmetrical electrolyte adsorption. Chemical Conversion of the Copolymer Films. The anhydride moieties allowed for simple and efficient modification of the polymer substrates by chemical reactions summarized in Figure 3, which were implemented in the reported work. Important conversions base on the high reactivity of the anhydride function toward amines leading to the formation of amides, optionally followed by formation of the stable five-membered cyclic imide upon annealing at 120 °C. Hydrolysis of the copolymer films rapidly and conveniently performed at conditions of steam sterilization (120 °C, saturated water vapor at 2 bar, 20 min) quantitatively converts the anhydride units into carboxylic acid groups. XPS data prove that no anhydride reactivity toward methionine was left after hydrolysis. In the FTIR-ATR spectra of a
57°
PPMA film (Figure 4), the antisymmetric carbonyl stretching band of the cyclic anhydride at 1778 cm-1 vanishes and a new carbonyl stretching band of the maleic acid at 1710 cm-1 appears. Note that hydrolysis of the copolymer films is reversible upon annealing at dry conditions for 2 h/120 °C. Although the hydrolysis and regeneration of anhydride moieties is already known in the literature,21,31 the rapid hydrolysis by steam sterilization of even very hydrophobic copolymers such as POMA could be demonstrated in the reported work including its convenient and defined adaptation for the conversion of the thin film copolymer coatings. Reaction of the copolymer films with 1,4-butanediamine generates a high frequency of amine groups22 suitable for further reactions such as binding of poly(ethylene oxide) diacid (PEO 600) (previously activated with N-ethyl-N′-(3dimethylaminopropyl)carbodiimide hydrochloride and Nhydroxysulfosuccinimide sodium salt), which was successfully used as a spacer system for flexible protein attachment. Unspecific protein adsorption could be greatly diminished by conversion of the copolymer films with a monofunctional poly(ethylene oxide)-amine as worked out with R-methoxy poly(ethylene oxide) ω-propyl-2-amine (MW ) 1000) at concentrations above 5 mM and incubation times of about 6 h. Further realized details of the options for chemical conversions, not elaborated in the subsequent sections of this article, comprise the reaction of maleic acid anhydride films with hydrazine resulting in the formation of hydrazide groups useful for subsequent binding of polysaccharides. Hydrazine modifications were tested by ATR-FTIR and successful immobilization and detection of fluorescence conjugated heparin (data not shown). Also, the copolymer films were successfully converted with butyl-hydroperoxide to create perester units allowing for initiation of radical polymerizations with a wide variety of monomers. Polymerization of acrylic acid on top of the copolymer films resulted in hydrogel layers of several micrometer thickness which could be adjusted in the cross-linking density to allow for efficient uptake and release of different biopolymers (fluorescence conjugated proteins were imaged in hydrogel layers, data not shown). Immobilization of Bioactive Molecules and Modulation of Their Functional Characteristics. Maleic anhydride copolymers enable direct covalent protein immobilization in neutral and mildly alkaline media as -amino groups of the lysine residues of proteins spontaneously react with anhydride groups under these conditions.33-37 Because no spontaneous formation of covalent links occurs with the diacid form of the copolymers, the covalent or noncovalent deposition of proteins can be easily controlled by hydrolysis or annealing at 120 °C.
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Figure 3. Survey of applied chemical conversions of maleic acid anhydride copolymer films.
Using that option, the anchorage of fibronectin to maleic anhydride copolymers was systematically varied to reveal conditions for the formation of cell-matrix adhesions by endothelial cells. Hydrophobic and hydrophilic polymer substrates, POMA and PPMA films, were coated with similar amounts of fibronectin (about 450 ng/cm2, corresponding to monolayer coverage38) at conditions of either covalent or noncovalent immobilization. Although all of the compared fibronectin coatings were effective in stimulating attachment of endothelial cells, the initial formation of cell-matrix adhesions was found to be controlled by the type of interaction between predeposited fibronectin and the underlying substrate (Figure 5). Covalent linkage and hydrophobic interactions of the predeposited fibronectin with the polymer films interfered with the rapid generation of focal and fibrillar adhesion contacts. This was apparently caused by the fact
Figure 4. ATR-FTIR spectra of PPMA films after annealing at 120 °C or hydrolysis, respectively.
Maleic Anhydride Copolymers
Figure 5. Reorganization of fibronectin adsorbed on POMA (left) and PPMA (right) by endothelial cells at 50 min after seeding. (Confocal laser scanning images; image size: 125 µm.) Note that the reorganization is enhanced on the less hydrophobic substrate, no reorganization occurs for covalently bound fibronectin on POMA and PPMA (not shown).
Figure 6. Endothelial cells growing on a POMA surface patterned with stripes of poly(ethylene oxide) with a periodicity of 90 µm. The inset shows the fidelity of the pattern by the immobilized rhodamine conjugated fibronectin (both scale bars: 300 µm).
that only weakly bound fibronectin could become readily reorganized by the adherent cells.38 Reaction with functional amines permits us to additionally influence unspecific protein adsorption at the copolymer films where conversion with R-methoxy poly(ethylene oxide) ω-propyl-2-amine (MW 1000) was found to be efficient in preventing protein adsorption in general. The protein resistance of grafted poly(ethylene) oxide can be used for creating 2d patterns by microcontact printing as pioneered in refs 39 and 40. We used similar procedures to create 2d proteinresistant micropatterns on the different maleic anhydride copolymer films. Figure 6 shows the patterned growth of endothelial cells on POMA due to the creation of a stripewise protein resistant pattern and subsequent fibronectin immobilization. Advantageously, this patterning could be performed with any of the different copolymers in either the hydrolyzed or anhydride state allowing for the additional modulation of the protein anchorage on the protein-binding segments (data not shown). Introduction of spacer functions provides another important option for the modulation of immobilized bioactive molecules based on maleic anhydride copolymer films. Modification of POMA films by 1,4-butanediamine and subsequent introduction of poly(ethylene oxide) diacid spacer functions was utilized for that purpose. Linkage of the spacer to amine groups required activation of the terminating carboxyl groups with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide sodium salt. The impact of the spacer for the accessibility of the immobilized molecule is demonstrated for the example of benzamidine derivatives applied as surface bound thrombin inhibitors. As shown in Figure 7, the thrombogenicity of the polymer films
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Figure 7. Improved hemocompatibility of surfaces modified by benzamidine derivatives attached via PEO spacers (PEO+) in comparison to direct binding of the inhibitor on a POMA surface (POMA+) indicated by decreased amount of platelet factor 4 (PF4). Additionally, activity of pure POMA surface (POMA), PEO modified POMA surface (PEO), and control from blood plasma (I.V.) is shown.
Figure 8. Options of maleic acid anhydride copolymers for surface engineeringsschematic summary.
upon incubation in human whole blood for 3 h is more efficiently reduced if the inhibitor molecule is presented by means of a poly(ethyleneoxide) spacer.29 Discussion Copolymers of styrene-, olefins-, vinyl acetate-, and alkyl vinyl ether-maleic anhydride were introduced commercially to fill the need for resins of low viscosity, water solubility, and high functionality. The copolymers have been later suggested for a variety of applications which include adhesives, detergents, anti-soil and paper sizing agents, and pharmaceuticals.21 Also, the use of the copolymers for direct covalent immobilization of proteins onto solid substrates was established by earlier studies.33-37 With this work, a versatile platform for bioactive coatings on solid supports was developed on the basis of thin films of different alternating maleic anhydride copolymers and their derivatives enabling the gradual variation of physicochemical surface characteristics combined with specific biomolecular functionalities as sketched in Figure 8. The copolymer films can be covalently attached to various substrate materials exhibiting smooth or structured surface topographies. The choice of very thin layers not exceeding the coverage of the substrate with a covalently linked polymer monolayer was found to be adequate for subsequent chemical conversion or biomolecule immobilization as stability problems or undesired structural variations within the copolymer layer could be minimized. The required amino moieties can be introduced in various ways, e.g., by silanization of SiO2 surfaces or low-pressure ammonia plasma treatment of polymer surfaces. The covalently attached thin films of the maleic anhydride copolymers can be varied in a wide range of physicochemical
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Figure 9. Summary of the surface engineering options based on maleic anhydride copolymer films to enable the functional modulation of bioactive elements at interfaces. Binding of biomolecules (e.g., proteins) can be controlled through (from left to right) physisorption, simple covalent binding, covalent binding via spacer functions, entrapment in hydrogels, and repulsion.
properties. The hydrophobicity and the density of the functional anhydride moieties can be altered by the intrinsic characteristics of the different comonomers (octadecene, styrene, propene, and ethylene). The degree of hydrophobicity of the copolymer does not only influence the interaction strength with subsequently immobilized biopolymers but it also importantly determines the structural characteristics of the copolymer films ranging from a hydrophobic water insoluble film (e.g., POMA) to water soluble copolymer brushes (e.g., PEMA). As demonstrated with the molecular models (Figure 1), the long alkyl chains of POMA can easily screen the polar anhydride groups resulting in an overall hydrophobic behavior. In contrast, the small ethylene residue in PEMA provides a more hydrophilic and water soluble copolymer as the anhydride moiety and the ethylene unit are rather similar in size. Although the variation of hydrophobic properties of the copolymers through the choice of the comonomer is important for unspecific biopolymer binding,41 the crucial characteristic of the maleic anhydride copolymers is the wellknown high reactivity of the anhydride moieties with primary amine groups and, to lower degrees, with alcohols.20,22,42 The conversions can either be performed in the dissolved state of the copolymers or by surface chemistry following film formation. The amide generated upon reaction of the anhydride groups with primary amines can be stabilized upon annealing at 120 °C to give the very stable five-membered cyclic imide. Using these reactions (summarized in Figure 3) and the intrinsic characteristics of the copolymers (due to the comonomer units), binding of bioactive molecules onto solid surfaces can be fine-tuned by maleic anhydride thin films according to the requirements of the desired activity (Figure 9). The key advantage of the approach consists of the wide variability of molecular settings available based on similar chemical structures which allows us to restrict the modification of the surface to any property of interest while keeping other characteristics invariant. This may help to unravel bio-interfacial phenomena at artificial materials in a more definite way. Two examples have been elaborated to demonstrate the potentialities of the suggested platform technology: (i) Control of cell-matrix adhesion through fibronectin anchorage on copolymer films. Components of the extracellular matrix are immobilized to solid supports to unravel the constraints of cell-matrix adhesions and to develop cell culture carriers. The characteristics of the immobilized matrix
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proteins may influence cell functionality with respect to cell adhesion, migration, triggering of downstream cell signaling processes, and even differentiation.9-11 The maleic anhydride copolymer system allowed us to vary the anchorage of the immobilized proteins in a very controlled way and, thus, to reveal the origin of differences in the cell behavior. Covalent and noncovalent immobilization onto the different copolymers was established at similar amounts and conformations of the layered fibronectin while inducing different binding strength of the protein to the substrate.38 As shown in Figure 5, more weakly attached fibronectin on the hydrolyzed PPMA film was more quickly incorporated in the assembly of fibronectin fibrils on the substrate surface in comparison with hydrolyzed POMA films demonstrating the pronounced effects achieved by fine-tuned variations in the characteristics of the copolymer films. (ii) The actiVity of coValently immobilized thrombin inhibitors is influenced by attachment through PEG spacers. POMA films were converted by covalent attachment of a synthetic low-molecular weight thrombin inhibitor.29 The in vitro hemocompatibility results obtained with those preparations upon incubation in whole blood for 3 h show that the same benzamidine inhibitor moiety linked to the surface with or without use of a poly(ethylene oxide) spacer was similarly active with respect to the plasmatic coagulation system (data not shown), whereas the thrombogenicity (see Figure 7) of the surfaces was more efficiently reduced for the surfaces bearing the poly(ethylene oxide) spacer. The decreased protein adsorption from whole blood, in addition to the specific inhibition of thrombin, may further enhance the hemocompatibility characteristics for the latter type of coating. In summary, thin films of maleic anhydride copolymers were elaborated as a versatile tool for the concise interfacial presentation of several bioactive molecules. The resulting options are very valuable both for the preparation of model substrates for fundamental studies on biointerfacial phenomena as well as for the controlled surface modification of a great variety of bulk substrates used in advanced products such as 3-D cell culture scaffolds for tissue engineering strategies, balloon catheters for cardiovascular intervention, or sensoric microarrays for molecular evaluation of complex biofluids. Acknowledgment. Contributions of M. Nitschke, R. Schweiss, C. Rauwolf, F. Simon, T. Osaki, and A. Baier (all at Institute of Polymer Research Dresden) to plasma surface treatments, ATR-FTIR measurements, molecular structure modeling, XPS measurements, electrokinetic measurements, and graft polymerization of poly(acrylic acid), respectively, are gratefully acknowledged. References and Notes (1) Kao, W. Y. J.; Hubbell, J. A. Murine Macrophage Behaviour on Peptide-Grafted Poly(ethylene glycol)-Containing Networks. Biotechnol. Bioeng. 1998, 59, 2-9. (2) Liesveld, J. L.; Broudy, V. C.; Harbol, A. W.; Abboud, C. N. Effect of stem cell factor on myelopoiesis potential in human Dexter-type culture systems. Exp. Hematol. 1995, 23, 202-9. (3) Zisch, A. H.; Schenk U, Schense; J. C., Sakiyama-Elbert; S. E., Hubbell, J. A. Covalently conjugated VEGF- -fibrin matrixes for endothelialization. J. Controlled Release 2001, 72, 101-13.
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