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Immobilization of Silver in Polypyrrole/Polyanion Composite Coatings: Preparation, Characterization, and Antibacterial Activity Milena Ignatova,†,‡ David Labaye,† Sandrine Lenoir,† David Strivay,§ Robert Je´roˆme,*,† and Christine Je´roˆme† Centre for Education and Research on Macromolecules (CERM), University of Lie` ge, Sart-Tilman, B6, B-4000 Lie` ge, Belgium, and Institut de Physique Nucle´ aire, Atomique et de Spectroscopie (IPNAS), Sart-Tilman, B15, B-4000 Lie` ge, Belgium Received June 3, 2003. In Final Form: July 28, 2003 Thin films of polypyrrole doped by polyanions have been deposited onto stainless steel and carbon fibers by anodic electropolymerization of pyrrole in the presence of an anionic polyelectrolyte in water. Films with a thickness smaller than 600 nm have been formed, which strongly adhere to the solid supports. The polymeric dopant has been selected for its ability to complex silver ions. Scanning electron microscopy, Rutherford backscattering analyses, and IR spectroscopy have confirmed that silver is actually immobilized in the films and provides them with an antibacterial activity against the Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus.
Introduction Stainless steel 316L is widely used to prepare implants dedicated to orthopedic surgery.1,2 The lower cost of carbon fibers, together with high mechanical performances, low weight, outstanding chemical resistance, and biocompatibility with blood, soft tissues, and bones, make them competitors for application in the biomedical prosthesis field.3,4 One possible limitation of stainless steel containing prostheses and medical devices is their bioinertness and, thus, their inability to stop bacteria proliferation in the case of infection.5 Then, the implanted biomaterial has to be removed. Therefore, there is a need for coatings with specific properties of anti-proliferation and anti-adhesive properties toward bacteria that would strongly adhere to stainless steel and carbon fibers. The “in vitro” compatibility of polypyrrole with mammalian cells is well-known.6 A unique combination of biocompatibility, chemical and thermal stability, ease of preparation, and electroactivity explains why polyaniondoped polypyrrole (PPy) has been considered for biomedical applications.7,8 * To whom correspondence should be addressed. Telephone: (32) 4-3663565. Fax: (32) 4-3663497. E-mail:
[email protected]. † University of Lie ` ge. ‡ Permanent address: Institute of Polymers, bl. 103-A, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. § Institut de Physique Nucle ´ aire. (1) Gristina, A. G.; Naylor, P. T. Implant Associated Infection. In Biomaterials Science: An Introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, CA, 1996; p 205. (2) Park, J. B. Metallic Biomaterials. In The Biomedical Engineering Handbook; Branzino, J. D., Ed.; CRC Press: Boca Raton, FL, 1995; p 537. (3) Longo, J. A.; Koeneman, J. B. In Biomaterials Engineering and Devices: Human Applications: Orthopedic, Dental, and Bone Graft Applications; Wise, D. L., Ed.; Humana Press, Inc.: Totowa, NJ, 2000; Vol. 2, p 203. (4) Donnet, J.-B.; Bansal, R. C. Carbon fibers applications. In Carbon Fibers, 2nd ed.; Lewin, M., Ed.; Marcel Dekker: New York, 1990; p 430. (5) Gristina, A. G.; Rovere, G. D.; Shoji, H.; Nicastro, J. F. J. Biomed. Mater. Res. 1976, 10, 273. (6) Williams, R. L.; Doherty, P. J.; Vince, D. G.; Grashoff, G. J.; Williams, D. F.; Dasgupta, M. K. Crit. Rev. Biocompat. 1989, 5, 221. (7) Collier, J. H.; Camp, J. P.; Hudson, T. W.; Schmidt, C. E. J. Biomed. Mater. Res. 2000, 50, 574.
There are several reports on the biological activity of polymers and copolymers that contain salicylic acid derivatives. Indeed, homopolymers of 4-vinylsalicylic acid and 5-vinylsalicylic acid and their copolymers with methacrylic acid show an antibacterial activity against Grampositive and Gram-negative bacteria.9 A methacrylic derivative of salicylic acid has been polymerized and copolymerized with vinyl pyrrolidone and hydroxyethyl methacrylate into (co)polymers with low toxicity, good analgesic and anti-inflammatory properties, and antiaggregation action on platelets.10,11 The chelating properties of the polyacrylic derivatives of 4- and 5-aminosalicylic acid toward metal ions must also be noted12,13 and increase the range of potential applications of salicylic acidcontaining polymers. For instance, silver ions known for remarkable antibacterial properties, very low toxicity, and desired biocompatibility14 could be immobilized in polymeric salicylic acid derivatives, so enhancing their antibacterial activity. Indeed, bioprosthetic materials prevented infection when loaded with silver.15 “In vivo” growth of Staphylococcus aureus on silver peritoneal catheters was reduced as result of silver release in the environmental tissues.16 In a previous paper,17 some of us reported on adherent coatings of poly(2-acrylamido-2-methyl-1-propanesulfonic (8) Garner, B.; Hodgson, A. J.; Wallace, G. G.; Underwood, P. A. J. Mater. Sci.: Mater. Med. 1999, 10, 19. (9) Vogl, O.; Tirrell, D. J. Macromol. Chem. 1979, A13 (3), 415. (10) San Roman, J.; Madruga, E. L.; Pargada, L. J. Polym. Sci., Polym. Chem. Ed. 1987, 25, 203. (11) San Roman, J.; Darias, V.; Bravo, L.; Sanchez Mateo, C. C.; Tello, M. L.; Abdallah, S. S.; Viva, J. M. Die Pharmazie 1992, 47, 867. (12) Kennedy, J. F.; Barker, S. A.; Nicol, A. W.; Hawkins, A. J. Chem. Soc., Dalton Trans. 1973, 1129. (13) Lange, R.; Ritter, H. Makromol. Chem. 1987, 188, 1641. (14) Denes, F.; Manolache, S. Immobilization of Active Biopolymers from Cold Plasma - Functionalized Surfaces for the Creation of Molecular Recognition and Manufacturing - Molecular Systems. In Polymeric Biomaterials, 2nd ed.; Dumitriu, S. Ed.; Marcel Dekker: New York, 2002; p 239. (15) Grier, N. Silver and its compounds. In Disinfection, Sterilisation and Preservation, 3rd ed.; Block, S. Ed.; Lea and Febiger: Philadelphia, 1983; p 375. (16) Dasgupta, M. K. Adv. Peritoneal Dial. 1994, 10, 195. (17) Leroy, D.; Martinot, L.; Debecker, M.; Strivay, D.; Weber, G.; Je´roˆme, C.; Je´roˆme, R. J. Appl. Polym. Sci. 2000, 77, 1230.
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acid) (PAMPS) and poly(2-acrylamidoglycolic acid)-doped PPy on stainless steel and platinum, able to complex uranyl ions and uranium. This paper deals with the coating of metals (mostly stainless steel 316L) and carbon fibers by polyanion-doped PPy, by a one-step anodic electrochemical technique. The thickness and chemical and physical properties of the film are controlled by the proper choice of the experimental conditions. The polyanion has a dual role of doping agent for PPy and complexing agents for Ag ions. Two polyanions, that is, PAMPS and poly(2-hydroxy-4-N-methacrylamidobenzoic acid) (PHMA), which is a polymeric derivative of salicylic acid, have been selected for complexing properties of silver ions and, thus, ultimately for antibacterial activity. The chemical composition and morphology of the coatings and their complexes with Ag+ on stainless steel and carbon fibers have been characterized by electrochemical techniques, IR spectroscopy, scanning electron microscopy (SEM), and Rutherford backscattering (RBS) techniques. The antibacterial activity of the electrodeposited films against the Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus has also been assessed.
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Materials. Pyrrole was freshly distilled in vacuo at 60 °C before use. Acryloyl chloride and acetonitrile were dried over calcium hydride and distilled under reduced pressure. N,NDimethylformamide (DMF) was dried over phosphorus pentoxide and distilled at 70 °C under reduced pressure. Tetraethylammonium perchlorate was heated in vacuo at 80 °C for 12 h prior to use. 4,4’-Azobis-(4-cyanovaleric acid) (ACVA), 2-acrylamido2-methyl-1-propanesulfonic acid (AMPS), 1,1′-azobis(cyclohexanecarbonitrile), and 4-amino-2-hydroxybenzoic acid (AHA) were of analytical grade, purchased from Aldrich and used without further purification. PAMPS was prepared by free radical polymerization ([M] ) 1 M), initiated by ACVA ([I] ) 3.6 × 10-4 M) in distilled water, at 70 °C for 24 h. The unreacted monomer (5 wt %) was removed by dialysis (Spectra/Por membrane tubing; molecular cutoff ) 6000-8000 Da, diameter ) 14.6 mm) against distilled water for 3 days. The final polymer was freeze-dried, the yield being 90%. PAMPS molecular weight was 7.3 × 105 g mol-1, as calculated by the [η] ) 2.11 × 10-5 × M0.8 equation,18 in a 5 M NaCl aqueous solution at 25 °C. 2-Hydroxy-4-N-methacrylamidobenzoic acid (HMA) was prepared by amidation of AHA with methacrylic anhydride at 0 °C in acetone.19 HMA (2 M) was homopolymerized in DMF at 80 °C, using 1,1′-azobis(cyclohexanecarbonitrile) as an initiator ([I] ) 8 × 10-3 M), in the presence of a chain-transfer agent, that is, 1-phenylethyldithio benzoate ([PEB] ) 0.04 M) synthesized as reported elsewhere.20 The polymer was further purified by repeated precipitation from DMF solution in diethyl ether and vacuum-dried at room temperature (yield ) 65%). 1H NMR (CDCl3) of PHMA, δ (ppm): 1.11 (d, 3H, Ph-CH(CH3)-);21 1.97 (d, 2H, -CH2-CH- from polymer chain), 2.88-2.92 (t, H, -CH2CH- from polymer chain), 7.17-7.81 (m, 14H, Ph-C(S)-S-, Ph-CH(CH3)-, benzyl rings from HMA moieties),21 8.00 (m, 2H, Ph-C(S)-S-),21 10 (s, H, -CONH-)-. Electrochemical Techniques. Cyclic voltammetry and controlled potential coulometry were carried out in double distilled water, previously deoxygenated by bubbling of nitrogen for 30 min. All the electrochemical experiments were conducted under nitrogen at room temperature in a closed cell, with either stainless steel plates (2 cm2) or carbon fibers (10 cm2 of polarized sample) as working electrodes and with a platinum foil as a
counter electrode. A platinum wire was used as a pseudo-reference electrode. An EG&G Princeton Applied Research Model 263A potential/galvanostat was used in the cyclic voltammetry. Coulometric measurements were recorded with a current integrator (type IG6-N, Tacussel). Stainless steel 316L and carbon fibers were washed with acetone and heptane and dried at 150 °C under vacuum before use. PPy/PAMPS and PPy/PHMA films were prepared by anodic polymerization of Py (0.5 M to saturation) in the presence of PAMPS and PHMA polyanions (0.1 M), respectively, as the conducting salts. The pH of the aqueous solution of PHMA was fixed at 5.0 before mixing with Py. The PPy/PAMPS films were prepared by controlled potential coulometry and at a constant anodic potential of 0.75 V with respect to the pseudo-reference. The current quantity was lying in the range from 300 to 1000 mC for stainless steel and from 5 to 70 C for carbon fibers. By using cyclic voltammetry (with the type IG6-N integrator, Tacussel), films of PPy/PHMA with different thicknesses were prepared by changing the current quantity. The thickness of the films was evaluated knowing that 1 C/cm2 results in a 2-µmthick PPy deposit.22 Typically, coatings of 0.4, 1.5, and 2.3 µm were deposited onto carbon fibers for antibacterial testing. After electrolysis, stainless steel and carbon fibers were washed thoroughly with distilled water and acetone and dried. They were silver loaded by dipping in a 0.05 M aqueous solution of AgNO3 at 25 °C in darkness for 3 h. The coatings were intensively washed with distilled water and dried. Measurements. IR spectra of PPy/polyanion, PPy/polyanion/ Ag+, and PPy/NO3- films were recorded with a Perkin-Elmer, Spectrum One FT-IR spectrophotometer at room temperature. PPy/PAMPS and PPy/PHMA electrodeposited films and their complexes with Ag+ were observed with a SEM microscope (Stereoscan 440 Leo). The thicknesses of the films onto carbon fibers was estimated from the increase in diameter of the uncoated fibers (average of 10 samples observed by SEM). Energydispersive X-ray (EDX) analysis (Thermo Noran Voyager III) was used to analyze the atomic composition of the surface. Adhesion of the films to stainless steel 316L was measured by an American Society of for Testing and Materials (ASTM) peeling test with adhesive ribbons (3M acrylic foam 4930) of increasing adhering strength.23 PPy/PAMPS or PPy/PHMA films electrodeposited onto carbon plates (2 cm2) were analyzed by RBS with 2 Me VR particles produced by a Van de Graaff accelerator. The backscattered particles were detected by an annular PIPS detector with an active detection area of 100 mm2 and a resolution of 19 keV. Antibacterial Assessment. The antibacterial activity of electrodeposited PPy/PAMPS/Ag+ films onto carbon fibers against the Gram-negative bacteria E. coli (DH 5R) was assessed by a viable cell-counting method.24,25 A freeze-dried ampule of E. coli was opened, and the culture was picked off with a micropipet and placed in 2 mL of nutrient LB broth, Miller (Luria-Bertani; pancreatic digest of casein 10.0 g/L, yeast extract 5.0 g/L, sodium chloride 10.0 g/L; pH 7.0), which was then incubated, at 37 °C, overnight. Then, 200 µL of the culture was placed in 100 mL of nutrient broth and incubated at 37 °C for 4 h. Upon appropriate dilution with sterilized 0.9% saline solution, a culture of about 107 cell/mL was prepared and used for antibacterial testing. Pieces of coated and uncoated fibers with a 4.15 × 10-3 m2 surface area were sterilized by UV irradiation (2 h) and exposed to the E. coli cell suspension (10 mL containing about 107 cells/mL). At a specified time, 1 mL of bacteria culture was added to 9 mL of sterilized 0.9% saline solution, and several decimal dilutions were repeated. The surviving bacteria were counted by the spreadplate method. At various exposure times, 0.1-mL portions were removed and quickly spread on the nutrient agar. After inoculation, the plates were incubated at 37 °C for 24 h, and the colonies were counted. Counting was triplicated for each experiment.
(18) Fisher, L. W.; Sochor, A. R.; Tan, J. S. Macromolecules 1977, 10, 949. (19) Elvira, C.; Roman, J. S. Polymer 1997, 38 (18), 4743. (20) Le, T. P.; Moad, G.; Rizzardo, E.; Thang, S. H. Polymerization with living characteristics. WO Patent 98 01478, 1998. (21) Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H. Macromolecules 1998, 31, 5559.
(22) Momma, T.; Komaba, S.; Osaka, T.; Nakamura, S.; Takemura, Y. Bull. Chem. Soc. Jpn. 1995, 68, 1297. (23) Lou, X.; Je´roˆme, C.; Detrembleur, C.; Je´roˆme, R. Langmuir 2002, 18, 2785. (24) Franklin, T. J.; Snow, G. A. Biochemistry of Antimicrobial Action; Chapman and Hall: London, 1981; p 58. (25) Kanazawa, A.; Ikeda, T.; Endo, T. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, 3031.
Experimental Section
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Figure 1. Cyclic voltammograms for PPy/PHMA films electrodeposited in water (v ) 20 mV/s), (A) onto carbon fibers (number of cycles n ) 20) and (B) onto stainless steel (n ) 10). In a second test, carbon fibers before and after coating with a PPy/PAMPS/Ag+ film were immersed in an E. coli containing solution, and the fibers were removed from the bacterial suspension of the strain 2 h later. Samples were fixed and dehydrated according to a procedure reported elsewhere.26 The (26) Maquet, V.; Martin, D.; Malgrange, B.; Franzen, R.; Schoenen, J.; Moonen, G.; Je´roˆme, R. J. Biomed. Mater. Res. 2000, 52, 639.
fiber surface was observed with a SEM Stereoscan 440 Leo equipped with an EDX detector (Thermo Noran Voyager III). The antibacterial activity against the Gram-negative bacteria E. coli (DH 5R) and Gram-positive bacteria S. aureus 749 (obtained from National Bank of Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria) of stainless steel and carbon fibers coated with a PPy/polyanion and PPy/polyanion/Ag+ film was evaluated by the modified Kirby-Bauer technique that consists
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of measuring an inhibition zone of bacterial growth around the film.27 The flat surface of the agar gel medium was inoculated with a suspension of a 24-h culture of E. coli or S. aureus containing about 106 cells/mL, after dilution with a sterile saline solution. Previously sterilized by UV irradiation for 2 h, coated and uncoated stainless steels were pressed onto bacteria-overlaid agar. One stainless steel plate was contacted with the culture medium in a Petri dish that was incubated at 37 °C for 24 h. The width of the inhibition zone of the bacterial growth was measured.
Results and Discussion Anodic Electrodeposition of PHMA-Doped PPy onto Stainless Steel and Carbon Fibers. PHMA-doped PPy films were electrodeposited in an aqueous solution (pH ) 5) by cyclic voltammetry from -0.8 to +0.8 V (vs a platinum wire used as a pseudo-reference electrode). Coatings of several thicknesses (300-600 nm on stainless steel and 300 nm to 1.1 µm onto carbon fibers) were prepared by changing the current quantity from 300 to 600 mC in the case of stainless steel and 2.5 to 7 C in the case of carbon fibers. Figure 1 shows typical curves for the potentiodynamic growth of a PHMA/PPy film on carbon fibers and stainless steel. The foot of the polymerization peak is at approximately 0.6 V on both the substrates. The first scan shows a low-intensity polymerization peak, which results from the formation of PPy nuclei. Whenever the potential scanning is repeated, the polymerization peak is shifted toward a less positive potential and its current density is increased (Figure 1), which indicates that more pyrrole rings and oligomers have reacted during the scan, in agreement with the nucleation process commonly observed for PPy electrosynthesis. The redox peaks characteristic of the PHMA/PPy film are observed at Ered ) 0.465 V/Pt and Eox ) 0.35 V/Pt. The resolution of the peaks is higher when carbon fibers (Figure 1A) are used rather than stainless steel (Figure 1B), consistent with differences in the surface characteristic features including roughness. PAMPS has been substituted for PHMA in the electropolymerization of pyrrole onto stainless steel by cyclic voltammetry. Under the same experimental conditions, the observations are qualitatively the same. The foot of the polymerization peak is at +0.46 V/Pt, and the redox potentials are only slightly shifted compared to the PHMA/ PPy counterpart. The thicknesses of the electroactive films range from 0.3 to 1 µm, the doping peak is at +0.06 V/Pt, and the dedoping peak at -0.40 V/Pt. In parallel, PAMPS/PPy films have also been electrodeposited by controlled potential coulometry. A stainless steel working electrode was dipped in a 0.5 M Py and 0.1 M PAMPS aqueous solution, polarized at a constant potential of +0.75 V, and 0.3-1-µm-thick films were formed in a time scale of 30-97 s, thus with a current density ranging from 300 to 1000 mC. A typical potentiostatic curve is reported in Figure 2A, which shows an initial slight decrease in the current intensity, followed by a rapid increase and ultimately a plateau, as a result of the nucleation and growth of PPy. A similar time dependence of the current intensity is observed at a potential of +0.75 V for the anodic electrodeposition of a PPy/PAMPS film onto carbon fibers (Figure 2B). Redox Properties of PAMPS-Doped PPy and the Complex with Ag+. Cyclic voltammetry of PAMPS-doped PPy films in a 1 M NaClO4 aqueous solution shows that the reduction occurs at quite a low cathodic potential (-0.52 V/Pt; Figure 3, curve A1) compared to a small doping agent (-0.2 V/Pt, for ClO4-), which is typical for (27) Traub, W. H.; Leonhard, B. Chemotherapy 1994, 40, 374.
Figure 2. Controlled potential coulometry for the synthesis of PPy/PAMPS films onto (A) stainless steel at 0.75 V/Pt for 32 s and (B) carbon fiber at 0.75 V/Pt for 67 s.
polyelectrolyte-doped PPy (Ered ) -0.7 V/Pt, for PPy/poly(styrene sulfonate) films).28 In this case, the insertion of the Na+ cation occurs (Scheme 1) rather than the ClO4anion incorporation. Whenever the film has been dipped in a 0.05 M aqueous solution of AgNO3 at room temperature for 2 h, the electroactivity is maintained, with, however, a significant shift of the reduction peak to less cathodic potential, that is, to -0.28 V/Pt (Figure 3B). This value is close to the reduction peak for the PPy/ClO4films (Ered ) -0.2 V/Pt),28 probably because Na+ cation exchange is replaced by an anion exchange (of NO3- or ClO4-) during the redox process (Scheme 1). These changes in the voltammograms of the films that contain additional small doping species Ag+ are an evidence that Ag+ is included in the PPy/PAMPS films. IR Spectroscopy of Polyanion-Doped PPy and the Complex with Ag+. The IR absorptions characteristic of the NH stretching and OH stretching of PHMA are observed in the 3200-3368-cm-1 region.19 The absorption at 3112 cm-1 is assigned to the aromatic CsH stretching. Two strong absorptions at 1668 and 1510 cm-1 are characteristic of the amides I and II, respectively. The shoulder at 1682 cm-1 is the signature of the carbonyl stretching of the carboxylic group. Bands at 1610, 1524, and 1394 cm-1 are assigned to the skeletal vibrations, involving CsC stretching of the aromatic ring. In reference to the IR spectrum of PPy/NO3- films, the broad band at 3140-3423 cm-1 is typical of the NsH stretching of the pyrrole ring.29,30 The CsC, CsN, and CdC stretchings and the NsH and CsH deformations can be found in the (28) Je´roˆme, C.; Martinot, L.; Je´roˆme, R. Radiochim. Acta 1998, 83, 61. (29) Jones, R. A. Heterocyclic Compounds Pyrroles, Part 1; Wiley: New York, 1990; Vol. 48, p 61. (30) Pouchert, C. J. The Aldrich Library of IR Spectra, 2nd ed.; Aldrich Chemical Co.: Milwaukee, WI, 1975.
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Figure 3. Cyclic voltammograms (v ) 20 mV/s) for (A) a PPy/PAMPS film and (B) a PPy/PAMPS/Ag+ film in an aqueous solution of NaClO4 (1 M): first (1) and second (2) scans. Scheme 1. Ion-Exchange between a NaClO4 Aqueous Solution and the Electroactive Films of (I) PPy/ PAMPS and (II) PPy/PAMPS/Ag+, Respectively
1100-1700-cm-1 range,31 whereas the absorption at 835 cm-1 results from the CsH wagging vibration of the hydrogen atoms adjacent to a ring compound.29 The IR spectrum of the PPy/PHMA films shows the absorptions characteristic of both the PHMA and PPy constituents. Indeed, the stretching vibration of the pyrrole ring is observed at 1376 cm-1.32 Moreover, the amide I absorption is shifted to a lower wavenumber by 13 cm-1 (1655 cm-1), whereas the amide II band is shifted to a higher wavenumber by 34 cm-1 (1544 cm-1) as compared to neat PHMA. The ionic interaction of the amide groups of PHMA and the protonated N of PPy is responsible for a decrease in the intensity ratio of the amide I and amide II bands of PHMA. Upon complexation of Ag+ by PPy/ PHMA films, the amide I absorption is shifted to a lower wavenumber and the amide II band is shifted to a higher wavenumber by 18 cm-1 (1562 cm-1), such that the two bands overlap each other. (31) Drzal, L. T.; Rich, M. J.; Madhukar, M.; Herrera-Franco, P. Characterization of Fiber-Matrix Adhesion in Composite Materials Conference Proceedings; ASM Press: Detroit, 1990; p 155. (32) Bazzaoui, M.; Martins, L.; Bazzaoui, E. A.; Martins, J. I. Electrochim. Acta 2002, 47, 2953.
IR spectra of PAMPS and pieces of PPy/NO3-, PPy/ PAMPS, or PPy/PAMPS/Ag+ films detached from the electrode have also been recorded. The main bands in the PAMPS spectrum are as follows: 3430-3090 cm-1 (-NH and OH stretching vibrations), 1652 cm-1 (-amide I), 1558 cm-1 (-amide II), and 1386 cm-1 (-CH2 bending).33 The absorption of the sulfonic acid groups is observed in the 1224-1175-cm-1 range.34 The IR spectrum of the PPy/PAMPS films shows the absorption for the amide I at 1641 cm-1 and for the amide II at 1553 cm-1. The occurrence of ionic interactions between the protonated pyrrole rings and the sulfonic acid groups of PAMPS is supported by the shift of the sulfonic acid absorption toward a higher frequency (1181 cm-1). The reflection-absorption IR spectrum for the PPy/ PAMPS/Ag+ complexes shows that a series of absorptions are shifted, that is, the amide I (downward, by 14 cm-1), the amide II (upward, by 14 cm-1), and the sulfonic acid (upward, by 43 cm-1) compared to PAMPS-doped PPy. These observations suggest that the amide groups and sulfonic acid groups from PAMPS residues are coordinated to Ag+. Morphology of the Films. As previously observed, substitution of small size dopants (e.g., perchlorates and nitrates) by polyanions, such as PHMA and PAMPS, results in smoother PPy films.35 Moreover, the EDX analysis shows typical peaks for oxygen, nitrogen, and carbon (and sulfur in case of PPy/PAMPS), which confirms that the PPy films electrodeposited onto stainless steel are doped by PHMA and PAMPS, respectively. The intensity of these peaks is essentially independent of the probed area, consistent with the homogeneity of the film compositions. Silver ions in the PPy/polyanion/Ag+ films are also detected by EDX at 3 keV (Figure 4), in agreement with previous IR analyses. Aggregates of silver particles can, however, be observed with a diameter of approximately 0.4 µm in PPy/PHMA/Ag+ films and approximately 1.7 µm in the PPy/PAMPS/Ag+ ones (Figure 4). These (33) Stoilova, O.; Koseva, N.; Manolova, N.; Rashkov, I. Polym. Bull. 1999, 43, 67. (34) Huglin, M. B.; Rego, J. M. Polymer 1990, 31, 1269. (35) Je´roˆme, C.; Martinot, L.; Je´roˆme, R. Radiochim. Acta 1998, 83, 61.
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Figure 4. SEM images of (A) untreated and (B, C) silver-containing PPy/PAMPS films onto stainless steel. Magnification: ×7500 (top), ×2500 (bottom). The surface analyzed by EDX is shown in part C.
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Figure 5. (A) SEM micrographs and (B) EDX analysis of carbon fibers coated with PPy/PHMA/Ag+ (number of cycles n ) 13). The surface area 1 was analyzed by EDX.
observations indicate that silver ions are locally reduced into elemental silver, Ag0, upon light exposure. This characteristic feature is actually desirable because it is known that atomic/metallic silver and colloidal silver are highly effective contact biocides.14,36 The successful electrodeposition of PPy/PHMA and PPy/ PAMPS onto stainless steel followed by the incorporation of silver ions has been extended to carbon plates and carbon fibers. For current quantities in the 5-20-C range, carbon fibers are coated by a continuous thin film of PPy/PAMPS. Larger current quantities (70 C) result in the partial coalescence of the fibers. The thickness of the coating can be estimated from the electron micrographs, at approximately 0.4 µm for a current quantity of 2.5 C, 1.5 µm when the current quantity is 10 C, and 2.3 µm when 20 C is used. In the case of PPy/PHMA coating, the current quantity was in the 2.5-7-C range. The thickness of the fiber coating was estimated by SEM at 0.3 µm for a current (36) Taylor, A. J.; Roberts, G. A. F.; Wood, F. A. Preparation of articles having a contact biocidal property. WO Patent O2 15698 A1, 2002.
quantity of 2.5 C, increasing up to 0.7 and 1.1 µm for current quantities of 5 and 7 C, respectively. The EDX analysis (Figure 5) of the PPy/polyanion/Ag+ coated fibers shows the characteristic peaks for N, O, C, S (in the case of PAMPS), and Ag whenever incorporated in the films. Moreover, some aggregates of silver particles onto the fiber surfaces are also detected by SEM. RBS Analysis of PPy/Polyanion/Ag+ Films. The RBS spectrum for a PPy/PAMPS/Ag+ film deposited onto a carbon plate shows all the characteristic peaks of the constitutive elements, that is, C, N, O, S, and Ag, incorporated by complexation. The thickness of the polymer film has been estimated at 550 nm from the width of the sulfur peak, which is in good agreement with the film thickness calculated from the linear relationship between the current quantity and the thickness of the electrodeposited polymer. Moreover, the Ag/S (1:2) and N/S (4:1) atomic ratios have been determined from the surface area of the respective peaks. It results that one silver atom has been incorporated into each two AMPS
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Table 1. Adhesion of the PPy/Polyanion Films Measured by the ASTM Peeling Test
films PPy/PAMPS PPy/PAMPS PPy/PAMPS PPy/PAMPS PPy/PAMPS PPy/PAMPS PPy/PHMA PPy/PHMA PPy/PHMA a
technique used for preparation the films
film thickness (nm)
adhesion (N/m)
cyclic voltammetry cyclic voltammetry cyclic voltammetry controlled potential coulometry controlled potential coulometry controlled potential coulometry cyclic voltammetry cyclic voltammetry cyclic voltammetry
400 600 800 400
not detacheda 1590 480 not detacheda
600
1680
800
570
400 600 800
1705 1350 465
Film remains attached to the electrode after peeling.
units and that one AMPS unit has been inserted in PPy for each three pyrrole units. More Ag is immobilized by the PPy/PHMA copolymer, for which an Ag/O atomic ratio of 1:4 has been measured, which corresponds to the binding of one silver atom per HMA unit. Adhesion of the PPy/Polyanion Films on Stainless Steel and Carbon Fibers. The peeling force required for detaching electrodeposited PPy/PHMA and PPy/ PAMPS films of different thicknesses from the solid substrate has been measured and reported in Table 1. This force increases when the film thickness is decreased, at least in the range of 800-400 nm. For the lower thickness, the PPy/PAMPS films are no longer detached from the electrode; the rupture then occurs at the polymer film/scotch tape interface, and the experimental force is 2000 N/m. These observations are independent of the technique used for the electrodeposition, that is, cyclic voltammetry and controlled potential coulometry. The PPy/PHMA films of 400 nm seem to be less adherent than the PPy/PAMPS counterparts. Nevertheless, adhesion is strong as far as the films are thin, which is in line with the physisorption of the chains in contact with the substrate. It is quite a problem to measure the adhesion of the films electrodeposited at the surface of the carbon fibers. The qualitative information that has been collected until now is the film resistances to a series of liquids, that is, distilled water, saline solution, and organic solvents including acetone and methanol, under sonication for 2 h. In all the cases, the film remains tightly bound to the fibers, which is a strong indication of the adhesion of the coating onto the substrate. Antibacterial Activity of the Coated Carbon Fibers and Stainless Steel Surfaces. The antibacterial activity against the Gram-negative bacteria E. coli of PPy/ polyanion/Ag+ films with a thickness of 400 nm onto stainless steel surfaces (2 cm2) and 300 nm onto carbon fibers (28.3 cm2) has been estimated from the width of the inhibition zone around the coated surfaces. Stainless steel surfaces, before and after coating with PPy/PAMPS and PPy/PHMA, do not inhibit the growth of E. coli. An inhibition zone of 8.5 mm is observed in the case of PPy/ PAMPS/Ag+ films and 5 mm for the PPy/PHMA/Ag+ ones. The Ag+ ions released from the films appear, thus, responsible for the antibacterial activity. The same general observation holds for carbon fibers, which are active against E. coli only when coated by silver-containing films (inhibition zone of 7 mm). Clinical studies indicate that among the species that dominate biomaterial-centered infections, S. aureus is one of the most common pathogens isolated from tissue and the environment around implant
Figure 6. Logarithm plot of the viable cell number of E. coli versus the exposure time, for uncoated carbon fibers and fibers coated by PPy/PAMPS/Ag+.
infections.37 That is why we also evaluated the antibacterial activity of the surface coatings against the Grampositive bacteria S. aureus by determing the width of the inhibition zone around the coated surfaces. The test shows that the antibacterial activity of the coatings against S. aureus is higher than that against E. coli. The same tendency as in the case of the Gram-negative bacteria E. coli was observed, that is, absence of the inhibibition zone around the uncoated stainless steel and fibers and around the coated surfaces without silver, in contrast to the welldefined inhibition zone around the silver-containing coated surfaces: approximately 10 mm, 8 mm, and 18 mm for PPy/PAMPS/Ag+ films, PPy/PHMA/Ag+ films, and silvercontaining coated fibers, respectively. The antibacterial activity against E. coli of PPy/PAMPS/ Ag+ films electrodeposited on carbon fibers has also been tested by the viable cell-counting method.24,25 For the test to be sensitive enough, carbon fibers have been used rather than stainless steel plates because of a much higher surface area. As shown by Figure 6, silver-immobilized fibers killed the bacteria within 120 min of contact in contrast to fibers uncoated or coated by a non-silver-loaded PPy/PAMPS film. This observation confirms that the antibacterial activity results from the silver ions release38-40 and that the effect is bactericidal rather than bacteriostatic. After 120 min, the three coatings have released enough Ag+ for killing all the bacteria (Figure 6). In the meantime, the effect is as important as the coating is thick and, thus, as the amount of incorporated silver is high. The surfaces of coated and uncoated carbon fibers have been exposed to E. coli and observed by SEM (Figure 7). Cells of E. coli can be seen at the surface of the uncoated fibers and never in direct contact with PPy/PAMPS/Ag+ (37) Foster, T.; Ho¨o¨k, M. In Infections Associated with Indwelling Medical Devices, 3rd ed.; Waldvogel, F., Bisno, A., Eds.; ASM Press: Detroit, 2000. (38) Nonaka, T.; Uemura, Y.; Enishi, K.; Kurihara, S. J. Appl. Polym. Sci. 1996, 62, 1651. (39) Slawson, R. M.; Lohmeier-Vogel, E. M.; Lee, H.; Trevors, J. T. Biometals 1990, 7, 30. (40) Rosenkranz, H. S.; Carr, H. S. Antimicrob. Agents Chemother. 1978, 2, 367.
Immobilization of Silver in Composite Coatings
Langmuir, Vol. 19, No. 21, 2003 8979
Figure 7. SEM micrographs of carbon fibers onto which E. coli was inoculated: (A) uncoated carbon fibers and (B) carbon fibers coated with PPy/PAMPS/Ag+. Magnification: ×10 000. (C, D) EDX analyses of different areas of the coated fibers.
films. To know whether the bacteria at the surface of the uncoated fibers are viable, these fibers have been pressed onto an agar gel and have been incubated at 37 °C for 24 h. Many bacteria colonies have been observed in the direct vicinity of these fibers, which is never the case when the fiber coating contains silver. The E. coli cells that adhere to the surface of uncoated carbon fibers are, thus, viable. After exposure for 2 h to the suspension of E. coli cells, the coating (1.3-µm thick) remains adhered to the carbon fibers as observed by SEM (Figure 7B). EDX analysis (Figure 7C,D) also indicates that only a part of the silver ions have been released in the bacteria suspension. The antibacterial activities against E. coli and S. aureus of well-adhering Ag+-containing films are tentatively explained by the penetration of the released Ag+ ions through the bacteria cell wall and their complexation with enzymes in the cell membrane, which results in the inhibition of the enzymatic activity and the bacteria death.38-40 Conclusions PPy electrodeposited onto stainless steel 316L plates and carbon fibers has been doped by polyanions selected for their capacity to complex silver ions. The adhesion of these films is as strong as the thickness is low, particularly below 600 nm. Silver ions containing PPy coatings (300-
400-nm thick) have a bactericidal activity against the Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus. The combination of good adhesion and antibacterial properties makes this type of new electrocoating promising for a variety of biomedical applications. Acknowledgment. M.I. gratefully acknowledges the Belgian Office for Scientific, Technical and Cultural Affairs for a post-doc fellowship at the University of Lie`ge. The authors thank this office for general support to CERM in the frame of the Poˆles d'Attraction Interuniversitaires: PAI 5/03 (Supramolecular Chemistry and Supramolecular Catalyst). C.J. is Chercheur Qualifie´ by the Fonds National de la Recherche Scientifique (FNRS). The authors are grateful to Dr. M. Galleni (ULg) for assistance in the microbiological tests with E. coli, to Dr. N. Markova (Institute of Microbiology, Bulgarian Academy of Sciences) for assistance in the microbiological test with S. aureus, to G. Moutzourelis (ULg) for the preparation of the bacteria cultures, to Dr. V. Maquet (CERM, ULg) for a fruitful discussion about the bacteria interaction with electrically conducting substrates, and to V. Raisi (Arcelor) for SEM micrographs and EDX measurements. LA034968V
