Materials Science and Engineering C 49 (2015) 534–540

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Silver release and antimicrobial properties of PMMA films doped with silver ions, nano-particles and complexes O. Lyutakov a,⁎, I. Goncharova b, S. Rimpelova c, K. Kolarova a, J. Svanda a, V. Svorcik a a b c

Department of Solid State Engineering, Institute of Chemical Technology, Prague, Czech Republic Department of Analytical Chemistry, Institute of Chemical Technology, Prague, Czech Republic Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Czech Republic

a r t i c l e

i n f o

Article history: Received 18 April 2014 Received in revised form 11 December 2014 Accepted 6 January 2015 Available online 8 January 2015 Keywords: Doped polymer Silver nanoparticles Silver helical complex Antimicrobial properties

a b s t r a c t Materials prepared on the base of bioactive silver compounds have become more and more popular due to low microbial resistance to silver. In the present work, the efficiency of polymethylmethacrylate (PMMA) thin films doped with silver ions, nanoparticles and silver–imidazole polymer complex was studied by a combination of AAS, XPS and AFM techniques. The biological activities of the proposed materials were discussed in view of the rate of silver releasing from the polymer matrix. Concentrations of Ag active form were estimated by its ability to interact with L-cysteine using electronic circular dichroism spectroscopy. Rates of the released silver were compared with the biological activity in dependence on the form of embedded silver. Antimicrobial properties of doped polymer films were studied using two bacterial strains: Staphylococcus epidermidis and Escherichia coli. It was found that PMMA films doped with Ag+ had greater activity than those doped with nanoparticles and silver–imidazole polymeric complexes. However, the antimicrobial efficiency of Ag+ doped films was only short-term. Contrary, the antimicrobial activity of silver–imidazole/PMMA films increased in time of sample soaking. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Wide use of antibiotics in common medical praxes has initiated development of significant bacterial resistance and it becomes a significant problem in health care. The increase of the resistance poses a serious problem in the prevention and treatment of bacterial biofilms and related infectious diseases. Dealing with bacterial resistance requires precautions that lead to development of new antimicrobial substances. For these reasons, close attention has been paid to Ag-based compounds which, in contrast to many antibiotics [1], do not give significant rise to microorganism resistance. Of course, the development of bacterial resistance to silver is a matter of time, but at the moment, there are only few reports in literature concerning bacterial resistance to Ag-compounds [2]. Antibacterial activity of different forms of silver, including ions (Ag+), nanoparticles (AgNPs) and recently silver in the form of polymeric complexes, has been reported [3–6]. (i) Silver ions are widely applied in some medical fields. However, Ag+ is unstable and can be easily reduced to Ag0, which limits its practical applications. One of the ways of solving this problem is protection of silver by a polymer matrix. Silver-impregnated polymers provide a significant antimicrobial

⁎ Corresponding author. E-mail address: [email protected] (O. Lyutakov).

http://dx.doi.org/10.1016/j.msec.2015.01.022 0928-4931/© 2015 Elsevier B.V. All rights reserved.

efficacy with a sustained release of Ag+ [7]. (ii) Silver nanoparticles (AgNPs) also exhibit a wide spectrum of antimicrobial activities. AgNPs were found to accumulate in the bacterial membrane and induce formation of “pits” in the cell wall of the bacteria. A membrane with such morphology exhibits a significant increase in permeability, resulting in death of the cell [8]. In short time after the scientific reports of anti-microbial properties of AgNPs were made, they were considered as a universal panacea, but more recent studies indicated that AgNPs are cytotoxic, genotoxic, and antiproliferative. Their future applications should be limited by the fact that they are equally toxic to both, mammalian and bacterial cells [9]. (iii) Silver complexes with the helical polymeric structure provide a way to realize antibacterial but noncytotoxic material. They show a wide spectrum of the effective antibacterial and antifungal activities [10]. Coordinating ligands of the silver(I) complexes play the role of a silver ion carrier (I) to the biological system [11], and thus they are more selective against bacteria. Additionally, silver complexes can effectively restrict development of antibiotic resistance bacteria in medical settings [12,13]. Due to its low cost, mechanical strength, and minimal inflammatory response, polymethylmethacrylate (PMMA) has become one of the most frequently used materials to fill denture and bone cements. However, the risk of infection significantly increases in cases where PMMA is used as a medical material [14]. Incorporation of antibiotics in PMMA introduces antimicrobial properties, but it worsens the mechanical properties of the material. Additionally, the development of the

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antibiotic resistant bacteria has become a serious risk. To overcome these problems, the utilization of silver-based materials as effective antimicrobial agents was proposed [15]. Silver can be introduced into PMMA in its different bioactive forms, including ions [16,17], oxide [18] and frequently used silver nanoparticles. The results of the numerous studies showed that Ag/PMMA based materials significantly reduced bacterial growth and prevented biofilm formation [16–19]. So far, the efficiency of silver-doped polymer has not been linked with the structure of a polymer matrix and/or utilization of the different silver forms. The antimicrobial properties of Ag/PMMA depend strongly on the rate of silver release from the polymer matrix. The ability to inhibit the growth or kill bacteria by released silver has been reported by several authors [14,20]. On the other hand, as it was described [21], silver was not detected even after 120 days of storage of Ag/PMMA samples in deionized water. In such cases, the antimicrobial activity was manifested only when the bacterial cell was in direct contact with the tested samples. These unexpected results could be attributed to the cross-linked nature of acrylic resin that was used in the experiments. On the other hand, the utilization of non-crosslinked PMMA led to the appearance of apparent inhibition zone around the sample boundaries. The observed results proved the silver release from the polymer matrix [22]. An additional parameter that can affect the efficacy of materials based on Ag/PMMA is the soaked medium. As an example, antibacterial activity of AgNO3 can be reduced by components of serum peptides and proteins [23]. Moreover, in the work [24] complete absence of Ag/PMMA antimicrobial properties in surrounding culture medium has been reported. It should be noted that authors used only 1% (w/w) of silver in PMMA. On the contrary, our previous results [25] indicated that only PMMA films containing at least 10% of Ag become effective. With regard to the silver forms, materials with AgNPs are one of the most popular. Nevertheless, in the case of nanoparticles, their action is associated with the formation of the reactive oxygen species (ROS) [26]. ROS showed an additional antibacterial effect and the AgNPs based materials have a synergetic double antimicrobial action. However, the formation of ROS is a limiting factor of AgNP application in vivo [27]. Introduction of silver ions in polymer matrices is also a possible perspective, but, these materials have low temperature and light stability. The other limitation is their high reactivity and low binding selectivity. On the other hand, implementing coordination complexes of silver enable to get rid off these limitations. The application of such type of compounds is a new way of the preparation of the Ag bioactive materials. Recent attention in their development has been focused on a combination of silver with different ligands with the aim to prepare stable materials with appropriate resistivity to external temperature and light treatment [28]. Another alternative way to improve stability of silver complexes is their introduction into a polymer matrix. Up to now, this strategy has not been reported. In the present work, we report about the incorporation of silver imidazole complex into PMMA, its release, and resulting antimicrobial properties. The obtained results and antimicrobial efficiency were compared with the results for AgNP/PMMA and Ag+/PMMA materials.

2. Experimental 2.1. Materials AgNO3 (≥ 99.0% purity) and imidazole (≥ 99% purity) were purchased from Sigma Aldrich (Czech Republic) and used without further purification. PMMA of optical purity was purchased from Goodfellow (Germany). Tetrahydrofuran (≥ 99.9% purity) and 1-methyl-2pyrrolidinone (≥ 99.0% purity) were obtained from Sigma Aldrich (Czech Republic). Chloroform (≥99.5% purity, Cl− concentration max. 0.000015%) was purchased from Lach-ner (Czech Republic).

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2.2. Sample preparation 2.2.1. Preparation of Ag–imidazole complex AgNO3 water solution (0.024 mol, 12 mL) was added to an imidazole solution (0.048 mol, 48 mL) in water, which was followed by adding of NaOH water solution (0.025 mol, 25 mL). A white precipitate immediately formed. It was collected on a Buchner funnel (Whatman No. 5) after 2 h of stirring, washed with 500 mL of water, 200 mL of EtOH, and 200 mL of acetone. Then, it was dried in vacuum (3.56 g, yield N 84%). Analytical data: Calculated for C3H3N2Ag as a monomeric unit (%); C, 20.6; H, 1.7; N, 16.0; Ag, 61.7. Found (%): C, 20.6; H, 1.7; N, 15.7; Ag, 60.7; total, 98.7. The characteristic IR bands in 1700–500 cm−1 region measured by ATR (cm−1): 1504(m), 1476(s), 1462(s), 1306(m), 1280(m), 1245(m), 1234(m), 1173(m), 1092(vs),946(m), 858(m), 828(m),776(s), 757(s), 748(s), 664(vs). 2.2.2. Preparation of PMMA solution 5 g of PMMA was immersed into 66.5 g of chloroform for 5 days with periodical stirring. Formation of homogeneous solution was checked through optical transparency and absence of solution opalescence. 2.2.3. Preparation of doped polymer films For preparation of Ag+ doped polymer films 0.061 g of AgNO3 was dissolved in 5 g of tetrahydrofuran and mixed with 5 g of 7% PMMA solution. After careful mixing for 0.5 h in a sealed flask at ambient temperature, the thin PMMA films containing 10% of Ag+ were deposited by spin-coating procedure (1000 rpm for 10 min) onto a glass substrate and dried under 50 °C for 2 h to remove the residual solvent. Then, the samples were rinsed with distilled water. The resulted samples contained 10% of Ag+ (calculated amount). For preparation of AgIm doped polymer films, 0.063 g of AgIm powder was dispersed in 5 g of tetrahydrofuran and mixed with 5 g of 7% PMMA solution. After 1 h sonication in a sealed flask at ambient temperature, thin films of doped PMMA were deposited by spin-coating procedure (1000 rpm for 10 min) onto a glass substrate and dried under 50 °C for 2 h to remove the residual solvent. Then, the samples were rinsed with distilled water. Elemental composition was determined by the standard methods and PE2400 CHN elementary analyzer was used. The Ag contents of the films were determined by atomic absorption spectroscopy. Result of the chemical analysis: Ag 9.80% (15.87% of AgIm). For preparation of AgNPs doped polymer films, 0.061 g of AgNO3 was dissolved in 7.2 g of n-methyl pyrrolidone and mixed with 5 g of 7% PMMA solution in chloroform. After 30 s spin-coating, thin films were rapidly deposited onto a hot plate (200 °C) until a uniform yellow coloring appeared. N-methyl pyrrolidone served both as a cosolvent to dissolve the oxidative products and as a reducing agent. The details of the silver reduction by n-methyl pyrrolidone were described in Refs. [29,30]. The obtained samples contained 10% of Ag. Silver conversion rate was determined by XPS method and total silver distribution: 85% of Ag0 (apparently in the form of AgNPs) and 15% of the unreacted Ag+. AgNP size and distribution are given in the Results and discussion section. All spin-coating procedures were carried out using a WS-650-23B spin-coater. Resulted film thickness was measured by profilometry (Hommel Tester T1000 profilometer) using scratch method and optimized by relative solvent concentration (see above) to be 1 μm. 2.3. Sample soaking For further analysis, fresh and soaked (72 h) samples were used. Soaking of the samples was applied with the aim to estimate the stability of antimicrobial properties of the prepared thin films. Additionally, water sorption by doped polymer films was also estimated by gravimetric

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measurements before and after sample soaking. In all cases soaking was performed in distilled water. 2.4. Measurements 2.4.1. Silver–imidazole complex characterization AgIm complex formation and release were confirmed by Fourier transform infrared (FTIR) spectroscopy in the attenuated total reflectance mode (ATR). FTIR-ATR spectra were recorded using a Nicolet 6700 spectrometer (Thermo Scientific, France) with a SMART ATR accessory device. For ATR measurements, the films were mounted on the ZnSe crystal for further spectrum recording with 128 scans and 4 cm− 1 resolution. Solid polymer films were measured before and after the extraction in water. The FTIR-ATR data presented thus far resulted from the measurement of more than twenty films and their extracts. For the peak height intensity ratio measurements, the spectra were corrected using a straight baseline in region 3500–600 cm−1. For the study of the extracting solution, prepared films where soaked in 5 mL of D2O and extracted solutions were taken for analysis in different time intervals (0, 6, 12, 24, 48, and 72 h). Release of the complex from the polymer matrix was monitored by a decrease (for the films) and an increase (in the case of extract) in characteristic AgIm absorption bands in the regions of 1550–1400 cm− 1 (ring stretching and C\H deformation vibrations), 1250–1000 cm−1 and 950–600 cm−1 (C\H deformation vibrations). For extracted solutions, the observed changes in the regions of 3500–3300 cm−1 and 1800–1600 cm−1 associated with partial \OD/\OH exchange in D2O solvent. 2.4.2. Water sorption Water sorption tests were performed by mass-change measurement. Polymer films were deposited on 2.5 × 2.5 cm, 0.1 mm thick glass substrate and weighed before and after 12 h of soaking in distilled water. After soaking, the samples were purged with a stream of air to remove “surface” water drops from the surface. 2.4.3. Surface topography For characterization of sample surface before and after soaking (72 h), the AFM technique was applied. Surface profile was examined in a tapping mode with Digital Instruments CP II set-up. Areas of 1 × 1 μm were analyzed and arithmetic deviation of surface roughness (Ra) was calculated using standard algorithm in SPM-Lab Analysis software. In each case, measurements were carried out on three identical samples at three different locations on each sample. 2.4.4. Silver concentration in PMMA films Depth profile of silver concentration in PMMA films before and after sample soaking in distilled water (72 h) was measured by XPS technique combined with plasma etching. An Omicron Nanotechnology ESCAProbeP spectrometer was used to measure X-ray induced photoelectron spectra (XPS) and determination of element concentration. The analyzed area had dimensions of 2 × 3 mm2. The concentration of silver was calculated in at.%. Electron beam etching was additionally applied between separated XPS measurements with the aim to estimate Ag depth profile. 2.4.5. Released silver concentration in distilled water Prepared samples were soaked in 10 mL of water. After different time intervals, the extracted solutions were taken and evaporated to the final volume of 1 mL, then nitric acid (0.5 mL) was added to avoid Ag ion absorption on flask walls. For each measurement the individual sample of 1 μm thick and with an area of 1 cm2 was used. The measurements were repeated three times for each point. Bioactivity of released silver was determined through interaction of Ag with L-cysteine. For silver leaching, distilled water was used. Applied technique [31] gives clear results regarding the presence and concentration of the “biologically active” form of silver. Formation of chiral,

optically active complexes consisting of Ag and L-cysteine was determined by electronic circular dichroism (ECD) spectroscopy. Measurements were performed after different times of silver release. The CD spectra were recorded on a Jasco J-810 spectrometer (Jasco, Japan) using a quartz cell with a path length of 1 cm for spectral regions of 190–450 nm. The conditions of the measurement included a scanning speed of 100 nm min−1, a bandwidth of 1 nm, the standard sensitivity setting, an integration time of 2 s for each spectral point and 5 accumulations. All of ECD spectra of the samples were baseline-subtracted using the spectrum of the solvent obtained under the same experimental conditions. 2.5. Antimicrobial tests The antibacterial effect of PMMA films doped with AgNO3, AgNPs, and AgIm was carried out using two environmental bacterial strains: (i) Escherichia coli and (ii) Staphylococcus epidermidis, similarly as in work [32]. E. coli (DBM 3138) and S. epidermidis (DB 3179) for antibacterial tests are from the Microbial Type Culture Collection (Institute of Chemical Technology, Prague). Both types of PMMA samples, pristine and soaked type (1 × 1 cm2), were immersed in 2 mL of distilled water for different time intervals (0.5, 6 and 24 h) under sterile conditions, the control of pH was performed in each case and it showed no change. Overnight cultures of E. coli or S. epidermidis derived from a single colony and cultivated in Luria-Bertani broth medium were used for the experiment. The sample extracts were inoculated with the 0.5 mL of bacterial suspension and 0.5 mL of extract; final concentration of 1000 colony forming unit mL−1 (CFU mL−1). Bacterial samples incubated only in the pure physiological solutions served as controls. The inoculated solutions were incubated at laboratory temperature in static conditions for 24, 36 and 48 h. Aliquots of 25 μL from all samples were placed on LB agar plates in multiple replicates. The growth of E. coli and S. epidermidis was evaluated after 12 h of growth at 37 °C. Each sample was prepared separately in a triplicate. 3. Results and discussion Silver conversion rates as well as created NP size and distribution were carefully characterized by XPS, TEM and UV–VIS as was described in our previous works [25]. Briefly, both conversion rate and nanoparticle size distributions are the functions of the initial silver concentration and applied temperature. Under the conditions applied in this work, the conversion rate of silver was 85% and the size distribution of created NPs had the center at 5.2 nm and 2.3 nm halfwidth of a Gaussian distribution. Fig. 1 presents the concentration profile of Ag over depth of PMMA films before (Fig. 1A) and after (Fig. 1B) soaking for 72 h in distilled water. The profiles are shown for PMMA doped with AgNO3, AgNPs and AgIm. Prepared films have a thickness of 1 μm and etching depth was chosen to be 900 nm that is close to full depth of films. The silver distribution was not homogeneous immediately after the film preparation. In the case of AgNPs, the preparation procedure includes film heating over 200 °C. On the other hand, PMMA has a glass transition temperature of 120 °C. Thus, at 200 °C polymer chains are movable and try to exclude AgNPs from the polymer bulk. This phenomenon is typical for the polymer with dopant that is not covalently attached to the polymer matrix. The AgNO3 doped films show low silver concentration at the near to surface area. This observation can be explained by fast and simple leaking of silver from polymer surface during the sample rinsing after the spin-coating. The AgIm containing samples that were not heated and include silver in the form of “large” complexes showed relatively homogeneous silver concentration along the sample depth. Fig. 1B shows corresponding Ag depth profiles after 72 h film soaking. In all cases, surface concentrations of Ag were equal to zero. It is also evident, that 72 h of soaking is enough for complete leaching of

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Fig. 1. Surface morphology of PMMA film doped with AgIm complex before (A) and after (B) sample soaking for 72 h in distilled water.

AgNO3. On the contrary, a residual amount of Ag is well apparent in the cases of AgIm and AgNPs. Ag concentration increases with increasing depth up to an approximately constant value. This constant value is greater for AgNPs, indicating that AgIm complexes diffuse from PMMA easier than AgNPs. Therefore, non-homogeneous distribution of silver after the sample soaking could be attributed to both static and dynamic conditions. Small and soluble AgNO3 can quickly diffuse from the polymer films. Large AgNPs and insoluble AgIm complexes diffused at much slower rate. As for AgNPs, their presence can be easily monitored by film transparency (absence of a yellow color). Full transparency of AgNPs doped polymer films can be achieved within two weeks by periodic water replacement or about two months in static conditions. The surface characterization of samples by AFM indicated changes that occurred during dopant releasing. In this work, the attention was focused onto AgIm–PMMA films. Fig. 2 gives the surface morphology before and after film soaking for 72 h. An apparent change in surface morphology is evident. Sample surface became rougher after soaking. The surface roughness, i.e. arithmetic deviation from ideally flat surface (Ra), increased almost twice. Corresponding values of Ra (and their standard deviations) for pristine PMMA and PMMA doped with AgNPs and AgNO3 are given in Table 1. The surface morphology of pristine PMMA and AgNO3 doped film before and after silver extraction remained almost constant. In AgNPs and AgIm containing films the surface roughness increased twice. Therefore, surface roughening can be attributed to silver dopant releasing and larger dopants caused more pronounced surface distortion. Fig. 3 gives the results of atomic absorption measurements of Ag concentration in solutions extracted from AgNO3, AgNPs, and AgIm doped PMMA. In all cases the concentration of released Ag increased rapidly during early stages of extraction (0–2 h) and achieved a constant value (AgNO3) or continued to increase (AgNPs, AgIm) slowly with time. The determined amounts of released silver were the highest in the case of AgNO3 and approximately equal for AgNPs and AgIm. It was also evident that Ag+ ions were released more rapidly. The slowest release was observed in the case of AgNPs. Inset in Fig. 3 shows more detailed information about the first stages of the Ag release. From the inset it was apparent that silver release from AgIm was delayed when compared to the immediate silver release in the cases of AgNPs and AgNO3. It was proposed that in this case, Ag release was driven by a complex mechanism that includes diffusion forces and swelling of the polymer. The basis for this assumption is the high content of watersoluble fraction in the polymer. With the aim to estimate the polymer swelling the gravimetric tests were performed and their results confirmed water sorption by PMMA–AgIm films. Samples of 1 μm thickness and of 1 cm2 area soaked up to 6.6 μg of distilled water. Controlled

Fig. 2. Silver depth concentration profile in PMMA films doped with AgNO3, AgNPs, and AgIm complex before (A) and after (B) silver extraction for 72 h in distilled water.

measurements performed on pure PMMA, PMMA/AgNPs and PMMA/ AgNO3 films showed no increase in sample weight. Probably, diffusion of water in the film was caused by the polymer swelling and increase in a free volume in polymer matrix. The free volume increase may facilitate the release of AgIm. Polymer swelling can occur due to loose packing of polymer chains in the AgIm/PMMA films. This structure develops due to the lack of heat treatment during preparation procedure and the high AgIm concentration. In the case of PMMA/AgIm, it was also questionable in which form is the silver has been released from PMMA. In particular, there are two possibilities: (i) destruction of complexes and (ii) diffusion of Ag+ and Im separately or release of holistic complexes. To verify the integrity of AgIm during its embedding and releasing from PMMA, IR spectroscopy of doped polymer films and extracted solutions was performed. The results are given in Fig. 4 (A — solutions; B — films). Bottom curves of both parts of Fig. 4 corresponded to pure AgIm complex and indicated typical IR peaks [31,33] for AgIm. For the better resolution the peaks of interest were marked by dashed rectangles. Both parts of Fig. 4 indicate conservation of complex structure after incorporation into polymer as well as after extraction. From Fig. 4A, it is evident that during extraction the concentration of AgIm in extracted solution increased. Simultaneously, the concentration of AgIm in PMMA decreased (Fig. 4B). In general, we can conclude that AgIm is indeed released in a complex form.

Table 1 Surface roughness (Ra) of pristine PMMA and PMMA films doped with AgNO3, AgNPs, and Ag–imidazole complex (AgIm) before and after silver extractions. Sample

Ra (nm)/before

Ra (nm)/after

PMMA PMMA–AgNO3 PMMA–AgNPs PMMA–AgIm

0.14 ± 0.01 0.21 ± 0.02 0.22 ± 0.02 0.18 ± 0.02

0.17 ± 0.02 0.25 ± 0.03 0.47 ± 0.03 0.34 ± 0.04

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Fig. 3. Time dependences of silver concentration in extracted solution for PMMA films doped with AgNO3, AgNPs, and AgIm complex.

Fig. 5. Relative concentrations of biologically active silver released from PMMA films doped with AgNO3, AgNPs, and AgIm complex over time.

The biological activity of silver is a function of many parameters, such as a silver form, an oxidation state, and the releasing kinetic. Absolute Ag concentration from atomic absorption measurement (Fig. 3) is useful but not a deciding parameter. The concentration of biologically active silver was determined by interaction with L-cysteine. The changes in the concentration of Ag active form in dependence on the extraction

time are shown in Fig. 5. As it could be expected, in all cases, “active” silver concentrations increased rapidly from zero and after certain time reached an approximately constant value. The quickest increase occurs in the case of AgNO3. In the same case this constant value is higher comparing to AgNPs and AgIm. This result was in good agreement with the results from absorption spectroscopy and XPS measurements (Figs. 2

Fig. 4. FTIR spectra indicated of AgIm releasing in PMMA films: A — spectra of extracting solution taken after different time intervals, B — spectra of thin PMMA films doped with AgIm taken after different extraction times.

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Table 2 Antimicrobial study (● — growth, — 50% growth and ○ — inhibition) of fresh (A) and soaked (B) samples of PMMA doped with AgNO3, Ag–imidazole (AgIm), and AgNPs. Staphylococcus epidermidis and Escherichia coli were employed as model bacterial strains and were in contact with the tested samples for 24, 36 and 48 h, the silver extraction times were 0.5, 6 and 24 h. Untreated bacteria in physiological buffer and pristine PMMA served as controls. A — fresh sample Bacteria treatment time

24 h

Ag extraction time

0.5 h

36 h

48 h

6h

24 h

0.5 h

6h

24 h

0.5 h

6h

24 h

Staphylococcus epidermidis Control ● PMMA ● ● AgNO3 AgIm ● AgNPs ●

● ● ● ● ●

● ● ● ● ●

● ● ○ ●

● ● ○ ●

● ● ○ ● ●

● ● ○ ○ ○

● ● ○ ○ ○

● ● ○ ○ ○

Escherichia coli Control PMMA AgNO3 AgIm AgNPs

● ● ● ● ●

● ● ● ● ●

● ●

● ●

● ●

● ●

● ● ○ ○ ○

● ●

● ●

● ● ● ● ●

○ ○

○ ○

36 h

48 h

24 h

36 h

48 h

24 h

36 h

48 h

Staphylococcus epidermidis Control ● PMMA ● ● AgNO3 AgIm ● AgNPs ●

● ● ● ● ●

● ● ● ● ●

● ● ○

● ● ○

● ● ○ ● ●

● ● ○ ○ ○

● ● ○ ○ ○

● ● ○ ○ ○

Escherichia coli Control PMMA AgNO3 AgIm AgNPs

● ● ● ● ●

● ● ● ● ●

● ● ●

● ● ● ● ●

● ● ● ● ●

● ● ● ○ ○

● ● ● ○ ○

● ● ● ○ ○

● ● ● ● ●

B — soaked sample Ag extraction time

0.5 h

Bacteria treatment time

24 h

● ● ● ● ●

6h

●

24 h

and 3). On the other hand, the comparison of absolute and biologically active silver concentrations in the case of AgNPs and AgIm gave inconsistent results. From AAS and XPS, there were evident, approximately equal concentrations of released Ag but according to Fig. 5 the concentration of biologically active silver was higher for AgIm. Despite the fact that the absolute amounts of released silver were equal, the biological activity of silver released from AgIm complex was higher than from AgNPs. As a final step, the primary antimicrobial activity of Ag doped polymer films was verified using a Gram-positive bacterial strain of S. epidermidis and a Gram-negative bacterial strain of E. coli. The antimicrobial tests were performed on “fresh” samples and samples soaked for 12 h in distilled water. Probes of the solutions were taken after 0.5, 6, and 24 h of silver extraction. After sampling, the probes were in contact with bacteria for 24, 36, and 48 h (37 °C) and observations of bacterial growth were performed after 12 h. The results of the antimicrobial tests are presented in Table 2. An empty symbol corresponds to complete growth inhibition of bacteria (both E. coli and S. epidermidis), see Fig. 6; a closed symbol indicates a case of fully surviving bacteria (the number of founded colonies corresponded to the number of CFU in a drop of water — 235 with the error of 10%), the half-closed symbols correspond to an intermediate case, where we have observed only moderate growth inhibition. Nevertheless, the number of CFU was significantly decreased. Noteworthy, the “fresh” samples of PMMA films doped with AgNO3 exhibited the best antimicrobial activity against S. epidermidis. Complete growth inhibition effect on S. epidermidis and the particular growth inhibition effect on E. coli occurred after bacterial

Fig. 6. Photos for illustration of inhibitory effect of silver doped PMMA samples on growth of E. coli. The full circles indicate growth (A, ●), the half full circles indicate 50% of growth (B, e) and the empty circles indicate inhibition effect (C, ○).

contact with solutions extracted only for 6 h. This result correlates poorly with the data of absorption spectroscopy, which indicates quick silver release (according to Fig. 3 silver concentration in solution takes constant value after 45 min of extraction). However, the correlation with the concentration of biologically active silver seems to be somewhat better (according to Fig. 5 concentration of biologically active silver has constant value after 4 h of extraction). Mildly decreased antimicrobial activity against S. epidermidis was observed in the case of AgNP– PMMA films. The particular influence on S. epidermidis growth was evident already after 6 h of extraction. However, these films seem to be more effective on a Gram-negative bacterial strain, E. coli. To fully inhibit the growth of E. coli, at least 24 h of extraction was necessary. The least active ones against S. epidermidis were AgIm-containing films. In the case of E. coli, their activity was comparable to that of AgNPs and improved over AgNO3. Like in the case of AgNO3 effectivity of AgNPs and AgIm doped films corresponded better with Fig. 5 than with Fig. 3. Absolute silver concentration reaches constant value after 3 h of extraction in the case of AgIm, and only slowly increases after the same time for AgNP case (Fig. 3). However, concentration of biologically active silver did not reach a constant value until 12 h of extraction.

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The “fresh” and soaked samples with AgNPs and AgNO3 had similar effect on S. epidermidis growth. Surprisingly, the activity of AgIm containing films against S. epidermidis increased after soaking. Probably, water sorption during the soaking resulted in a more facile release of silver during the following extractions. It was also observed that soaked PMMA–AgNO3 films completely lost their effectivity against E. coli. The soaked AgNPs and AgIm containing films, however, conserved their effectivity against E. coli. Additionally, the effectivity of PMMA–AgIm increased after the soaking. The obtained results indicated that the proposed thin silver doped polymer coatings are perspective and highly effective antibacterial coatings and fillings for the utilization with the aim to treat bacteria colonization and to reduce the likelihood of infection. 4. Conclusion In the present paper, the relation between the ability of silver release from PMMA films doped with Ag ions, nanoparticles, and imidazole polymeric complex and their time-dependent antimicrobial activities was studied. Our result showed that short-term antimicrobial efficiency of Ag+/PMMA materials was higher compared to samples containing AgNPs and AgIm. However, the antimicrobial efficiency of AgNO3 doped PMMA films was significantly reduced after 72 h of soaking. The highest total released silver concentration was found in the case of AgNPs doped polymer films. In the case of AgIm, the concentration of the released silver was enhanced by swelling of doped polymer films. Additionally, it was found that AgIm conserves structure of the complex after embedding into polymer matrix and during its release. According to the result of microbiological tests, the biological potency of released silver from the polymer matrix changes in a row AgNO3 doped PMMA films N AgIm doped N AgNPs doped films. The antimicrobial activity of doped polymer films also depended on the form of embedded silver. Based on our experiments, it was shown that AgNO3 doped PMMA films has shorter-termed activity compared with AgIm or AgNPs doped films and general trend in the long-term activity was described as AgNPs doped films N AgIm doped PMMA ≫ AgNO3 doped PMMA. Unexpectedly, it was found that the antimicrobial activity of AgIm films increased after soaking. Thus, Ag complexes were shown as perspective agents that save their antimicrobial properties in polymer matrix and had no limitation associated with application of cytotoxic AgNPs. Additionally, AgIm can be protected from the external environment by incorporating it into the polymer matrix. Acknowledgments This work was supported by the GACR under the project P108/12/ G108. References [1] T. Okabe, K. Saito, Y. Otomo, Antimicrobial activity and safety of hinokitiol, Fragr. J. 17 (1989) 74–79. [2] R.Y. Pelgrift, A.J. Friedman, Nanotechnology as a therapeutic tool to combat microbial resistance, Adv. Drug Deliv. Rev. 65 (2013) 1803–1815. [3] R. Kumar, H. Munstedt, Silver ion release from antimicrobial polyamide/silver composites, Biomaterials 26 (2005) 2081–2088. [4] K. Nomiya, S. Takahashi, R. Noguchi, S. Nemoto, T. Takayama, M. Oda, Synthesis and characterization of water-soluble silver(I) complexes with L-histidine (H(2)his) and (S)-(−)-2-pyrrolidone-5-carboxylic acid (H(2)pyrrld) showing a wide spectrum of effective antibacterial and antifungal activities. Crystal structures of chiral helical polymers [Ag(Hhis)](n) and {[Ag(Hpyrrld)](2)}(n) in the solid state, Inorg. Chem. 39 (2000) 3301–3311. [5] J. Siegel, M. Polivkova, N. Slepickova Kasalkova, Z. Kolska, V. Svorcik, Properties of silver nanostructure-coated PTFE and its biocompatibility, Nanoscale Res. Lett. 8 (2013) 1–10. [6] J. Siegel, K. Kolářová, V. Vosmanská, S. Rimpelova, J. Leitner, V. Svorcik, Antibacterial properties of green-synthesized noble metal nanoparticles, Mater. Lett. 113 (2013) 59–63.

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