Fibronectin Exchange at a Constant Surface Concentration

In a first set of experiments, interfacial FN exchange was studied at various compositions of the protein solution applied for the displacement. Homoexchange by FN as well as heteroexchange by human serum albumin (HSA) was measured by fluorescence confocal laser scanning microscopy (LSM), which was supported by high-performance liquid chromatography (HPLC)-based amino acid quantification. Based upon preceding studies (Salchert et al. 2003), the adsorbed amount of FN onto the polymeric surfaces was quantified by HPLC-based amino acid analysis. Time- and concentration-dependent adsorption experiments revealed that the surface coverage has almost reached a steady state at the chosen solution concentration of 50 |g/ml after 1 h of adsorption. Figure 3 indicates the quantity of adsorbed protein on the three copolymer surfaces.

The adsorbed amount of FN implies no simple side-on adsorption of the protein. An assumptive monolayer, supposing a side-on adsorption of a molecule with two arms of 60 nm length and 2.5 nm in diameter (Hae-berli 1998), is calculated to be 0.25 |gcm2. However, in a realistic scenario,

Fig. 3. Initially adsorbed surface concentration (r) of fibronectin (FN) on POMA, PPMA, and PEMA from a solution concentration of 50 |g/ml after 1 h of adsorption

adsorbed protein layers cannot be considered to create a homogeneous structure. The amount of FN gained maybe attributed to the heterogeneity in the orientation of the adsorbed protein molecules, because a heterogeneous protein layer could combine both, side-on and end-on orientation in a random distribution.

FN exchange was studied immediately following the adsorption process only interrupted by rinsing with PBS. The kinetics of the surface coverage of rhodamine-conjugated FN, as determined by LSM measurements, showed distinct differences for the compared polymer films and exchange solutions. Both homo- and heteroexchange solutions were applied at a concentration of 50 ^g/ml. The FN surface concentration during exchange is exemplarily shown on POMA for hetero- and homoexchange in Fig. 4A, and for heteroexchange by HSA at the three copolymer surfaces in Fig. 4B.

FN exerted a bigger impact on the displacement of the preadsorbed FN than HSA on POMA. In addition, the heteroexchange demonstrates the impact of the physicochemical characteristics of the polymer substrates, with an increasing exchange occurring on the more polar surfaces. The higher amount of FN exchanged on the hydrophilic surfaces (PEMA, PPMA) correlates with a stronger anchorage of FN at hydrophobic surfaces. Noticeable in Fig. 4isthatthereisafastdeclineduringthefirstfewhoursofobservation and an attenuation of the curves towards the later stages. We detected this as a general trend in all exchange experiments, which is in agreement with earlier published findings (Huetz et al 1995; Wertz and Santore 2002). Huetz et al. (1995) reported data indicating a similar behaviour for a model system of immunoglobulin molecules displaced by fibrinogen on a silica surface. The exchange mechanism was classified into a fast exchange step, followed by a slower step independent of the solution containing the molecules. The latter is apparently in contrast to our observation. Nevertheless, we have been able to describe the exchange process with two exponential functions on the basis of this theoretical approach with a coverage-dependent exchange kinetics giving, dr 1

dt t where r and t are surface coverage and desorption time constant, respectively. Assuming two protein species, fast (A) and slowly (B) desorbing, leads to:

ta tb

The results of the exponential fits to the measured exchange kinetics are summarised in Table 2. It is interesting to note that protein exchange

Fig. 4. A Displacement of adsorbed FN by FN and human serum albumin (HSA) on POMA (circles) and PPMA (triangles), and B byHSAonPOMA, PPMAandPEMA. Relativeamounts of preadsorbed protein remaining after incubation with different protein solutions were measured by laser scanning microscopy (LSM). The exponential fit (Eq. 2) is shown exem-plarily in A (solid lines)

Fig. 4. A Displacement of adsorbed FN by FN and human serum albumin (HSA) on POMA (circles) and PPMA (triangles), and B byHSAonPOMA, PPMAandPEMA. Relativeamounts of preadsorbed protein remaining after incubation with different protein solutions were measured by laser scanning microscopy (LSM). The exponential fit (Eq. 2) is shown exem-plarily in A (solid lines)

Table 2. Summary of the fit parameters of the exponential approach describing hetero- and homoexchange of fibronectin (FN) on maleic anhydride copolymer surfaces

Copolymer Heteroexchange by HSA Homoexchange by FN

TA(h) TB(h) Ta (|g/cm2) Tb (|g/cm2) taW 13(h) Ta (|g/cm2) rB (|g/cm2)

was observed for FN on the hydrophobic POMA, which is in contrast to the findings of several earlier findings (Capadona et al. 2003; Pettit et al. 1992) on various different hydrophobic surfaces. However, desorption of small fractions of adsorbed protein layers was also reported in the literature (Wertz and Santore 2002). This confirms that protein adsorption phenomena do not simply depend on any single surface property, like hydrophobicity, but depend on a balance of several interactions.

In addition we were able to reliably convert the relative amount of adsorbed protein into absolute amounts with the aid of fluorescent calibration beads (for details see Renner et al. 2004). The conversion allows a more valuable comparison between the exchange kinetics of the protein amounts on both polymer surfaces and a rescaling to the quantitative surface coverage by the HPLC data. The calibrated absolute fluorescence intensities were shown to correlate well with the HPLC data by introducing the ratio between the amounts adsorbed onto PPMA and POMA (APP:APO). We gained values, which differ in the range of the mean error (Table 3), and concluded that the HPLC data corresponds to the converted absolute amount. Based on this correlation, the exchange kinetics measured by LSM could be rescaled to reflect the dynamic change of the surface coverage quantitatively.

Starting from there, the homo- and heteromolecular exchange of FN on the different copolymers could be analysed in more detail. The results shown in Fig. 5 for the homomolecular exchange indicate an exchange accompanied by additional adsorption on both polymer surfaces. The HPLC data indicate that although after the initial adsorption time of 1 h the amount of adsorbed FN increases further, it does so very slowly. This process can be attributed to conformational changes, reorganisation of the adsorbed protein layer or exchange of protein with adsorption in different orientations. Such processes are already known from the literature to occur over long time scales (Calonder et al. 2001; Haynes and Norde 1994; Nygren 1993; Wertz and Santore 1999). The exchange process is illustrated by the LSM data indicating the amount of remaining FN from the initial adsorption step with a more intense exchange on the hydrophilic PPMA surface.

A similar behaviour was measured for the heteromolecular exchange of FN on the two copolymer surfaces (Fig. 6). On the hydrophilic PPMA subTable 3. Comparison of high-performance liquid chromatography (HPLC) data and converted absolute laser scanning microscopy (LSM) data using the mass or intensity ratios

Appma:^poma

FN

HSA

LSM

0.89 ± 0.2

0.46 ±0.17

HPLC

0.92 ±0.15

0.62 ±0.1

Fig. 5. A Homomolecular exchange of FN on POMA and B on PPMA, as measured by high-performance liquid chromatography (HPLC; grey bars) and LSM (circles and solid lines)

strate, more FN is displaced by HSA. Again, an increase in the total amount of adsorbed protein could be observed, which was attributed to conformational changes and exchange processes, as observed for the homomolecular system. The exchange kinetics clearly indicate a higher affinity of FN towards hydrophobic surfaces. Furthermore, the data given in Fig. 6 provide further evidence for the good agreement between HPLC and LSM analyses and proof of the reliability of the LSM measurements. While the influence of the fluorochrome on the adsorption and the exchange properties of the proteins might be critical in the LSM experiments, the agreement with the HPLC data of the unlabelled proteins indicate that the label has no influence on the adsorption and the exchange processes at the analysed settings. While HPLC provides more quantitative data, this ex situ method is limited to lower numbers of time points.

Fig. 6. A Heteromolecular exchange of FN by HSA solutions on POMA and B PPMA, as measured by HPLC (bars) and LSM (circles and solid lines)

The results can be qualitatively summarised as follows: (1) homo- and heteroexchange of FN occur on surfaces that exhibit different degrees of polarity, and (2) FN exhibits a higher affinity to the hydrophobic substrates as compared to the more hydrophilic and more negatively charged substrates. Extending this qualitative picture, a multivariate regression analysis was applied in order to reveal the relevance of interaction parameters in the complex setting of different exchange processes on different copolymer surfaces (Renner et al. 2004). The linear model used therein could already elucidate several important features of the analysed processes, like the significant influence of the protein solution applied in the displacement experiments on ta and tb and of the substrate hydrophobicity on the exchange and non-exchanged amounts rA and rB. However, the simplicity of the linear approach and the assumed interaction schemes did not allow for a satisfactory description of the complex protein interaction processes during protein exchange on solid substrates. In a more restricted approach with a smaller number of variables, a convincing trend of the time constants ta and tb of the FN heteroexchange following the anhydride concentration of surfaces (used as a measure of surface polarity) can be shown (Fig. 7). As the balance of the polar maleic acid groups and the hydrophobic components of the comonomers dominates the copolymer surface characteristics, a major impact of this parameter on protein-substrate interactions can be expected. Matching this expectation, a correlation of the FN anchorage -

Fig. 7. FN anchorage strength measured in terms of the time constants ta (A) and tb (B) of FN heteroexchange by HSA versus the anhydride concentration of the maleic anhydride copolymer surfaces.

expressed by the exchange time constants ta and tb - to the surface polarity - characterised by the anhydride density - is observed.

In view of this result we may conclude that protein displacement experiments allow for the characterisation of protein-substrate interactions and permit the establishment of a quantitative link between the derived features of the exchange processes and the substrate characteristics.

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