Gradated Substrate Physicochemistry

Addressing the very obvious need to establish structure-property relationships in biointerfacial processes, several approaches for the preparation of materials with controlled variation of the surface characteristics have been suggested over the last few years. In our approach, alternating maleic acid anhydride copolymers have been used as covalently attached coatings in order to switch the chemical reactivity of solid substrates towards biopolymers and to precisely adjust their physicochemical characteristics. Towards that aim, alternating maleic acid anhydride copolymers (Trivedi and Culbertsen 1982) were deposited as thin films on carrier materials. Through the choice of the comonomer and via conversions of the reactive anhydride moieties, a wide variety of surface parameters can be adjusted. While earlier works already made use of the high reactivity of maleic anhydrides for several surface modification concepts (Chapman et al. 2000; Jenkins et al. 2000), we have been able to demonstrate additional options of maleic anhydride copolymer coatings for the gradual variation of physico-chemical surface characteristics with specific biomolecular functionalities and lateral constraints independent of the underlying substrate (Pompe et al. 2003b; Schmidt et al. 2002).

In the experiments reported herein, the latter features of alternating maleic anhydride acid copolymers were used to vary the polar-non-polar balance of the molecules as well as the structural characteristics of the macromolecules in polar and non-polar environments and, through this, the surface free energy and charge density of the copolymer films. It is hypothesised that the choice of comonomer influences the non-specific biopolymer binding (Malmsten 1998). To restrict the interactions with biopolymers to non-covalent ones the high reactivity of the anhydride moieties can be "switched off" by hydrolysis, creating surfaces that are characterised by the interplay between carboxylic acid groups and the features of the comonomer subunits.

To cover a broad range of interaction strengths, the following copolymers were utilised: -poly(octadecene-aZi-maleic anhydride) (POMA), -poly-(propene-aZi-maleic anhydride) (PPMA) and -poly(ethylene-aZi-maleic anhydride) (PEMA).

Molecular images of the characteristic repeating units allow the visualisation of the decreasing ratio of the size of the non-polar comonomer

Fig. 1. A,B Molecule models of the repeating units of different copolymers: POMA (top), PPMA (bottom left) and PEMA (bottom right)

side chain and the polar anhydride group in the different copolymers in the order PEMA < PPMA < POMA (Fig. 1). Already from these images a tendency of the extended alkyl chains of POMA to self-assemble can be anticipated.

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 copolymers - realised 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 functionali-sation of the substrates was achieved on SiO2 surfaces (glass coverslips or silicon wafers) by silanisation with 3-aminopropyl-dimethylethoxy-silane. Polymer substrates were amino-functionalised by surface-selective, low-pressure plasma treatments in ammonia atmospheres (Nitschke et al. 2002; Tusek et al. 2001) or by oxygen plasma modification with subsequent amino-silane functionalisation.

The film thickness of the dry copolymer coatings was determined to be between 3 and 6 nm by ellipsometry for covalently attached layers (Table 1). The concentrations of accessible anhydride moieties of about 1014 /cm2 were estimated from x-ray photoelectron spectroscopy (XPS) data via determination of the sulphur and nitrogen content of the surface after a reaction with 50 mM methionine amide hydrochloride in buffer solution for 1 h (Table 1). The determination assumes a density of 2.85 g/cm3 of the detected SiO2 from the silicon substrate and a mean attenuation length of the XPS signal in the substrate and the surface layer of about 3 nm (Mark et al.

Table 1. Physicochemical parameters of the maleic anhydride copolymer thin films used

Parameter

POMA

PPMA

PEMA

Thickness1 (±0.5 nm)

3.5 nm

3.0 nm

4.5 nm

Anhydride surface density2

9x1013/cm2

3x1014/cm2

7x1014/cm2

Water contact angle3 (±3 °C)

100°

38°

26°

1 Determined by ellipsometry

2 Determined by XPS after methionine amid conversion 3Measurement of hydrolysed copolymer surfaces

1 Determined by ellipsometry

2 Determined by XPS after methionine amid conversion 3Measurement of hydrolysed copolymer surfaces

1987; Pompe et al. 2005b). Within this routine the elemental compositions (C, N, S, Si) of the layer components (SiO2, maleic anhydride copolymer, methionine amide) were fitted to the XPS data in a least-square sense. The aminosilane surface concentration was set to a fixed value of 5 x 1014 /cm2, which was determined independently on surfaces without copolymer films using the same method. Data collected using this approach reflect the anhydride density accessible for the reaction with amines from aqueous solutions, which is of interest in our set-up. The estimated anhydride surface concentrations for POMA, PPMA and PEMA agree well with the expectation of higher values for smaller comonomers (Table 1). Furthermore, the ratio of calculated anhydride to comonomer units shows the better accessibility of anhydride moieties for the more hydrophilic copolymers. The same tendency manifests itself in differences in the hydrophobicity of the copolymer films (Mark et al. 1987). A decreasing mass content of the polar anhydride component leads to a decrease in the surface energy. This was confirmed by measurements of the advancing water contact angles, which revealed differences in the hydrophobicity of the films related to the size of the hydrocarbon structure in the comonomer units (Table 1).

The physicochemical surface characteristics of the covalently attached thin films of the maleic anhydride copolymers were found to be graduated over a wide range in a well-defined manner. The variation in the degree of hydrophobicity and the density of functional anhydride moieties suggests not only an influence on the interaction strength with biopolymers, but furthermore, a variation in the structural characteristics of the copolymer films ranging from a hydrophobic, water-insoluble film (POMA) to point-attached, water-soluble copolymer chains (PEMA). The copolymer film characteristics under physiological buffer conditions were studied in a different set of experiments, as reported elsewhere (Pompe et al. 2005b). The swollen film thickness was determined from deflection-separation curves of scanning force microscope measurements and cross-checked by investigations with a quartz crystal microbalance with dissipation measurements (QCM-D). As expected, POMA exhibits no swelling, in contrast to PPMA and PEMA, which swell by a N3/5 scaling known from free polymer chains in a good solvent, or also termed a "mushroom" regime in the context of polymer brushes (Binder 2002). It was concluded that the copolymer chains are coupled to the amino groups on the substrate surface with a rather low frequency. The individual chains seem to have only one or very few grafting points to the surface, so they still behave like free copolymer chains with respect to the scaling to their molar mass. Changing the pH value to pH 3.0 led to a shrinkage in film thickness, which can be attributed to the change in ionisation of the primary and secondary carboxylic acid groups (pKa1 = 3.0 and pKa2 = 7; Dubin and Strauss 1967,1970; Osaki and Werner 2003; Sugai and Ebert 1986). There exists a sensitive balance of repulsive electrostatic forces of the polar acid groups and attractive hydrophobic interactions of the non-polar alkyl moieties.

It follows, that not only the pH value but also the ionic strength can affect the degree of swelling of the copolymer films. As shown exemplar-ily in Fig. 2 for a PPMA film in different concentrations of phosphate-buffered saline (PBS) solution, this dependency is very pronounced in the range between 10-1 M and 10-3 M, but can be neglected above 10-1 M or below 10-3 M. While below 10-3M the chains are probably almost fully stretched, the negligible shrinkage above 10-1 M can be attributed to the fully screened charges of the carboxylic acid groups. Because of this low degree of swelling under physiological conditions (10-1 MPBS) onlyminor or negligible influences of the compression of expanded polymer chains or protein penetration within the copolymer layers are expected for the protein exchange experiments reported below.

100 150 200 250

time [min]

Fig. 2. Thickness of swollen films of hydrolysed PPMA at different ionic strengths of phosphate-buffered saline solutions, measured using a quartz crystal microbalance with dissipation measurements. The ionic strength at the different time points is indicated by the molar concentration of the buffer solution

100 150 200 250

time [min]

Fig. 2. Thickness of swollen films of hydrolysed PPMA at different ionic strengths of phosphate-buffered saline solutions, measured using a quartz crystal microbalance with dissipation measurements. The ionic strength at the different time points is indicated by the molar concentration of the buffer solution

Based on this knowledge of the physicochemical characteristics of the above-described set of copolymer thin films, the interactions directing protein anchorage and displacement could be analysed for the prominent example of surface-bound FN. The results obtained are reported in the subsequent sections.

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