Intrinsically Antibacterial Materials Based on Polymeric Derivatives of

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Biomacromolecules 2008, 9, 2530–2535

Intrinsically Antibacterial Materials Based on Polymeric Derivatives of Eugenol for Biomedical Applications Luis Rojo,*,† Jose. M. Barcenilla,‡ Blanca Va´zquez,† Ramoń Gonza´lez,‡ and Julio San Romań† Institute of Polymer Science and Technology, CSIC, and CIBER-BBN, C/ Juan de la Cierva 3, 28006 Madrid, Spain, and Institute of Industrial Fermentations, CSIC, C/ Juan de la Cierva 3, 28006 Madrid, Spain Received May 22, 2008; Revised Manuscript Received July 15, 2008

Infections are the most common cause of biomaterial implant failure representing a constant challenge to the more widespread application of medical implants. This study reports on the preparation and characterization of novel hydrophilic copolymeric systems provided with antibacterial properties coming from eugenol residues anchored to the macromolecular chains. Thus, high conversion copolymers were prepared from the hydrophilic monomer 2-hydroxyethyl methacrylate (HEMA) and different eugenol monomeric derivatives, eugenyl methacrylate (EgMA) and ethoxyeugenyl methacrylate (EEgMA), by bulk polymerization reaction. Thermal evaluation revealed glass transition temperature values in the range 95-58 °C following the order HEMA-co-EgMA > PHEMA > HEMA-co-EEgMA and a clear increase in thermal stability with the presence of any eugenyl monomer in the system. In vitro wettability studies showed a reduction of water sorption capacity and surface free energy values with increasing the content of eugenol residues in the copolymer. The antimicrobial activity of copolymeric discs was evaluated by determining their capacity to reduce or inhibit colony formation by different bacterial species. All eugenyl containing materials showed bacteria growth inhibition, this one being higher for the EEgMA derivative copolymers.

Introduction Infections play an important role in all surgery fields as dental restorative implants, orthopedics, and so on. Consequently, in recent years, infections are the most common cause of biomaterial implant failure and represent a significant challenge to the more widespread application of medical implants.1-3 An approach to prevent these biomaterial related infections is to provide the biomaterial with some antimicrobial properties by functionalizing it with different basteriostatic functional groups such us amine groups, ammonium groups, and so on.4 Studies have been made on the introduction of biologically active groups to monomers, followed by their polymerization.5 This is a unique approach to achieve intrinsically antibacterial materials which do not release any bactericidal components due to their stabilization against volatilization, dissolution and diffusion. The main advantage of these systems is that they present a longer antibacterial action and, at the same time, that the use of reactive monomers, fixatives, toxic solvents, or other additives for stabilization is avoided. In consequence, antimicrobial biopolymers may be incorporated into fibers, resins, oils and many other medical products for uses as meshes, gels, ointments, and so on, and would be a promising solution for many problems associated to the treatment of different diseases as periodontal infections, bacterial colonisation of implants, and so on. In this sense, polymeric biocide materials have been developed in the recent years for specific applications as coatings for artificial implants, active insoluble antibiotics, and so on.6 Another approach to obtain materials with antibacterial properties is the immobilization of water-soluble or emulsible disinfectant onto * To whom correspondence should be addressed. Tel.: +34 915 622 900, Ext 432. Fax: +34 915 644 853. E-mail: [email protected]. † Institute of Polymer Science and Technology. ‡ Institute of Industrial Fermentations.

macromolecular material surfaces. In this case, the anchorage of soluble antimicrobial agents onto the matrix may be a simple way to achieve the release of the agent from the material in a wet environment. The main advantage of this approach is the potential capacity to release and control the doses of the released active product. However, both strategies also present some disadvantages to be used in biomedical applications and further modifications are needed to achieve clinically useful materials. For instance, some of the main challenges of the intrinsically antibacterial materials concern with the control of the hydrolysis and lost of chemical stability in body environments. For systems of the latter approach, the main drawback lies in the difficulty of controlling the adequate active dose of the soluble principle and its possible excretion through the organism. Eugenol is a well-known antimicrobial essential oil found in cloves. In the biomedical field it is mainly used in dentistry as a root canal sealer. Eugenol containing materials have been shown to inhibit the growth of a range of microorganisms, including facultative anaerobes commonly isolated from infected root canals.7 Eugenol has also been shown to inhibit the growth of Escherichia coli,8 Penicillium citrinium,9 and human herpes virus in vitro.10 However, eugenol containing materials present serious disadvantages due to the fact that the release of some concentrations of eugenol can produce tissue irritation and induce inflammatory reactions over the oral mucous membrane. Moreover, eugenol is a strong antioxidant and a potent free radical inhibitor, which makes necessary a complete cleaning of the dental scenery when a restorative composite based on the free radical polymerization of the components is applied. The covalent linking of eugenol to macromolecular chains is advantageous as it reduces the migration of eugenol to the surrounding tissues and improves its hydrolytic stability.

10.1021/bm800570u CCC: $40.75  2008 American Chemical Society Published on Web 08/15/2008

Antibacterial Materials for Biomedical Applications

Figure 1. Structure of the eugenyl derivative monomers and their respective copolymers with 2-hydroxyethyl methacrylate (HEMA).

The use of acrylic polymers (such us PHEMA, PMMA, etc) as biomaterials is widely extended due to their easy and economical manufacture as well as their optimal mechanical properties and adequate temporal stability. However the polymers derived from this family do not present specific bioactivity, playing only structural and filling functions. A continuous challenge in our days consists of the functionalization of these acrylic systems by the incorporation of bioactive residues. Recent works of our research group have led to the development of new eugenol derivative copolymeric systems which offer an interesting strategy to provide with biological properties the acrylic systems.11,12 It has been demonstrated that the eugenol derivative monomers do not produce the inhibitory effect of eugenol against free radical polymerization11 and high molecular weight copolymers can be obtained. Taking all those antecedents into consideration, the aim of this study was the preparation and characterization of novel hydrophilic copolymeric systems provided with intrinsically antibacterial properties coming from eugenol residues anchored to the macromolecular chains. These systems have potential application as antibacterial implants in different biomedical fields, for example, intraocular lenses and other ocular implants in ophthalmology, or permanent dental cements in dentistry, to mention some of them. Thus, high conversion copolymers of eugenyl methacrylate (EgMA) and ethoxyeugenyl methacrylate (EEgMA), with the hydrophilic monomer 2-hydroxyethyl methacrylate (HEMA), have been prepared and characterized. Their in vitro wettability has been studied in conjunction with their antibacterial activity. The antimicrobial activity of the copolymers has been evaluated by determining their capacity to reduce or inhibit colony formation by different bacterial species.

Materials and Methods Materials. Eugenol (Acros) was used as received without further purification. HEMA (Aldrich) monomer was purified by distillation under reduced pressure. Eugenyl monomers derivatives, eugenyl methacrylate (EgMA) and etoxyeugenyl methacrylate (EEgMA) were synthesized as reported previously.11 2,2′-Azobisisobutyronitrile (AIBN; Merck) was recrystallized from methanol (mp 194 °C). The solvents dimethyl sulfoxide (DMSO; Scharlau) and n-hexane (SDS) were purified by standard procedures. Copolymers Preparation. High conversion poly(hydroxyethyl methacrylate) (PHEMA) and p(HEMA-co-EgMA) and p(HEMA-coEEgMA) copolymers from feed HEMA/eugenyl monomer molar ratios of 80:20 and 50:50 were obtained by bulk radical polymerization using AIBN as a radical initiator (1 wt % with respect to monomers). A total of 250 mg of the corresponding monomer or monomer mixture was

Biomacromolecules, Vol. 9, No. 9, 2008

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introduced in polypropylene capped cylinders molds of 15 mm of diameter. The reaction medium was satured with N2 atmosphere for 15 min and then the tube was capped and heated at 70 °C for 2 h. Subsequently, the temperature was reduced to 60 °C and the reaction was allowed to proceed for 46 h. The polymerized samples were extracted from the molds, thoroughly washed with hexane to remove residual monomers and filed with sandpaper to obtain 1 mm thickness discs. Finally, the samples were dried under vacuum until constant weight. Chemical structures of the monomers and copolymers obtained are given in Figure 1. Copolymers Characterization. ATR-FTIR spectra of all the copolymers and the corresponding homopolymers were recorded on a Perkin-Elmer Spectrum One spectrophotometer with an ATR attachment. The soluble fraction of each material was isolated by Soxhlet extraction in toluene for 48 h. After the treatment, samples were dried under vacuum and the percentage of soluble fraction (Fsol) was calculated by using eq 1

Fsol(%) ) [100 × (Wi - Wf) ⁄ Wi]

(1)

where Wi is the initial weight of the sample before the treatment and Wf is the dried weight of the sample after treatment. Three samples of each material were tested and results were averaged. Glass transition temperatures (Tg) of the copolymers and the corresponding homopolymers were measured using differential scanning calorimetry (DSC) with a Perkin-Elmer DSC7 interfaced to thermal analysis data system TAC 7/DX. Films of dry samples (15-20 mg) were placed in aluminum pans and heated from -20 to 150 °C at a constant rate of 10 °C/min. Tg was taken as the midpoint of the heat capacity transition. Two samples were tested for each material. Thermogravimetric diagrams were obtained in a thermogravimetric analyzer using a TGA Q500 (TA Instrument) apparatus, under dynamic nitrogen at heating rate of 5 °C/min in a range of 40-600 °C. Samples were tested in duplicated. Wettability. Swelling experiments were performed on polymeric discs of 15.0 mm diameter and 1 mm thickness. Discs of polymers were accurately measured, weighed and introduced in borate buffered solution (H3BO3 2.5 g/L; Na2BO4 · 10H2O 0.15 g/L; NaCl 8.0 g/L) at 37 °C where they were kept until they reached equilibrium. The values of the percentage of hydration degree (H) were determined gravimetrically using eq 2

H(%) ) [100 × (Weq - Wi) ⁄ Wi]

(2)

where Weq is the weight of the wet sample at equilibrium and Wi is the initial weight of the dry sample. Three samples of each material were evaluated and the results were averaged. Contact angles were measured on nonfiled polymer surface discs by using the sessile drop technique and employing liquids with known surface tension: water (γL ) 72.8 mN/m2) and methylene iodide (γL ) 51.8 mN/m2). γL refers to the total surface free energy of the liquid. A minimum of 10 drops were applied on each sample. The surface free energy (γS) of the polymer and copolymers was calculated by the Fowkes’13 and Owens’ method.14

γs ) γds + γps

(3)

(1 + cos θ)γ1 ⁄ 2 ) (γds γdl )1⁄2 + (γps γpl )1⁄2

(4)

where θ is the contact angle, γs and γl are the surface free energy of solid and liquid, respectively, and γsd, γsp, γld, and γlp are the dispersive and polar components of γS of solid and liquid, respectively. Antibacterial Assays. Escherichia coli (DH5R) and Streptococcus mutans (CECT479) strains were used as gram-negative and grampositive models, respectively. Precultures were grown overnight at 37 °C in Brain-Heart Infusion (BHI; Difco Laboratories, U.S.A.) or Luria Broth (LB, Yeast extract 0.5%; tryptone 1%; NaCl 1%, all supplied by Difco Laboratories, U.S.A.), until stationary phase. Concentration of bacterial suspension was determined by measuring absorption at 600 nm (A600; Boecos-22 spectrophotometer) and interpolating the A600

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Rojo et al. Table 1. Values of Glass Transition Temperature (Tg), Soluble Fraction (Fsol), and TGA Data for Degradation of Eugenyl Containing Copolymers system PHEMA P(HEMA-co-EgMA) PEgMA p(HEMA-co-EEgMA) PEEgMA

Tmaxc T50%d composition Fsol (%) [S.D.]b Tg (°C) (°C) (°C) (%) 100 80:20 50:50 100 80:20 50:50 100

3 [0.6] 1 [0.8] HEMA-co-EEgMA. Tg values, and therefore flexibility, were strongly dependent on the content of eugenyl moieties present in the copolymer according with results previously reported.19 HEMA-co-EgMA copolymers exhibited values of Tg above the observed for the HEMA homopolymer with a trend to increase with the EgMA content in the copolymer, which reflects the greater rigidity of the EgMA unit. For HEMA-co-EEgMA copolymers, on the other hand, values of Tg decreased with the content of EEgMA units in the copolymer, indicating that the bulky group of the eugenol residue separated from the backbone through an oxyethylene group contributed to the flexibility of the side group. The thermal stability of the copolymers was evaluated by thermogravimetric analysis and results are shown in Figure 3. The thermogravimetric curves of PHEMA showed a broad degradation step around 380 °C ascribed to the random chain scission of the main chain leading to depolymerisation of the acrylic polymer. However the eugenol containing copolymers reduced the range of this step and underwent the depolymerization at higher temperatures. The 50% of eugenyl moieties in the copolymer showed practically single-step decomposition, which can be attributed to the total random scission of the main chain. The presence of the eugenyl monomer provided a clear increase in thermal stability, which is reflected in the values of the temperature for maximum rate of decomposition and


Figure 3. TGA and DTA curves of eugenyl derivative copolymers obtained by bulk polymerization: upper, HEMA-co-EgMA systems; lower, HEMA-co-EEgMA systems.

temperature for 50% weight loss as shown in Table 1. These results confirm that the eugenyl side residue is very active as scavenger of the free radicals produced in the thermal decomposition mechanism at temperatures higher than 250 °C, and it seems clear that this effect could be kept in a similar way in the physiological medium where the eugenyl residues could contribute to the stabilization and inhibition of the free radicals produced by the decomposition of hydroperoxide compounds associated to inflammatory processes. A similar behavior was found previously for the polyacrylic derivative of vitamin E.20 Wettability of Copolymeric Systems. Wettability of the copolymeric systems was studied to determine the influence of the eugenyl moieties present in the copolymers on the hydrophilicity of the systems, which is clearly manifested in their interaction with the medium. Wettability was evaluated by swelling experiments in borate buffered solution of pH ) 7.4 and 37 °C and through characterization of the surfaces properties. Hydration degrees of the copolymers are shown in Table 2 along the corresponding values of the respective homopolymers reported in the literature.19,21 Both eugenyl containing copolymers showed a reduction of water sorption capacity with increasing the content of eugenol residues. This reasonable trend is associated to the increase of the hydrophobicity with the addition of this monomer containing a substituted aromatic ring. In addition to swelling, the hydrophilicity of the surface is specifically important in the material-medium interactions. Thus, surface properties of copolymer discs were assessed by contact angle measurements considering two liquids of opposite polarity, water and methylene iodine. Values of contact angle (θ) are summarized in Table 2 along with those of surface free


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energy (γS). Water contact angle for PHEMA was in the range of the hydrophilic surfaces (47°). In the case of eugenyl containing copolymers, the presence of the eugenyl monomers, EgMA and EEgMA, reduced the surface free energy values from 61 mN/m for the PHEMA, to values below 50 mN/m, raising significantly the hydrophobic character of the corresponding copolymeric surface with the amount of the eugenyl residue. Comparing both eugenyl derivative systems, HEMA-co-EgMA and HEMA-co-EEgMA, the lower value of the EgMA derivatives (40 mN/m) indicates its higher hydrophobic character. According with those results, wettability of the eugenyl derivatives copolymers decreased in the order PHEMA > HEMAco-EEgMA > HEMA-co-EgMA. Antibacterial Assays. The antibacterial effects of both monomers derived from eugenol and those of HEMA and eugenol were studied against S. mutans and E. coli, both commonly found in dental and medical surgery field. Tables 3 and 4 summarize the size of the halo produced around the different monomers in both the hole plate and the standard diffusion assays. It can be seen that eugenol, EgMA, and EEgMA presented antibacterial effects showing inhibition halos in both hole plate and agar diffusion methods. In opposite to that, no halo was detected for HEMA monomer, neither for the DMSO solution used as a blank. The antibacterial effect of the eugenyl monomers against the two strains showed appreciable differences with respect to the eugenol sample. Activity of eugenol and other phenolyc compounds is associated to the alteration of membrane permeability of the microbial cells and the consequent block of ionic pumps as proton and potassium ions.22 This activity is mainly due to two factors, the presence of the hydroxylic group coupled to a delocalized electron system23 and the hydrophobicity of the compounds.24 In spite of the chemical modification of the eugenol molecule to obtain the R,β-unsaturated esters, EgMA and EEgMA, these new derivatives present a good hydrophobic balance and a certain proton exchange capacity. That fact together with the 4-allyl group make those compounds maintain the inhibitory capacity of colony growing. Furthermore, the ester compounds could be hydrolyzed by intracellular microbial enzymes,25 thus generating intracellular eugenol. On the other hand, the acrylic residue did not have appreciable contribution to the activity, as indicated by the absence of inhibitory halos for the hydroxyethyl methacrylate samples. Due to the hydrophobic character of the EgMA and EEgMA derivatives, they could diffuse through the cytoplasm membranes where the inhibitory capacity of each compound depends on the degree of interaction with the cell functions. EgMA samples presented lower inhibition capacity showing reduced halos compared to those of eugenol. However, the eugenol residue in EEgMA presented a higher mobility, which could contribute to a higher interaction degree with the lipid bilayer in the membranes causing changes in their physicochemical properties and altering protons flow through membranes.26 These higher interactions induce a greater inhibition capacity resulting in higher inhibition halos. Antibacterial activities of copolymers were determined by an agar contact method for which the samples are bulk discs of the corresponding material. Figure 4 shows the results of this assay where no inhibition zones around the discs were produced by any of the experimental materials, indicating that there was no elution of any antibacterial component, such as residual monomers, from the bulk samples. However, the surfaces of the materials directly exposed to the medium are capable of inhibiting bacterial growth, or at least delaying colony

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Rojo et al.

Table 2. Hydration Degree, Contact Angle, and Solid Surface Free Energy Components for High Conversion HEMA-co-EgMA and HEMA-co-EEgMA Copolymersa system PHEMA p(HEMA-co-EgMA) PEgMA p(HEMA-co-EEgMA) PEEgMA

composition

Η (%) [S.D.m]

θ (H2O) [S.D.n]

θ (CH2 I2) [S.D.n]

d γS (mN/m) [corr.]

d γS (mN/m)

p γS (mN/m)

100 80:20 50:50 100 80:20 50:50 100

50b 18.3 [0.4] 6.8 [0.2] 0.2c [0.05] 18.0 [1.3] 6.2 [0.2] 1.7c [0.3]

46.8 [1.1] 75.6 [0.9] 85.8 [0.9] 86.8 [0.9] 76.4 [1.0] 75.9 [0.8] 75.8 [2.7]

33.8 [3.4] 37.2 [1.3] 42.5 [1.6] 48.2 [2.1] 36.6 [0.2] 39.3 [2.9] 35.9 [0.7]

61.2 [99.6] 45.6 [98.8] 40.1 [99.5] 35.2 [99.2] 46.0 [99.5] 44.1 [99.7] 46.1 [98.6]

42.5 40.3 38.0 35.2 41.3 39.0 41.6

18.7 5.3 2.1 2.2 4.7 5.2 4.5

a S.D. ) standard deviation, n ) 20; m ) 3; θ(H2O) ) water contact angle; θ(CH2I2) ) methylene iodide contact angle; and γS ) surface free energies with their dispersive (γSd) and polar (γSp) components. b Schiraldi et al.21 c Rojo L. et al.19

Table 3. Inhibition Zone Diameters Produced by the Different Test Solutionsa inhibition zones (mm) blank

eugenol

HEMA

EgMA

EEgMA

N.D.

19 [1]

N.D.

7 [2]

22 [1]

a

Determined by the Hole Plate Method against Streptococcus mutans (CECT 479) cultivated at 24 h and 37 °C. DMSO, 1 M. Solvent used as blank. N.D. ) not detected. Standard deviations are in brackets (n ) 3).

Table 4. Inhibition Zone Diameters Produced by the Different Test Solutionsa inhibition zones (mm) blank HEMA eugenol EgMA EEgMA S. mutans (CECT 479) E. coli (DH5R)

N.D. N.D.

N.D. N.D.

22 [2] 25 [2]

10 [1] 10 [2]

14 [2] 13 [1]

a Determined by the standard agar diffusion method against S. mutans (CECT 479) and E. coli (DH5R) cultivated at 24 h and 37 °C. N.D. ) not detected. Standard deviations are in brackets (n ) 3). DMSO, 1 M. Solvent used as blank.

formation on them. Thus, a notable inhibition was observed for bacteria incubated in contact with eugenyl containing copolymers. This inhibitory effect was higher for EEgMA derivatives than for EgMA derivatives. In contrast, bacterial growth inhibition was not observed for PHEMA bulk discs and colonies were formed both beneath and around them. Activity of polymeric materials carrying immobilized antimicrobial agents that cannot be leached and do not form inhibition halo, has been previously reported in the literature.27 Similarly, eugenyl derivative copolymers showed a clear growing inhibition against E. coli and S. mutans but no halo

formation, supporting the hypothesis that the bacteriostatic effect is caused by immobilized agents. It is important to notice that the effect observed consists of the reduction of colonies growth on the agar zone beneath the polymer samples. According to that, dental resins and other restorative materials containing those eugenyl acrylic derivatives would eventually contribute to the reduction or even the inhibition of bacteria growing at the interfaces between materials and the surrounding tissues, for example, in the secondary caries formation process between the dentine surface and the restorative material. Antibacterial materials with immobilized molecules showed their activity only against bacteria which come into contact with them, so that its effect does not reach distant areas from the material. Furthermore, the antibacterial effect of the immobilized component is mainly bacteriostatic as the agent cannot penetrate through the cell wall or membrane unlike free antimicrobials.28 The copolymers studied in this work owe their activity to the eugenyl residue pendant out of the polymeric main chain. However, their activities depend on the contact between those residues and the bacteria. The intensity of this contact depends on several factors as nonspecific interactions resulting from Van der Waals and electrostatic forces and short-range forces as dipole-dipole and hydrophobic interactions. These interactions are related to the polymer humectability and therefore to the free surface energy of the different materials. Several research groups have analyzed the relationship between surface free energy and bacterial interaction. According to them, higher values of surface free energy (higher hydrophilicity) induce lower bacterial adhesion on the surfaces while a certain hydrophobic character improves the polymer-bacteria interactions,29,30 although high hydrophobic degrees hinder the

Figure 4. S. mutans (left) and E. coli (right) colony formation (brown areas beneath and surrounding the polymeric specimens) observed after 48 h of incubation at 37 °C. Arrows indicate the end of the polymer disk specimens.


cellular adhesion. Many studies carried out with HEMA derivatives copolymers indicate an optimum bacterial adhesion to surfaces with water contact angles ranged from 65° to 90°, showing lower adhesion levels with the increase of the HEMA content in the corresponding copolymers.31,32 According to that, copolymers reported in this work show higher inhibitory character for increasing contents of the eugenyl derivatives. The higher the hydrophobic character of those copolymers, the higher the interaction with the bacteria, resulting in a greater accessibility to the eugenyl moieties responsible of the bacteriostatic activity. Following the same trend observed for the monomers, EEgMA derivative copolymers present higher flexibility in the macromolecular chains and better humectabillity than EgMA copolymers, providing an effective contact between bacteria and the copolymer surface and, therefore, showing a greater inhibitory character on microbial growth. On the other hand, previously in vitro studies carried out in our research group have demonstrated that both monomer and copolymer derivatives of eugenol present good biocompatibility behavior when they are in contact with human cellular cultures of fibroblasts.19 All these results open a novel approach to achieve biofunctionalized acrylic systems with applications in different biomedical fields such us dentistry, orthopedic surgery, ophthalmology, and so on. The novelty of these systems in comparison with other eugenol containing materials, mainly used in dentistry, lies in the covalently anchorage of the eugenol molecule to the macromolecular structure, avoiding its migration to the surrounding tissues and improving its hydrolytic stability, but maintaining the beneficial properties of eugenol and, therefore, exploiting the applications of the eugenol containing materials in the field of dentistry and extending them to other fields such as orthopedic surgery and ophthalmology. In this sense, new autopolymerizing formulations bearing the eugenyl derivative monomers have being developed, presenting intrinsically antibacterial activity against different microorganisms commonly found in both dental and osseous cavities. Moreover, due to the high refractive index of these functionalized materials, they could also been employed as intrinsically antibacterial ocular implants to obtain very thin and flexible implants to be used as intraocular lenses.

Conclusions Novel intrinsically antibacterial acrylic hydrophilic copolymers containing different eugenyl derivatives were obtained by bulk at high conversion presenting a slightly cross-linked structure which thermal stability, flexibility, wettability and antibacterial properties strongly dependent on the content and type of the eugenyl derivative present in the copolymer. Thus, the presence of the extender separating the active 4-allyl methoxy phenol residue from the inactive polymer backbone provided networks of enhanced flexibility, hydrophilicity, and colonies growing inhibition on the copolymer surface compared with the systems that have the eugenyl residue directly linked through an ester group to the main macromolecular chains. We would like to highlight that both copolymeric systems with immobilized eugenol residues show their bacteriostatic activity against bacteria when the surface of the materials is in contact


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with the microorganims, and this fact would exploit the applications of eugenol through new antimicrobial materials for biomedical applications. Acknowledgment. This research was supported by Comisioń Interministerial de Ciencia y Tecnologıá, CICYT, (MAT200763355) and NoE EXPERTISSUES (UE Contract No. 5002832).

References and Notes (1) Gottenbos, B.; van der Mei, H. C.; Klatter, F.; Nieuwenhuis, P.; Busscher, H. J. Biomaterials 2002, 23, 1417–1423. (2) Neut, D.; van De Belt, H.; Van Horn, J. R.; van der Mei, H. C.; Busscher, H. J. Biomaterials 2003, 24, 1829–1831. (3) Woo, G. L. Y.; Yang, M. L.; Yin, H. Q.; Jaffer, F.; Mittelman, M. W.; Santerre, J. P. J. Biomed. Mater. Res. 2002, 59, 35–45. (4) Kenawy, E. R.; Abdel-Hay, F. I.; El-Magd, A. A.; Mahmoud, Y. J. Bioact. Compat. Polym. 2005, 20, 95–111. (5) Vogl, O.; Tirrell, D. J. Macromol. Sci. Chem. 1979, 13, 415–439. (6) Tashiro, T. Macromol. Mater. Eng. 2001, 286, 63–87. (7) Kaplan, A. E.; Gonzalez, M. I.; Macchi, R. L.; Molgatini, S. L. Dent. Traumatol. 1999, 15, 42–45. (8) Blaszyk, M.; Holley, R. A. Int. J. Food Microbiol. 1998, 39, 175– 183. (9) Vazquez, B. I.; Fente, C.; Franco, C. M.; Vazquez, M. J.; Cepeda, A. Int. J. Food Microbiol. 2001, 67, 157–163. (10) Benecia, F.; Courreˆges, M. C. Phytother. Res. 2000, 14, 495–500. (11) Rojo, L.; Va´zquez, B.; Parra, J.; Lo´pez-Bravo, A.; Deb, S.; San Romań, J. Biomacromolecules 2006, 7, 2751–2761. (12) Rojo, L.; Va´zquez, B.; San Romań, J.; Deb, S. Dent. Mater. 2008 doi: 10.1016/j.dental.2008.04.004. (13) Fowkes, F. M. Ind. Eng. Chem. 1964, 56, 40–52. (14) Owens, D. K.; Wendt, R. C. J. Appl. Polym. Sci. 1969, 13, 1741– 1747. (15) Bauer, A. W.; Kirby, W. M.; Sherris, J. C.; Turck, M. Am. J. Clin. Pathol. 1966, 45, 493–496. (16) Everall, N. J., Chalmers, J. M., Griffiths, P. R., Eds.; In Vibrational Spectroscopy of Polymers: Principles and Practice; Wiley: Chichester, England, 2007. (17) Tyagi, A. K.; Choundary, V.; Varma, I. K. Eur. Polym. J. 1992, 28, 419–422. (18) Rahim, E. A.; Sanda, F.; Masuda, T. J. Macromol. Sci. 2004, 41, 133– 141. (19) Rojo, L.; Borzacchiello, A.; Parra, J.; Deb, S.; Va´zquez, B.; San Romań, J. J. Mater. Sci. Mater. Med. 2008, 19, 1467–1477. (20) Ortiz, C.; Va´zquez, B.; San Romań, J. J. Biomed. Mater. Res. 1999, 45, 184–191. (21) Schiraldi, C.; D’Agostino, A.; Oliva, A.; Flamma, F.; De Rosa, A.; Apicella, A.; Aversa, R.; De Rosa, M. Biomaterials 2004, 25, 3645– 3653. (22) Sikkema, J.; De bont, J.; Poolman, B. Microbiol. ReV. 1995, 59, 201– 222. (23) Ultee, A.; Bennik, M. H. J.; Moezelaar, R. Appl. EnViron. Microbiol. 2002, 68, 1561–1568. (24) Weber, F. J.; de Bont, J. A. M. Biochim. Biophys. Acta 1996, 1286, 225–245. (25) Tamm, C. FEBS Lett. 1974, 48, 7–20. (26) Ben Arfa, A.; Combes, S.; Preziosi-Belloy, L.; Gontard, N.; Chalier, P. Lett. Appl. Microbiol. 2006, 43, 149–154. (27) Isquith, A. J.; Abbot, E. A.; Walters, P. A. Appl. Microbiol. 1972, 24, 859–863. (28) Imazato, S. Dent. Mater. 2003, 19, 449–457. (29) Good, R. J.; Islam, M.; Baier, R. E.; Meyer, A. E. J. Dispersion Sci. Technol. 1998, 19, 1163–1173. (30) Absolom, D. R.; Lamberti, F. V.; Policova, Z.; Zingg, W.; Van Oss, C. J.; Neumann, A. W. Appl. EnViron. Microbiol. 1983, 46, 90–97. (31) Harkes, G.; Feijen, J.; Dankert, J. Biomaterials 1991, 12, 853–860. (32) Hogt, A. H.; Dankert, J.; Feijen, J. J. Biomed. Mater. Res. 1986, 20, 533–545.

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Intrinsically Antibacterial Materials Based on Polymeric Derivatives of

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