Polyrhodanine

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Communications Synthesis and Antimicrobial Properties of Novel Silver/ Polyrhodanine Nanofibers Hyeyoung Kong and Jyongsik Jang* School of Chemical and Biological Engineering, College of Engineering, Seoul National University, 56-1 Shinlim Kwanak, Seoul 151-742, Korea Received May 22, 2008; Revised Manuscript Received July 18, 2008

Silver nanoparticle-embedded polyrhodanine nanofibers were synthesized by chemical oxidation polymerization. Silver ions were reduced to silver nanoparticles by oxidizing rhodanine monomer and simultaneously complexed with the rhodanine due to coordinative interactions, resulting in the formation of silver nanoparticle-embedded polyrhodanine nanofibers. The synthesized silver/polyrhodanine nanofiber was found to have the excellent antimicrobial activity against Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, and Candida albicans. The modified Kirby-Bauer test demonstrated that the silver/polyrhodanine nanofiber had better antimicrobial efficacy than silver sulfadiazine.

Introduction Metal-embedded conducting polymers have been extensively explored due to their diverse applications such as optical and electrochemical sensors, a catalysis, and an organic electroluminescent device.1-4 Among them, silver-polymer nanocomposite has been considered as a promising material for the field of electric device, olefin transport membrane, and antimicrobial agent.5-12 Interestingly, silver ion can be used as an oxidant in the chemical oxidation polymerization during synthetic process of silver-conducting polymer nanomaterials.13-17 Rhodanine derivatives have been applied for antiviral, antibacterial, antihistaminic, and anticorrosion agents.18-22 Furthermore, they are also used for detecting metal ions because rhodanine molecule has metal-binding functional groups such as thioamide and amide.23,24 Recently, Kardas¸ and co-worker demonstrated that electrochemical polymerization of rhodanine proceeded by virtue of the coupling reaction between nucleophilic methylene group and secondary amine group.25 It has been reported that the nanofibrous structure as an antimicrobial scaffold provides higher cell attachment compared to other structures.26,27 In particular, silver-impregnated polymer nanofibers have been developed as a new class of biomedical materials by releasing biocidal silver with a protective polymer barrier against infection.28 From this point of view, it is anticipated that silver/polyrhodanine nanofibers have enhanced antimicrobial efficacy originated from the combined antimicrobial activity of silver and polyrhodanine nanofiber. However, the fabrication of silver/polyrhodanine nanofiber has not been reported despite its potential application. Therefore, it is desirable to develop a simple and reliable method to synthesize silver/polyrhodanine nanofibers for potential antimicrobial application. Herein we report the fabrication of novel silver/polyrhodanine nanofibers and their antimicrobial efficacy. Chemical oxidation * To whom correspondence should be addressed. Tel.: (+82) 2-8807069. Fax: (+82) 2-888-1604. E-mail: [email protected].

Figure 1. Chemical structure of rhodanine monomer.

polymerization of rhodanine monomer proceeded using silver ion as an oxidant and the silver ion was simultaneously reduced to silver nanoparticles.29 Consecutively, silver formed the complex with rhodanine monomers owing to strong coordinative interactions. As a result, silver nanoparticle-embedded polyrhodanine nanofibers were synthesized without complicated procedures. The synthesized silver/polyrhodanine nanofibers have been shown to have potent antimicrobial efficacy against Gramnegative/Gram-positive bacteria and yeast.

Experimental Section Materials. Rhodanine, silver nitrate (AgNO3), and silver sulfadiazine were purchased from Aldrich (Milwaukee, WI) and used without further purification. For the bacterial test, Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), and Candida albicans (ATCC 10231) were purchased from Fisher Company. Fabrication of Silver Nanoparticle-Embedded Polyrhodanine Nanofiber. In a typical procedure, AgNO3 (18 mM) was dissolved in absolute ethanol (200 mL) and rhodanine monomer (7 mM) was added into the silver nitrate solution. Consecutively, silver-rhodanine complex was formed due to coordinative interactions between silver and electrondonating groups of rhodanine, and the chemical oxidation polymerization of rhodanine proceeded at 25 °C for 24 h. The chemical structure of the rhodanine monomer is presented in Figure 1. Vigorous magnetic stirring was induced for high shear flow during the fabrication process of polymer nanofibers. Under this experimental condition, polyrhodanine nanofibers with silver were generated in the flow direction.30,31 After polymerization, the synthesized silver/polyrhodanine nanofiber was obtained by centrifugal precipitation and washed with distilled

10.1021/bm800574x CCC: $40.75  2008 American Chemical Society Published on Web 09/05/2008

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Figure 2. (a) FE-SEM and TEM (inset) images of synthesized silver/ polyrhodanine nanofibers and (b) FT-IR spectra of silver/polyrhodanine (red dashed-line) and rhodanine monomer (black solid-line).

water and ethanol several times to remove the residual reagents. For further characterization, the nanofiber was dried under vacuum at 25 °C for 3 days. Antimicrobial Tests. Microorganisms were cultivated in sterilized LB broth and then incubated overnight at 37 °C with a shaking incubator. The microorganism suspensions employed for the tests contained from 106 to 107 colony forming units (CFU). For the kinetic test, each 3 mg of solid-state silver/polyrhodanine nanofiber was prepared in three sterilized test tubes (1.5 mL) and inoculated with 1 mL of each microorganism suspensions (E. coli, S. aureus, and C. albicans). In the test tube, 50 µL volumes were chosen as a function of contact time (min) and cultured in LB agar plates. The LB agar plates were kept at 37 °C for 24 h and the number of survival colonies was counted. For the test of surface coating on glass, 1 mL of 1 wt % silver/ polyrhodanine solution in methanol was coated on a glass slide.32 Aqueous suspension of E. coli (106 CFU/mL) was sprayed on the coated glass. Uncoated glass was prepared for comparison and also sprayed by the E. coli suspension. After air-drying for 5 min, autoclaved LB broth (which was cooled to 40 °C) was added to the glasses and solidified. The glasses were incubated at 37 °C for 24 h and the bacterial colonies were inspected. For the modified Kirby-Bauer test, equal amounts of silver/ polyrhodanine nanofiber and silver sulfadiazine were pelletized by hydraulic press.11 Sample pellets were placed on each E. coli, S. aureus, and C. albicans growth LB agar plates and incubated overnight at 37 °C. The zone of inhibition against three microorganisms was analyzed compared to the silver sulfadiazine. Characterization. Photographs of transmission electron microscopy (TEM) were obtained with a JEOL JEM-200CX. Acceleration voltage

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Figure 3. (a) XPS graph and (b) UV-vis spectra of synthesized silver/ polyrhodanine nanofibers in 1 M NaOH solution. In the UV-vis spectra, red dashed-lines are obtained by the peak deconvolution.

for TEM was 200 kV. Field-emission scanning electron microscopy (FE-SEM) images were obtained using a JEOL 6700 at an acceleration voltage of 5 kV. In the sample preparation of TEM and SEM characterizations, Ag/polyrhodanine nanofibers were dispersed in absolute ethanol and cast onto copper grid. Fourier transform infrared (FT-IR) spectra were recorded on a Bomem MB 100 spectrometer (Quebec, Canada) in the absorption modes at resolution of 4 cm-1 and 32 scans. The UV-vis spectra were taken at 25 °C with a PerkinElmer Lambda-20 spectrometer. X-ray photoelectron spectroscopy (XPS) data was obtained using Sigma Probe electron spectroscope. Energy dispersive X-ray (EDX) analysis was performed by a Philips CM-20 microscope coupled with an EDX facility. Size exclusion chromatography was carried out using Agilent 1100 series.

Results and Discussion Chemical oxidation polymerization was performed using silver ion (Ag+) as an oxidant (see Supporting Information, Scheme S1). In the synthetic procedure, silver ions were reduced to silver nanoparticles by oxidizing rhodanine monomer, leading to the polymerization of rhodanine. Simultaneously, silverrhodanine complex was formed under vigorous magnetic stirring due to coordinative interaction. Under the vigorous magnetic stirring, polyrhodanine nanofiber was generated in a flow direction by the shear force30,31 and silver coordinated with sulfur or oxygen atoms of two rhodanine molecules, resulting in linear complexes.33 As a result, silver nanoparticle-embedded polyrhodanine nanofibers were obtained via a one-step procedure.

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Figure 4. (a) Photographs of surviving colonies on the Ag/polyrhodanine coated and the uncoated glass slide after spraying E. coli suspension. (b) The plot of % reduction versus contact time (min) of the silver/polyrhodanine nanofiber on E. coli, S. aureus, and C. albicans. The % reduction was calculated as % reduction ) (A B)/A × 100 (where A is the number of surviving microbial colonies in the blank solution and B is the number of surviving microbial colonies in the silver/polyrhodanine nanofiber).

FE-SEM image displays the synthesized silver/polyrhodanine nanofibers with the diameter of about 30 nm and the length of

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about 5 µm (Figure 2a). The TEM image (inset in Figure 2a) indicates that silver nanoparticles of about 3 nm are embedded in the polyrhodanine nanofiber. Figure 2b represents the FT-IR spectra of pristine rhodanine and synthesized silver/polyrhodanine nanofiber. In the FT-IR spectrum of rhodanine, the peak at 3080 cm-1 is assigned to NH vibration band and the band at 1714 cm-1 is due to CdO stretching vibration. It is noteworthy that the NH vibration band of rhodanine disappears in the silver/ polyrhodanine nanofiber owing to the oxidation of rhodanine molecule. In addition, new peaks at 1560 cm-1 of CdN stretching vibration and at 1650 cm-1 of CdC stretching vibration appear in the spectrum of synthesized silver/ polyrhodanine. Furthermore, the CsO stretching peak at 1180 cm-1 intensively increases relative to the peak of pristine rhodanine. The FT-IR spectra show that the polymerization of rhodanine proceeds over carbon and nitrogen atoms by forming CdC, CdN, and CsO bonding. This result is consistent with the recent research of polyrhodanine which confirms the formation of polymer chain over carbon and nitrogen atoms.25 Conversion % of rhodanine to polyrhodanine was about 84% and the relative weight ratio of silver to polyrhodanine in the final product was calculated to be approximately 2:1 from the EDX characterization. To obtain the molecular weight and molecular weight distribution of synthesized polyrhodanine, size exclusion chromatography was carried out and the result showed that the silver/polyrhodanine nanofiber had an average molecular weight (Mw) of 70000 and polydispersity index (Mw/Mn) of 5. To characterize embedded silver nanoparticles, XPS analysis was performed in Figure 3a. The XPS spectrum indicates that two peaks are observed at 368 and 374 eV with 6.0 eV separation, corresponding to Ag 3d5/2 and Ag 3d3/2 binding energy of Ag0, respectively.31 Therefore, it can be concluded that silver ions are reduced to silver nanoparticles during the chemical oxidation polymerization. Furthermore, the surface chemical composition of silver/polyrhodanine could be calculated by the XPS spectroscopy (see Supporting Information, Figure S1). XPS result showed that the surface consists of C, O, N, S, and Ag elements and the atomic concentration of Ag/S was about 1.2. In other words, the relative molecular ratio of silver to rhodanine was approximately 2.4:1 in the surface of silver/polyrhodanine nanofiber. The silver/polyrhodanine nanofi-

Figure 5. Photograph images of the Kirby-Bauer plates of silver/polyrhodanine nanofiber (a, b, and c) and silver sulfadiazine (d, e, and f) against E. coli, S. aureus, and C. albicans (yeast).

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Table 1. Modified Kirby-Bauer Test of Silver/Polyrhodanine Nanofiber and Silver Sulfadiazinea zone of inhibition (mm) sample

E. coli

S. aureus

C. albicans

Ag/polyrhodanine silver sulfadiazine

30 17

37 16

25 18

a

Both pellet sizes are about 13 mm in diameter.

ber has three absorbance peaks at 360, 400, and 460 nm by peak deconvolution in the UV-vis spectroscopy (Figure 3b). It has been reported that the pristine rhodanine has a n-π* transition peak near 340 nm in 1 M NaOH solution.25,34 Thus, the observed peak at 360 nm can be explained by the red-shifted n-π* transition of rhodanine, and the red-shift indicates that Ag-binding rhodanine molecules are formed by the complex formation.35,36 On the other hand, the characteristic peak of silver appears at 400 nm37 and the absorbance peak near 460 nm originates from the polymer backbone of polyrhodanine.25 Judging from these data, it was evident that the silver nanoparticle-embedded polyrhodanine nanofiber was successfully fabricated using the chemical oxidation polymerization. It is anticipated that the synthesized silver/polyrhodanine nanofibers have potent antimicrobial efficacy because of both antimicrobial silver and polyrhodanine nanofiber. The antimicrobial surface coating on a glass slide was tested against Gramnegative bacterium E. coli.32 The glass slide was coated with the silver/polyrhodanine nanofiber and the uncoated glass slide was also prepared for the comparison. Then, E. coli suspension was sprayed on the glass slides. The bacterial growth under LB agar was visualized after overnight incubation. The growth of E. coli was observed on the uncoated glass slide, while the coated glass inhibited the bacterial growth (Figure 4a). The inhibited growth of E. coli on the coated glass represents the antimicrobial surface of silver/polyrhodanine nanofiber. To further antimicrobial investigation, the antimicrobial kinetics was performed against Gram-negative E. coli, Gram-positive S. aureus, and yeast (C. albicans).38-40 A total of 3 mg of silver/ polyrhodanine nanofiber was inoculated with each 200 µL (106-107 CFU/mL) volume of aqueous microbial solution and the % reduction of microbial colonies was obtained as a function of contact time (Figure 4b). As the contact time increases, the % reduction increases and approaches an asymptotic value. Finally, no microbial growth is detected after 60 min against all three microorganisms. This result shows that the silver/ polyrhodanine nanofiber has effective antimicrobial efficacy toward Gram-negative and Gram-positive bacteria, as well as yeast. In addition, the antimicrobial kinetic test was also performed against pure silver nanoparticle and polyrhodanine homopolymer (see Supporting Information, Figure S2). From this test, it was shown that silver/polyrhodanine nanofiber had better antimicrobial efficacy than pure silver and polyrhodanine homopolymer due to the combined antimicrobial activity of silver and polyrhodanine nanofiber. The contact antimicrobial property of the silver/polyrhodanine nanofiber was investigated by the modified Kirby-Bauer technique on the lawn of E. coli, S. aureus, and C. albicans.11,38 In the silver/polyrhodanine nanofiber, silver with low-molecular weight polyrhodanine can be released and diffused into the lawn of microorganisms to exhibit contact antimicrobial activity. To compare the release and diffusion of silver, silver sulfadiazine was used as a control antimicrobial agent because it contained biocidal silver. For the test, an equal amount of silver compounds was made into a pellet with a size of about 13 mm by

hydraulic press and placed on each lawn of microorganisms in agar plates. The clear zone of inhibition around the pellet was observed after a 24 h incubation. Antimicrobial agents released from the pellets play a pivotal role in inhibiting microbial growth, leading to a clear zone around the pellets. Figure 5 shows that the clear zones of inhibition of silver/polyrhodanine are larger than that of silver sulfadiazine against three microorganisms. The diameter of zone of inhibition was measured and shown in Table 1. The zone of inhibition of silver/ polyrhodanine nanofiber is twice as large as that of silver sulfadiazine against E. coli and S. aureus. In the case of C. albicans, the diameter of the zone of inhibition for the silver/ polyrhodanine nanofiber is about 25 mm, whereas that of silver sulfadiazine is about 18 mm. This result indicates that the silver/polyrhodanine nanofiber has the enhanced contact antimicrobial efficacy than silver sulfadiazine.

Conclusion In conclusion, silver nanoparticles-embedded polyrhodanine nanofiber has been synthesized using a simple chemical oxidation polymerization. Silver nitrate was bound to rhodanine monomer by the complex formation and simultaneously reduced to silver nanoparticle by oxidizing the rhodanine monomer, resulting in the chemical oxidation polymerization. The fabricated silver/polyrhodanine nanofiber has been evaluated as an antimicrobial agent and shows the enhanced contact antimicrobial efficacy compared to silver sulfadiazine against Gramnegative, Gram-positive bacteria, and yeast. These results suggest that the silver/polyrhodanine nanofibers have potential for use in biomaterial applications requiring sterilization. Acknowledgment. This work was supported by the “SystemIC2010” project of the Ministry of Commerce, Industry and Energy, Republic of Korea. Supporting Information Available. The reaction scheme of the synthesis of Ag/polyrhodanine nanofiber, the XPS spectrum, and an additional antimicrobial kinetic test. This material is available free of charge via the Internet at http://pubs.acs.org.

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