Biomimetic Poly(methyl methacrylate)-Based Terpolymers - American

Adherence of Staphylococcus aureus, responsible for major foreign body infections, was ... implanted materials via adhesion proteins such as fibronect...
4 downloads 0 Views 57KB Size
Download PDF
Biomacromolecules 2002, 3, 63-68

63

Biomimetic Poly(methyl methacrylate)-Based Terpolymers: Modulation of Bacterial Adhesion Effect Sandrine Berlot,† Zoubida Aissaoui,‡ Graciela Pavon-Djavid,† Joel Belleney,‡ Marcel Jozefowicz,† Ge´ rard He´ lary,† and Ve´ ronique Migonney*,† Laboratoire de Recherches sur les Macromole´ cules, FRE 2314, Universite´ Paris 13, 99 Avenue J-B Cle´ ment, 93430, Villetaneuse, France, and Laboratoire de Chimie Macromole´ culaire, UMR 7610, Universite´ Pierre et Marie Curie, 4 Place Jussieu, 75252 Paris Cedex 05, France Received June 29, 2001; Revised Manuscript Received October 30, 2001

Adherence of Staphylococcus aureus, responsible for major foreign body infections, was assessed onto functionalized poly(methyl methacrylate)-based terpolymers bearing sulfonate and carboxylate groups and onto poly(methyl methacrylate) as control. These terpolymers, have been synthesized by radical copolymerization of methyl methacrylate, methacrylic acid, and sodium styrene sulfonate by varying the ratio R ) [COO-]/[COO- + SO3-] from 0 to 1 and keeping ionic monomer content between 7 and 18%. Adsorption of fibronectin onto poly(methyl methacrylate) was shown to dramatically promote bacterial adherence, whereas a strong inhibition of bacteria adherence was observed onto functionalized terpolymers containing both carboxylate and sulfonate groups. When terpolymers were predominantly functionalized by carboxylate groups, bacteria adherence was favored and reached values close to those obtained for poly(methyl methacrylate). These results have been related to the distribution of the anionic groups along the macromolecular chains, creating active sites responsible for specific interactions with fibronectin and inducing modifications of its conformation. The conformation of the adsorbed adhesive protein was then suggested to have an influence on the availability of its interaction sites to bacteria adhesins and therefore on modulation of bacteria adherence. Inhibition of Staphylococcus aureus adherence by functionalized poly(methyl methacrylate)-based terpolymers is of great interest in the field of biomedical implants and especially in the case of ophthalmic applications. Introduction Implant-associated infection is a medical problem which induces complications and may result in amputation, osteomyelitis, or death. This is the major cause of implantation failure when antimicrobial treatment is not effective. For instance, Staphylococcus aureus (S. aureus), one of the most feared pathogens, cannot be cleared without removal of the implant. It was found that bacteria adherence to implanted material was strongly influenced by protein deposition onto the surfaces.1,2 Indeed, every surface in contact with body fluids is rapidly coated with a monolayer of host plasma and matrix proteins.3 The ability of S. aureus to adhere to implanted materials via adhesion proteins such as fibronectin or fibrinogen is a major factor for initiating foreign body infection.1,4,5 Specific domains of these proteins, also called bacterial adhesins, may interact with bacterial surface proteins.6 Therefore, a number of host matrix proteins such as fibronectin, fibrinogen, and vitronectin, which can bind S. aureus and adsorb onto the implant surfaces, are implicated in bacterial adherence to biomaterials and medical device related infection. * To whom correspondence should be addressed. E-mail: [email protected]. † Laboratoire de Recherches sur les Macromole ´ cules, FRE 2314, Universite´ Paris 13. ‡ Laboratoire de Chimie Macromole ´ culaire, UMR 7610, Universite´ Pierre et Marie Curie.

Among these proteins, the role of fibronectin was shown to be predominant in promoting S. aureus adherence onto polymers2,7,8 and metallic surfaces such as stainless steel, pure titanium, and titanium alloys.9 However, the first studies devoted to prevention of prosthetic device associated infection did not take the adsorption of fibronectin into account. Immobilization of antibiotics or antimicrobial agents for permanent activity or controlled release of active molecules were suggested solutions.10,11 Recently, several modifications of polymer surfaces were demonstrated to decrease protein adsorption and indirectly prevent bacterial adherence. Many authors showed that heparin-coated biomaterials reduced bacteria adherence whatever the nature of the polymer backbone.12-15 However, the main difficulty was to graft heparin to the polymer surface while keeping the bacteriostatic properties. Another approach consisted of modifications of the hydrophilic surface properties of polymers. Indeed, low interfacial tension in biological fluids with hydrophilic coating has often been suggested to significantly decrease the adsorption of proteins. For instance, a significant reduction of bacterial adherence was observed onto poly(ethylene terephthalate) surfaces modified by poly(ethylene oxide).16 Hydrophilic polymers such as poly(vinyl pyrrolidone) or poly(hydroxyethyl methacrylate) when grafted onto hydrophobic polymer surfaces have been demonstrated to reduce both protein adsorption and bacterial attachment.17,18

10.1021/bm015580m CCC: $22.00 © 2002 American Chemical Society Published on Web 12/01/2001

64

Biomacromolecules, Vol. 3, No. 1, 2002

The new approach was to graft ionic groups to polymeric backbones, to induce specific interactions with the living system’s components19,20 and particularly with adhesive proteins. Randomly distributed ionic groups such as sulfonate and carboxylate were demonstrated to specifically interact with adhesive proteins.21 The objective of this work was to study the initial attachment of S. aureus onto functionalized poly(methyl methacrylate) (PMMA)-based terpolymers bearing sulfonate and carboxylate groups. We have chosen PMMA-based terpolymers because PMMA has been widely used in biomedical applications or devices such as bone cement,22 intraocular lenses,23 and contact lenses.24 Thus, Staphylococci are the most common organisms isolated from postoperative endophthalmitis; bacteria are introduced in eyes through irrigation solution25 or by adhering to the implant.26 PMMA was also considered as a reference polymer in the study of foreign body infection.27 PMMA-based terpolymers were prepared by radical copolymerization of methyl methacrylate, methacrylic acid, and sodium styrene sulfonate. The adherence of S. aureus has been studied on terpolymers and PMMA in shape of films previously adsorbed with fibronectin. Materials and Methods Synthesis of Functional Terpolymers. Radical terpolymerizations of methyl methacrylate, methacrylic acid, and sodium styrene sulfonate were performed at the Laboratoire de Chimie Macromole´culaire headed by J.-P. Vairon, Universite´ Pierre et Marie Curie, Paris, France. Methyl methacrylate (MMA) and methacrylic acid (MA) commercial products from Aldrich were purified by distillation under reduced pressure. Sodium styrene sulfonate (NaSS) provided by Fluka and the initiator 2,2-azobisisobutyronitrile (AIBN) were purified by recrystallization in methanol. Dimethyl sulfoxide (DMSO), the common solvent of the three monomers, was purified by distillation on CaH2 under reduced pressure immediately prior to terpolymerization. Radical terpolymerizations of MMA/MA/NaSS or MMA homopolymerization were performed under nitrogen at 70 °C in DMSO solution (1 mol/L), using AIBN as thermal initiator (0.6 mol % with respect to monomer feed). Initial compositions in ionic groups were limited to 17% in order to avoid swelling of the terpolymers in buffer medium. Polymers were precipitated in water, then dried under vacuum and Soxhlet extracted with various solvents: dichloromethane, acetone, or water. Characterization of the Terpolymers. Final compositions of the terpolymers were determined by 1H NMR in deuterated DMSO previously distilled on calcium hydride (CaH2) under reduced pressure. Terpolymers were dried under vacuum at 70° for 12 h. Spectra were recorded using a Bruker AC200 equipped with a 5 mm dual probe and an Aspect 3000 computer. Typical conditions were as follows: spectral width 4000 Hz, 16 K data points, flip angle 20°, relaxation delay 4 s. A zero filling (32K) was applied prior to Fourier transform leading to a digital resolution of 0.24 Hz/pt.

Berlot et al.

Preparation of Lenses. Lenses were prepared by dissolving 10 g of terpolymer in 100 mL of DMSO. Solutions were then poured on polyamide molds (1.6 mm diameter). After an overnight evaporation at 80 °C under vacuum, lenses were detached and extensived washed with aqueous saline solutions: 1.5 M NaCl, 0.15 M NaCl, distilled water, and calcium- and magnesium-free Dulbecco phosphate buffer saline solution (PBS, 1X, Gibco Laboratories). Lenses were sterilized by UV exposure (254 nm) for 15 min on each side and stored in phosphate buffer solution (PBS) at -20 °C under sterile conditions. Bacterial Strain. Bacterial strain S. aureus Cowan I (purchased from ATCC No. 12598) was used for adherence experiments. Bacteria were kept at -80 °C in skim milk (Difco Laboratories), containing 10% glycerol (Sigma). Bacteria were transferred from cryotubes with a sterile loop to inoculate agar plate. The inoculum was widely spread in order to obtain well-isolated single colonies and was overnight incubated at 37 °C. Then, the plate was kept at 4 °C. Before each experiment, a well-isolated representative colony selected from the agar plate was overnight incubated with 1 mL of Mueller Hinton Broth medium (MHB, SanofiPasteur) at 37 °C. Saturated bacterial suspension was centrifuged at 3500g for 15 min. The supernatant was discarded and 1 mL of fresh MHB was added to the pellet. Solution is then vortexed in order to ensure the suspension of bacteria. Eighty microliters of fresh bacterial suspension was added to 66 µL of [3H]thymidine (Amersham, specific activity 1 mCi/mL) in 1 mL of MHB. The solution was incubated for 3 h at 37 °C to reach the exponential phase of bacterial growth. After the incubation period, bacterial suspension was centrifuged twice at 3000g for 15 min to remove unbound radioactivity. Ca2+ and Mg2+ supplemented PBS was then added to the pellet in order to obtain suitable bacterial dilution. One hundred microliters of [3H] radiolabeled bacterial suspension was added to 2 mL of scintillation fluid, and the radioactivity was measured with an automatic β-liquid scintillation counter (1214 Rackbeta LKB from Wallac). Bacterial concentrations were measured for each experiment by measuring viable cells by colony forming units (CFU) counting on agar plates. Staphylococcus aureus Attachment on Fibronectin-Coated PMMA Adherence Assay in Correlation to Fibronectin (Fn) Concentration. The attachment of S. aureus Cowan I to polymers was measured following the described experimental procedure derived from Vaudaux et al.27,28 (1) Passivation of the PMMA lenses: lenses (2.5 cm2 total surface) were incubated for 30 min at room temperature with PBS solution containing 0.4 g/L bovine serum albumin (0.4 g/L BSAPBS), under stirring, to avoid nonspecific adsorption of fibronectin. (2) Adsorption of fibronectin for 1 h at 37 °C under stirring with concentrations of Fn-PBS solutions varying from 0 to 25 µg/mL. (3) Elimination of unbound proteins by two washings with 0.4 g/L BSA-PBS. (4)

Biomimetic PMMA-Based Terpolymers

Figure 1. Schematic representation of MMA/MA/NaSS random terpolymer.

Addition of 1 mL of washed-log phase [3H]thymidine radiolabeled S. aureus Cowan I followed by an incubation for 60 min at 37 °C. Adhesion medium was 0.4 g/L BSAPBS supplemented with (1 mM) Ca2+ (0.5 mM) Mg2+. (5) PBS washings of lenses and β counting to estimate the number of attached bacteria.29 Attachment of S. aureus onto the Fn-coated lenses was averaged and normalized to the number of adherent CFUs/ cm2. Percent of adherence was calculated as follows: adherence % ) [cpm(adherent)/cpm(inoculated)] × 100 Correlation of Adherence Assay to Bacterial Concentration. The optimal bacterial size was determined by incubating Fn-coated PMMA lenses with various dilutions of log-phase cultures (metabolically radiolabeled). Polymer lens was incubated with 1 mL of purified Fn in PBS (20 µg/mL initial concentration that corresponds to 140 ng/cm2 adsorbed) for 60 min at 37 °C. Bacterial adherence was then assayed by incubating the Fn-coated PMMA lens with 1 mL of radiolabeled cultures (0.25 × 103 to 0.5 × 106 cpm/mL corresponding to 0.25 × 105 to 0.5 × 108 CFUs/mL). Staphylococcus aureus Attachment onto Fn-Coated PMMA-Derived Terpolymers. Bacterial attachment was tested on PMMA-based terpolymers as follows: Terpolymer lens (2.5 cm2 total surface) was coated with 0.4 g/L BSA in PBS for 30 min at room temperature under stirring. Terpolymers were incubated with 20 µg/mL of purified Fn in PBS for 60 min at 37 °C. Bacterial adherence was assayed by incubating Fn-coated lenses with 1 × 107 CFUs from log-phase cultures. Lenses were then twice rinsed with PBS and transferred to scintillation vials before β counting. To get mean values, we summed duplicate samples and normalized to adherent CFUs/cm2. By considering 100% of adherence onto PMMA, the inhibition of bacterial adherence exhibited by each terpolymer was expressed as the ratio of its adherence percentage and PMMA adherence percentage. Statistical Analysis. Results are expressed as mean values ( standard error of duplicate experiments. Data were analyzed by using Newman-Keuls multiple comparison tests. Results Synthesis and Characterization of Functional Terpolymers. Terpolymers were prepared by radical terpolymerization of MMA, MA, and NaSS in DMSO as schemed in Figure 1. Polymerization reactions were stopped after total

Biomacromolecules, Vol. 3, No. 1, 2002 65

conversion of the monomers and checked by GC and 1H NMR. Integration peaks corresponding to the three monomer units MMA, MA, and NaSS (Figure 2) allowed the determination of terpolymer composition. The initial feed in monomers and the final composition of the terpolymers determined after precipitation in water and washings with different solvents were reported in Table 1. The total contents in ionic groups (MA + NaSS) was maintained below 17 mol % (initial feed) in order to prevent water and buffer medium solubilization of the terpolymer. For ionic groups contents higher than 15%, high swelling of terpolymers was observed. To check that all the carboxylic groups were carboxylated at pH 7.4, apparent pKa values of poly(methacrylic acid) (PMA) were determined in both DMSO/water mixture and water to be pKa(DMSO) ≈ 8.4 and pKa(H2O) ≈ 5.5. The apparent pKa value of carboxylic acid groups in terpolymers could only be determined in DMSO/water due to its insolubility in water; the mean pKa value for all of the terpolymers was calculated and found equal to 8.46 ( 0.5. By extrapolation, the pKa value of carboxylic groups from MA units in the terpolymers was approximated to 5.5 in water. Given that experiments were carried out at pH 7.4, carboxylic acid groups of terpolymers were estimated to be 95-100% carboxylated. The comparison between initial feed in monomers and final compositions of the terpolymers after precipitation and washings with water shows a good agreement. This observation suggests that the macromolecular chain compositions are homogeneous until almost complete conversion of the monomers.30 From the reactivity ratios determined for the three couples of monomers MMA/MA, MMA/NaSS, and MA/NaSS, Belleney et al.30 showed that the chains become richer in NaSS at the end of the reaction. In the case of heterogeneous compositions, precipitation and washings with water would have eliminated the chains rich in NaSS units due to the high solubility of poly(sodium styrenesulfonate) (PNaSS) in this solvent. However, NaSS contents are systematically lower than 10% (Table 1) limiting its influence on physical and mechanical properties of the polymer. This is not the case for MMA units, which are largely predominant in terpolymers. To remove chains too rich in MMA units, terpolymers have been washed with PMMA solventss acetone or dichloromethane. Compositions of the terpolymers after extraction (see Table 1) are quite puzzling. When the initial feed in monomers was compared to final compositions for Z10 and Z11 terpolymers, it showed that dichloromethane washings had no effect. This solvent does not seem to extract chains rich in MMA units, indicating that their compositions were homogeneous. Whereas if washed with acetone, MMA content in Z8 and Z9 terpolymers increased when compared to initial feed. The extraction yields after washing with acetone or dichoromethane were higher than 50%, indicating that both solvents solubilized all the chains (rich or not in ionic groups) especially in the case of dichoromethane. Surprisingly chains rich in ionic groups seem to be predominantly eliminated in the case of acetone extraction. The

66

Biomacromolecules, Vol. 3, No. 1, 2002

Berlot et al.

Figure 2. Typical 1H NMR spectrum (200 MHz) of a terpolymer in DMSO-d6. Table 1. 1H NMR Determined Final Composition of Terpolymers initial feed (mol %) composition of terpolymers (mol %) refs MMA MA NaSS

MMA

MA

NaSS

R

Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9 Z10 Z11

89.5 93 92 93 90 88.2 81.2 94.4 90.7 85 84

3.5 5 6 2 8 6.9 11.9 3.6 5 10 14

7 2 2 5 2 4.8 6.9 2 4.3 5 2

0.33 0.71 0.75 0.28 0.8 0.59 0.63 0.64 0.54 0.66 0.88

93.1 92.5 92 90 90 88 85 85 88 85 84

2.3 5 6 5 8 7 10 10 7 10 14

4.6 2.5 2 5 2 5 5 5 5 5 2

consequence could be that the compositions of the chains are highly heterogeneous for terpolymers Z8 and Z9. Lenses were prepared by dissolving the terpolymers referenced in Table 1 in DMSO solvent, pouring solutions in molds, drying, and eliminating the solvent under vacuum. Lenses are transparent when detached. In water, lenses swelled with an the uptake close to 30%. Hydration led to homogeneous lenses presenting no shape alteration. Bacterial Adherence on PMMA. The first step was the preadsorption of PMMA lenses with various radiolabeled [125I]fibronectin solutions. Lenses were incubated for 1 h at 37 °C with [125I]Fn solutions varying from 1 to 25 µg/mL. After PBS washings, adsorbed fibronectin onto the surface was calculated and found in the range of 10 to 140 ng/cm2. The Fn-adsorbed lenses were incubated with bacteria dilutions varying from 2.5 × 104 to 5 × 107 CFU/mL. Adherence of S. aureus Cowan I was obtained by using radiolabeled bacteria according to the above-described procedure. Fixed CFU values were plotted versus incubated CFU giving a linear variation. The slope of the straight line gives the percentage of adherent bacteria (Figure 3). In the domain of bacteria dilutions used in the assays, polymeric surfaces were not saturated; values of adherent bacteria

Figure 3. Adhesion of S. aureus Cowan I on PMMA, influence of the amount of total inoculated radioactivity.

Figure 4. Adhesion of S. aureus Cowan I on PMMA, influence of adsorbed Fn concentration.

percentages were calculated per cm2 in each experiment to take the surface of lenses into account. For instance, bacteria adherence calculated on the lenses precoated by 140 ng/cm2 of fibronectin was found to 0.9 ( 0.1% as previously observed and mentioned by Vaudaux et al.27 Percent of adhered S. aureus Cowan I per cm2 of lenses versus adsorbed Fn is shown in Figure 4. In the absence of Fn, S. aureus adherence was around 0.05%/cm2; this very low value has been previously observed by many authors.7,27 Increasing the amount of adsorbed fibronectin favors the adherence of bacteria until a plateau value is reached at 140

Biomacromolecules, Vol. 3, No. 1, 2002 67

Biomimetic PMMA-Based Terpolymers Table 2. Percentages of Adherence and Inhibition Compared to PMMA as Control for Functionalized PMMA-Based Terpolymersa refs

R ) COO-/ (SO3- + COO-)

% adherence/ cm2

std error

% inhibition/ PMMA

PMMA Z1 Z4 Z9 Z6 Z7 Z8 Z10 Z2 Z3 Z5 Z11

0.28 0.33 0.54 0.59 0.63 0.64 0.66 0.71 0.75 0.8 0.88

0.90 0.03 0.02 0.08 0.10 0.07 0.09 0.05 0.03 0.02 0.02 0.74

0.1 0.005 0.006 0.010 0.010 0.005 0.010 0.016 0.001 0.001 0.003 0.010

0 97 98 92 89 93 90 95 97 98 98 18

a

Standart error of the average of fours determinations.

ng of adsorbed Fn/cm2. The maximal adherence value equals 0.9%/cm2, which is 18 times higher than the adherence in the absence of Fn. To evaluate the influence of the terpolymer compositions on bacterial attachment, we chose working within the range of the plateau value (140 ng/cm2). To keep from bacterial saturation on the polymeric surface (Figure 3), all the experiments were performed at (1-2) × 107 CFU/ mL. Adherence percentages of S. aureus Cowan I onto the terpolymers, characterized by the ratio R ) COO-/(COO+ SO3), are reported in Table 2. Results show that the adherence percentages are very low (0.015 ( 0.006 and 0.05 ( 0.04%/cm2 mean adhesion ( standard error, p < 0.001 Newman-Keuls test) whatever the R ratio value when compared to PMMA (0.9 ( 0.1%/cm2). This allowed us to calculate the inhibition percentages of bacteria adhesion when compared to PMMA as control. Fn-coated terpolymers exhibited very high inhibition percentage of S. aureus Cowan I adherence (between 89 and 98%) as far as R values stayed lower than 0.88. Terpolymer with an high COO- content (Z11) was suggested to favor bacteria attachment as indicated by the observed low inhibition percentage of adherence (18%). It is surprising that terpolymers Z3 and Z2 with high contents in carboxylate groups have so drastic an effect on preventing attachment compared to terpolymer Z11. This could be explained by the low content in total ionic groups of these two terpolymers (lower than 9%). Discussion The aim of this work was to synthesize functionalized PMMA-based polymers able to prevent the attachment of S. aureus Cowan I. To reach this goal, 11 terpolymers were synthesized by radical polymerization of MMA and two monomers bearing carboxylate and sulfonate groups. Terpolymers were precipitated in water, and final compositions in ionic groups determined by 1H NMR have been found to vary from 7 to 18.8 in mol %. To remove chains with too high MMA unit content, which is the main comonomer, extraction with PMMA solvents (acetone or dichloromethane) was achieved. The compositions of the “extracted” terpoly-

mers showed no selectivity in the extraction of chains richer in hydrophobic monomer. Terpolymers and PMMA as polymer control were used to prepare films in shape of lenses that were coated with Fn. In the case of PMMA, results showed that the presence of Fn-coating significantly promoted S. aureus Cowan attachment onto the surface. Moreover, adherence of S. aureus Cowan I onto PMMA is directly related to the amount of adsorbed Fn. Attachment was found to reach a plateau at 0.9%/cm2. This is 18 times higher than the attachment value in the absence of Fn. These results are consistent with previous studies suggesting that Fn may favor S. aureus infection by promoting attachment to implanted foreign bodies.5,7,13 S. aureus Cowan I attachment was then studied on Fn coated-functionalized PMMA-based terpolymers, and the results were compared to those obtained on PMMA as control. Modulation of S. aureus Cowan I adherence was observed on terpolymers of various compositions characterized by R ) COO-/COO- + SO3-. Terpolymers with R ratio values between 0.28 and 0.8 led to high inhibition percentages of S. aureus attachment. This observed low adherence value was independent of the ionic groups content (COO+ SO3-) for the same R value. For instance, inhibition percentages of Z2 terpolymer (R ) 0.71, 7% ionic groups) and of Z7 terpolymer (R ) 0.63, 18.8% ionic groups) when compared to PMMA were respectively 97 and 93%. The main result is the presence of both sulfonate and carboxylate groups, which interact specifically with Fn and prevent its interaction with bacteria leading to the observed bacteriostatic properties. Another point that should be considered is the variation of the biological properties with the homogeneity of the ionic groups distribution along all the chains. Recent results showed that the biological properties seem to be independent of this factor.31 The fact that Z8 and Z9, which present heterogeneities in sulfonate and carboxylate groups compositions due to acetone extraction, give high inhibition adherence percentage (close to 92%) confirms this hypothesis. The parameter which controls the interaction between the living system (here bacteria) and the material is the distribution of the ionic groups along the macromolecular chains. Whatever the terpolymer tested, the radical terpolymerization of the three monomers, MMA, MA, and NaSS gave random distribution as shown by Belleney et al.30 The synthesis process was assumed to induce the formation of active sites that interact specifically with Fn; the sites involve MA, NaSS, and MMA units, the latter being there only as spacer to impose precise distances between carboxylate and sulfonate functions. For high contents in carboxylate groups, bacterial adhesion was increased to a value (0.74 ( 0.01%) close to the adherence percentage observed on PMMA. This result should be confirmed by testing copolymers containing only MMA and MA units. The observed adherence inhibition may be related to modifications of the conformation of Fn when adsorbed onto polymeric surfaces, as already suggested to explain modifications of cell proliferation and differentiation.31,32 Modifications of absorbed Fn conformation were described on

68

Biomacromolecules, Vol. 3, No. 1, 2002

cross-linked polystyrene bearing sulfonate and carboxylate ionic groups; this effect was also found to depend on the COO- and SO3- ratio.34 The authors suggested that conformational changes involved the heparin-binding domains of Fn; under physiological conditions, three separated heparinbinding domains can be identified on each Fn subunit. Both of them are located in the amino-terminal part35,36 and in the carboxy-terminal domain37-39 of the molecule. These two domains were shown to be responsible for S. aureus attachment to Fn. With conformational changes of Fn having a great influence on the availability of these domains, they strongly modified the potential binding to bacteria. We have hypothesized that Fn adsorption onto terpolymers of appropriate chemical compositions left unavailable its Nterminal and/or C-terminal domains that are essential for staphylococcal adhesion. In conclusion, functionalized PMMA-based terpolymers bearing COO- and SO3- groups were synthesized by radical polymerization and characterized. Attachment of S. aureus Cowan I was evaluated onto terpolymers previously adsorbed with Fn, and a strong inhibiting effect of bacterial adhesion was observed. This inhibition of S. aureus attachment by functionalized PMMA-based terpolymers is of great interest in the field of biomedical implants and especially for ophthalmic applications. However, low adherence of bacteria may not always be correlated with inhibition of growth.40 We showed that both sulfonate and carboxylate functions randomly distributed on a chain of polymer can prevent attachment of bacteria. The next step will be the effect of these functional groups on the kinetics of S. aureus Cowan I proliferation. Depending on the results and by taking into account that these terpolymers have the property to inhibit lens epithelial cell proliferation, one major complication after cataract operation,41 this new bioactive material could be used to make intraocular implants. Acknowledgment. The authors thank the Creavis Society (Marl, Germany) for financial support of this work. References and Notes (1) Vaudaux, P.; Yasuda, H.; Velazco, M. I.; Huggler, E.; Ratti, I.; Waldvogel, F. A.; Lew, D. P.; Proctor, R. A. J. Biomater Appl. 1990, 5, 134. (2) Vaudaux, P.; Suzuki, R.; Waldvogel, F. A.; Morgenthaler, J. J.; Nydegger, U. E. J. Infect. Dis. 1984, 150, 546. (3) Andrade, J. D. Hlady, V. Ann. N. Y. Acad. Sci. 1987, 516, 158. (4) Vaudaux, P.; Pittet, D.; Haeberli, A.; Huggler, E.; Nydegger, U. E.; Lew, D. P.; Waldvogel, F. A. J. Infect. Dis. 1989, 160, 865. (5) Francois, P.; Vaudaux, P.; Foster, T. J.; Lew, D. P. Infect. Control Hosp. Epidemiol. 1996, 17, 514. (6) Foster, T. J.; McDevitt, D. FEMS Microbiol. Lett. 1994, 118, 199. (7) Herrmann, M.; Lai, Q. J.; Albrecht, R. M.; Mosher, D. F.; Proctor, R. A. J. Infect. Dis. 1993, 167, 312. (8) Franc¸ ois, P.; Schrenzel, J.; Stoerman-Chopard, C.; Favre, H.; Herrmann, M.; Foster, T. J.; Lew, D. P.; Vaudaux, P. J. Lab. Clin. Med. 2000, 135, 32.

Berlot et al. (9) Delmi, M.; Vaudaux, P.; Lew, D. P.; Vasey, H. J. Orthop. Res. 1994, 12, 432. (10) Kamal, G. D.; Pfaller, M. A.; Rempe, L. E.; Jebson, P. J. R. JAMA 1991, 265, 2368. (11) Tebbs, S. E.; Elliott, T. S. J. J. Antimicrob. Chemother. 1993, 31, 261. (12) Vaudaux, P.; Avramoglou, T.; Letourneur, T.; Lew, D. P.; Jozefowicz, J. J. Biomater. Sci. Polym. Ed. 1992, 4, 89. (13) Pascu, C.; Hirmo, S.; Ljungh, A.; Wadstro¨m, T. Microbiol. Lett. 1996, 45, 263. (14) Arciola, C. R.; Radin, L.; Alvergna, P.; Cenni, E.; Pizzoferrato, A. Biomaterials 1993, 14, 1161. (15) Arciola, C. R.; Caramazza, R.; Pizzoferrato, A. J Cataract RefractiVe Surg. 1994, 20, 158. (16) Desai, N. P.; Hossainy, S. F. A.; Hubbel, J. A. Biomaterials 1992, 13, 417. (17) Kritinsson, K. G. J. Med. Microbiol. 1989, 28, 249. (18) Jansen, B. Clin. Mater. 1990, 6, 65. (19) Lowe, A. B.; Vamvakaki, M.; Wassall, M. A.; Wong, L.; Billingham, N. C.; Armes, S. P.; Lloyd, A. W. J. Biomed. Mater. Res. 2000, 52, 88. (20) Lee, H. J.; Park, K. D.; Park, H. D.; Lee, W. K.; Han, D. K.; Kim, S. H.; Kim, Y. H. Colloids Surf., B 2000, 18, 355. (21) Jozefowicz, M.; Jozefonvicz, J. Biomaterials. 1997, 18, 1633. (22) Ishihara, K.; Biocompatible polymers. Part 3.3; Tsuruta, T., Hayashi, T., Ishihara, K., Kataoka, K., Kimura, T., Eds.; Biomedical Applications of Polymeric Materials; CRC Press: Boca Raton and Ann Arbor, 1993; p 143. (23) Werner, L. P.; Legeais, J. M. J. Fr. Ophthalmol. 1998, 21, 515. (24) Refojo, M. F. Encyclopedia of Polymer Science and Technology; Interscience: New York, 1978; Vol 1, pp 195-219. (25) Sherwood, D. R.; Rich, W. J.; Jacob, J. S.; Hart, R. J.; Fairchild, Y. L. Eye 1989, 3, 308. (26) Dilly, P. N.; Sellors, P. J. J. Cataract RefractiVe Surg. 1989, 15, 317. (27) Vaudaux, P. E.; Waldvogel, F. A.; Morgenthaler, J. J.; Nydegger, U. E. Infect. Immun. 1984, 45, 768. (28) Vaudaux, P. E.; Francois, P.; Proctor, R. A.; McDevitt, D.; Foster, T. J.; Albrecht, R. M.; Lew, D. P.; Wabers, H.; Cooper, S. L. Infect. Immun. 1995, 63, 585. (29) Vaudaux, P.; Pittet, D.; Haeberli, A.; Lerch, P. G.; Morgenthaler, J. J.; Proctor, R. A.; Waldvogel, F. A.; Lew, D. P. J. Infec.t Dis. 1993, 167, 633. (30) Belleney, J.; Helary, G.; Migonney, V. Eur. Polym. J., in press. (31) El Khadali, F.; He´lary, G.; Pavon-Djavid, G.; Migonney, V. Biomacromolecules 2002, 3, 0000. (32) Underwood, P. A.; Steele, J. G.; Dalton, B. A. J. Cell Sci. 1993, 104, 793. (33) Garcia, A. J.; Vega, M. D.; Boettiger, D. Mol. Biol. Cell 1999, 10, 785. (34) Stanislawski, L.; Serne, H.; Stanislawski, M.; Jozefowicz, M. J. Biomed. Mater. Res. 1993, 27, 619. (35) Ruoslahti, E.; Hayman, E. G.; Engvall, E.; Cothran, W. C.; Butler, W. T. J. Biol. Chem. 1981, 256, 7277. (36) Mosher, D. F. Prog. Hemostasis Thromb. 1980, 5, 111. (37) Hayashi, M.; Yamada, K. M. J. Biol. Chem. 1981, 256, 11292. (38) Yamada, Y.; Kleinman, H. K. Curr. Opin. Cell Biol. 1992, 4, 819. (39) Sakata, N.; Jakab, E.; Wadstrom, T. J. Biochem. (Tokyo) 1994, 115, 843. (40) Gottenbos, B.; Van der Mei, H. C.; Busscher, H. J. J. Biomed. Mater. Res. 2000, 50, 208. (41) Latz, C.; Migonney, V.; Pavon-Djavid, G.; Rieck, P.; Hartmann, C.; Renard, G.; Legeais, J. M. Graefes Arch. Clin. Exp. Ophthalmol. 2000, 238, 696.

BM015580M