Preparation of Polyethylene Composites Containing Silver(I

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Preparation of Polyethylene Composites Containing Silver(I) Acylpyrazolonato Additives and SAR Investigation of their Antibacterial Activity Fabio Marchetti,*,†,‡ Jessica Palmucci,*,† Claudio Pettinari,§,‡ Riccardo Pettinari,§,‡ Mirko Marangoni,† Stefano Ferraro,† Rita Giovannetti,† Stefania Scuri,∥ Iolanda Grappasonni,∥ Mario Cocchioni,∥ Francisco José Maldonado Hodar,⊥ and Roberto Gunnella# †

School of Science and Technology, Chemistry Section, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC) Italy ICCOM, CNR 62032 Camerino, Italy § School of Pharmacy, Chemistry Section, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC) Italy ∥ Research Centre for Hygienistic, Health and Environmental Sciences, School of Pharmacy, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino (MC) Italy ⊥ Departamento de Química Inorgánica, Universidad de Granada, Av. Fuentenueva s/n, 18071 Granada, Spain # School of Science and Technology, Physics Section, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino (MC) Italy ‡

S Supporting Information *

ABSTRACT: Novel composite materials PEn (n = 1−9) have been prepared by an easily up-scalable embedding procedure of three different families of Ag(I) acylpyrazolonato complexes in polyethylene (PE) matrix. In details, PE1−PE3 composites contain polynuclear [Ag(QR)]n complexes, PE4−PE6 contain mononuclear [Ag(QR)(L)m] complexes and PE7−PE9 are loaded with mononuclear [Ag(QR) (PPh3)2] complexes, respectively (where L = 1-methylimidazole or 2-ethylimidazole, m = 1 or 2, and HQR = 1-phenyl-3methyl-4-RC(O)-5-pyrazolone, where in detail HQfb, R = −CF2CF2CF3; HQcy, R = −cyclo-C6H11; HQbe, R = −C(H)C(CH3)2). The PEn composites, prepared by using a 1:1000 w/w silver additive/polyethylene ratio, have been characterized in bulk by IR spectroscopy and TGA analyses, which confirmed that the properties of polyethylene matrix are essentially unchanged. AFM, SEM, and EDX surface techniques show that silver additives form agglomerates with dimensions 10−100 μm on the polyethylene surface, with a slight increment of surface roughness of pristine plastic within 50 nm. However, the elastic properties of the composites are essentially the same of PE. The antibacterial activity of all composites has been tested against three bacterial strains (E. coli, P. aeruginosa and S. aureus) and results show that two classes of composites, PE1−PE3 and PE4−PE6, display high and persistent bactericidal and bacteriostatic activity, comparable to PE embedded with AgNO3. By contrast, composites PE7−PE9 exhibit a reduced antibacterial action. Contact and release tests in several conditions for specific migration of Ag+ from plastics, indicate a very limited but time persistent release of silver ions from PE1−PE6 composites, thus suggesting that they are potential antibacterial materials for future applications. Instead, PE7−PE9 almost do not release silver, only trace levels of silver ions being detected, in accordance with their reduced antibacterial action. None of the composites is toxic against higher organisms, as confirmed by D. magna test of ecotoxicity. KEYWORDS: composite materials, Ag(I) acylpyrazolonato additives, polyethylene, AFM analysis, SEM analysis, antibacterial activity, contact and release tests

1. INTRODUCTION

attracted considerable research interest for potential applications ranging in burn treatment,7,8 joint arthroplasty,9 bacterial colonization,10,11 and water treatment.12 Among the different approaches

Microbial contamination of polymeric materials plays an important role in the transmission of infectious diseases.1−4 Microorganisms have in fact a strong ability to survive on ordinary polymeric materials, and some strains can stay alive on various polymers for more than 90 days. In response to the microbial challenges, the development of antimicrobial plastics5,6 has © XXXX American Chemical Society

Received: August 4, 2016 Accepted: October 11, 2016

A

DOI: 10.1021/acsami.6b09742 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces for antimicrobial functionalization of plastics, two main strategies can be underlined: one involves a chemical coating procedure to create a surface with appended cationic moieties, such as quaternary ammonium fragments;13−17 another is based on the encapsulation of antimicrobial agents which exert their biocidal action by slow release from the plastic.18 In the latter approach silver nanoparticles (AgNPs),19,20 silver nanohybrid 21−23 or silver(I) based substances24−28 are by far the most used and investigated, due to silver broad-spectrum antibacterial performances and relatively lower toxicity to human cells than that to microbial cells.29 Particularly AgNPs have received in the last decades a growing interest and have found commercial applications.30 However, many problems persist in their safe use, as very little is still known about the metabolism, clearance and toxicity of nanoparticles. Still not defined with certainty is the nature of the optimal targets of certain infections and the ideal drug levels for therapeutic activity at the target site of pathogens.31 On the other hand, concerning the use of silver(I) compounds as biocides, very few of them combine light and thermal stability, chemical inertness and water insolubility to an appreciable antibacterial activity. So that it is still a great challenge to design and prepare new silver-based antimicrobial materials with high activity and durability.32 Many materials that kill bacteria by releasing bactericidal agents are in fact eventually depleted of the active substance because of its release into the surrounding solution within a short time. Our group has been involved in the silver coordination chemistry for many years,33−36 and our recent studies led us to prepare silver complexes37,38 with high Gram-positive and Gram-negative antibacterial activity. In particular, our research is devoted to prepare composite plastics with antibacterial properties, intended for the production of objects with low intrinsic value, such as filters, bottles and containers. To this goal, the simple addition of additives during the extrusion process is the best option to limit the increase of price of composite with respect to non additioned plastic. In previous works we have investigated the structural diversity and antibacterial activity of silver coordination polymers with dipyrazoles39 and acylpyrazolones,40 a family of β-diketones with a pyrazole fused to the chelating arms, which are low cost and ease to prepare and modify.41,42 With some acylpyrazolones we reported a number of mono- and polynuclear Ag(I) complexes and investigated their potentials as antibacterial additives for the preparation of composite polyethylene plastics,40 whose antibacterial activity is based on a very limited but persistent release of silver(I) ions within a long time. This is a very interesting feature, as it has been recently recognized that the extensive release of the Ag+ ions or AgNPs from materials loaded with them can lead to environmental hazards43 and toxicity for humans,44 while also promoting the development of resistant microbial strains.45 In continuation of previous research, we have recently synthesized novel mono- and polynuclear Ag(I) complexes with different acylpyrazolone ligands, of composition [Ag(QR)]n, [Ag(QR)(L)m] and [Ag(QR) (PPh3)2], bearing different acyl -C(O)R moieties (HQ in general = 4-acyl-5-pyrazolone; HQfb: R = −CF2CF2CF3; HQcy: R = −cyclo-C6H11; HQbe: R = −C(H)C(CH3)2) and nitrogen- (L = 1-methylimidazole or 2-ethylimidazole, m = 1 or 2) or phosphorus-based (triphenylphosphine) ancillary donor ligands (Figure 1).46 These ligands were chosen to modulate the physicochemical parameters and the biocidal activity of their silver complexes. In details, we have introduced lipophilic substituents in R position of the acyl moiety (Figure 1) to increase the adhesion toward the polyethylene matrix, thus improving the

Figure 1. Molecular structures of Ag(I) complexes 1−9.

homogeneity of the composite material. All the silver complexes were shown to be air, moisture, and light stable. Additionally they are insoluble in water and thermally stable well above 150 °C. Their intrinsic antibacterial activity was assessed and, while the polynuclear 1−3 and the mononuclear 4−6 displayed a high and almost steady in time antibacterial activity, the phosphinecontaining complexes 7−9 show a reduced efficiency in inhibiting the growth of Escherichia coli and Staphylococcus aureus. Such a different behavior was rationalized through a combination of DFT theoretical computations and experimental techniques and related to the degree of saturation of silver and to the presence of different ancillary ligands.46 Here we have employed the above-mentioned silver complexes 1−9 (Figure 1) as additives to embed in polyethylene (PE), with the aim to obtain novel low cost composite materials PEn (n = 1−9) with good antimicrobial performances. In addressing this goal, we have tried to use the minimum amount of additive, to reduce its impact on the mechanical characteristics of the polyethylene matrix and on the cost of the composite material. Polyethylene is currently the major volume polymer produced globally, with a total over 90 million metric tons per annum.47 It was chosen in this study because it is a readily available, inert and cheap common plastic material, extensively used in packaging and containers for food and safe drinking water, due to its flexibility, easy processability, thermal stability, environmental recyclability, and inexpensive properties.48 An extensive investigation of the bulk and surface properties of the PEn composites has been undertaken to identify the modifications of polyethylene matrix after the embedding process. Hence, the antibacterial activity of the composite materials toward selected bacterial strains has been evaluated and the different outcomes discussed in terms of structural differences of the embedded silver complexes. In particular we decided to test the biocidal activity of our composites against a Gram-positive strain, Staphylococcus aureus, and two Gramnegative strains, Pseudomonas aeruginosa and Escherichia coli. They have been chosen due to their isolation from human infections and because they represent well-known pathogens of medical relevance. They are difficult to treat and eradicate worldwide, predominantly due to their resistance to multiple antibiotics treatments. In detail, S. aureus is typically associated with skin infections, responsible for many hospital- and community-acquired infections and occasionally causes food poisoning.49 P. aeruginosa is a pathogen able of causing several infections in humans, including cystic fibrosis, urinary tract infections and surgical wounds.50 E. coli is usually related to food poisoning, diarrhea, and hemolytic uremic syndrome.51 Moreover, the E. coli strain is the reference recommended by CLSI (Standards). Finally, we have investigated the effects on bacterial B


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ACS Applied Materials & Interfaces membrane permeabilization by contact with the composites and quantified the silver ion release capacities of the composite materials in aqueous media, according to EU Legislation on the migration of chemicals from plastic materials,52 with the aim to confirm the mechanism of antibacterial action and demonstrate the relationship between typology of silver additive and silver release in aqueous media, as quantified by the amount of free silver ions present in water.

2. RESULTS AND DISCUSSION 2.1. Synthesis of the Silver Complexes. Silver complexes 1−9 were prepared and characterized according to previous synthetic procedure.46 They were obtained as colorless or pale yellow powders with granules dimensions within the range 10−100 μm (Figure S1 in Supporting Information). Their molecular structures are shown in Figure 1: complexes 1−3 are inorganic coordination polymers with coordination number three on silver, whereas 4−9 are molecular monomers where the silver coordination number ranges from two to three in 4−6 and four in 7−9. It is worth of mention that complexes 1−9 are soluble in dmso and 4−9 also in chlorinated solvents and slightly in acetonitrile, but all of them are insoluble in water. 2.2. Synthesis of the Composite Materials. The polyethylene (PE) composite materials PEn (n = 1−9) were prepared by embedding the silver complexes 1−9 in a PE matrix. The embedding process is a very simple procedure, easily reproducible and up-scalable even in the industry of plastics, where the reversible thermoplastic properties of PE are appropriately employed. Briefly, after mixing and grinding PE grains and the powdered biocidal additive in the correct w/w ratio, the solid mixture is heated at ca. 150 °C, well above the melting point of PE (98 °C) and the resulting liquid is maintained under heating and mechanical stirring for half hour. Then, the mixture is left to cool and to harden in the form of solid thin disks or squares of thickness 0.5 mm. In principle, we have prepared three different composites through embedding the silver complex 1 in PE by using three different additive/PE weight ratios as follows: PE1a (1:500 w/w), PE1b (1:1000 w/w) and PE1c (1:2000 w/w). The combination of surface characterization and antibacterial tests showed that PE1b with the 1:1000 w/w ratio was the most suitable for killing bacteria within 24 h, without using an excess of Ag(I) additive. Based on this, all PEn composites (n = 1−9) were prepared by using the 1:1000 w/w additive/PE ratio. It is worth of mention that AgNPs, massively investigated in the last years as antimicrobial additives for a number of plastics, are generally introduced in the polymeric matrix in a weight ratio much greater than 1:1000.19,53 2.3. Characterization of the Composite Materials. The PEn composites were characterized by FT-IR spectroscopy and TGA analyses. The FT-IR spectra and TGA curves of PE1, PE4, and PE7 are compared to PE0 in Figures 2a and b, respectively. As expected, with a 1:1000 w/w concentration of the silver additives in the polyethylene matrix, the composite materials do not display any sensible change of their infrared or thermal properties with respect to unloaded PE. In particular, the FT-IR spectra of PEn are almost identical to PE0, which displays the typical strong absorptions in the 3000−2850 cm−1 region due to C−H stretching, those around 1470 cm−1 due to the C−H scissoring and those at ca. 720−730 cm−1 due to long-chain methyl rock. Therefore, the embedding of the silver additives does not modify the internal structure of polyethylene.54 At so low concentrations in the polymeric matrix, the infrared absorptions of the additives were not detectable. To check eventual

Figure 2. Comparison of (a) FT- IR spectra of composites PE1 (red), PE4 (blue), and PE7 (purple) with respect to unloaded PE0 (black), and (b) TGA curves of PE1 (red), PE4 (blue), and PE7 (purple) with respect to those of PE0 (black).

modifications in their structure upon embedding, we have prepared PE3 composites loaded with higher amounts of the silver compound 3 and recorded their IR spectra. Only with a composite additioned with 2.5% of 3 (1:40 of 3:PE weight ratio) it was finally possible to observe most of the IR bands of the silver additive (Figure 3).

Figure 3. Comparison between the infrared spectra of unloaded PE0 (black), of composite PE3 (red) at 2,5% weight ratio, and of the silver compound 3 (blue).

The pattern of absorption bands in PE3, due to the silver additive, displays a main difference with respect to that in the spectrum of pure 3:46 the band at 1606 cm−1, likely due to νas(CO), slightly shifts at 1614 cm−1 upon embedding (Figure 3), which suggests some weak van der Waals interactions with the polyethylene matrix. The surface characterization of PE1a, PE1b, PE1c, PE6, and PE7, chosen as representative composites, was carried out through atomic force microscopy (AFM)55 and scanning electron microscopy (SEM) investigations. AFM is a powerful tool for the study of surface topography since it can be provide high-resolution 3D images of the surface of materials without any sample pretreatment. In particular, here we have used AFM to characterize the surface topography and roughness C


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Figure 4. Contact mode AFM pictures of different polyethylene samples of PE1, with different amounts of silver complex 1: (a) 0% (PE0, unloaded PE); (b) 0.05% (PE1c); (c) 0.1% (PE1b); (d) 0.2% (weight ratio) (PE1a).

of PE samples after the embedding process of silver additives. Images were taken by AFM contact mode microscopy and a tip at 20 kHz and 0.07 N/m by using constant voltage mode and 30 mV set point and lock-in feedback. Time base of 30 ms per point was used to minimize noise. In Figure 4 the evolution of the Ag complex 1 embedding into the pure polyethylene (PE0), by using different additive/polyethylene weight ratios, is reported. In addition to an overall loss of polycrystallinity after Ag(I) complex 1 embedding (visible as a fine texture in the pristine polyethylene PE0 in Figure 5), the formation of deeper valleys

Figure 6. AFM force−distance curves in a microscope for the pristine PE0 and the most additioned PE1a sample.

Figure 5. Higher resolution image showing loss of crystallinity from (a) the pristine PE0 to (b) the most additioned PE1a sample.

with respect to pristine smooth surfaces of polyethylene samples was observed, with a significant increase in slope and evolution of less flat surfaces. The elastic properties of pristine PE0 and of the most additioned PE1a (1:500 w/w additive/PE) were evaluated by the slope of the force−distance curves of the system “tip-sample” with the AFM microscope. As it can be seen from Figure 6, the system shows hysteresis when the measurements are repeated but the linear elastic properties are the same for the two samples with less than 5% indetermination in the slope value.56 Hence, we can consider that the elastic features of additioned PEn samples, with only 1:1000 w/w additive content, are essentially unaltered with respect to pristine PE0. In Figure 7 the 3D AFM picture of the surface of PE6 is reported, showing an agglomerate of the silver additive on the surface of PE. The size of the agglomerate, as visualized in the AFM image, and from the cross section profile, is about 10 μm of diameter, which is consistent with the scale of values (10−100 μm) found by SEM investigations (see below). An analysis of morphology of the PEn samples was carried out to check eventual changes of the surface roughness. We have undertaken this analysis because this parameter could influence the bacterial adhesion; in particular, it has been reported that a slight increase

Figure 7. Three-dimensional AFM image of the surface of PE6, displaying an agglomerate of the silver additive 1.

in surface roughness of ca. 0.04−1.24 μm can produce a substantial increase in bacterial adhesion of the plastic surface, while a successive increases in surface roughness in the range 1.86−7.89 μm results in a decrease in bacterial attachment.57,58 In our PEn samples, however, the microsurface roughness values reported in Table 1 are not considerably different between before and after the silver complexes embedding. Therefore, we can exclude that the surface roughness could influence the antibacterial activity on the loaded PEn samples in the present D


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Table 1. Morphology and Grain Size of Selected PEn Composites (Values between Brackets Are the Std Deviations), area is 11 nm2 Ra (average roughness), nm Rq (rms), nm

PE0

PE1a

PE1b

PE1c

PE6

PE7

0.12 (±0.08) 0.14 (±0.09)

49.92 (±5.40) 62.96 (±6.61)

16.34 (±2.08) 20.34 (±2.83)

0.16 (±0.08) 0.19 (±0.08)

15.68 (±1.89) 20.46 (±1.62)

20.29 (±3.71) 25.04 (±4.15)

are within 200 and 500 μm. By contrast, using a much lower additive/PE ratio (PE1c with 1:2000 w/w), the dimensions of conglomerates fall below 20 μm and the separation between them increases (Figure 11). In all composites the lower limits for the dimensions of conglomerates is however in the micrometric scale and is likely related to the starting dimensions of powdered granules of the additives. 2.4. Antibacterial Activity of the Composite Materials. The activity of the composites against E. coli, P. aeruginosa and S. aureus, the first two chosen as models of Gram-negative and the latter as a model of Gram-positive bacterial strains, was assessed by enumerating the total colony forming units (CFUs) after incubation for 24 h at 37 °C. A preliminary investigation was carried out on composites PE1a, PE1b, and PE1c, in order to find the most suitable additive/PE weight ratio for the sequential preparation of PEn composites with good antibacterial activity and low additive concentration. Figure 12 shows the trends of growth inhibition by PE1a−PE1c composites against the three bacterial strains used. The mean CFU/mL values for all experiments performed in triplicate, with their standard deviations, are reported in Table S1 (Supporting Information). The composite PE1c, prepared with 1:2000 w/w ratio, showed the lowest antimicrobial activity against all three bacteria strains within 24 h. Whereas, the composite PE1b, having a 1:1000 w/w displayed essentially the same efficiency of the most additioned PE1a. The superior agglomeration degree of the silver additive 1 in PE1a (Figure 10) evidently reduces its efficiency in delivering silver ions and also the antibacterial performances are lower than expected, being practically the same of PE1b.Based on such results, we decided to prepare the composites PEn by using the 1:1000 additive/PE weight ratio. Then, also the antibacterial activity of PEn composites was investigated against the abovementioned bacterial strains (E. coli, P. aeruginosa, and S. aureus). Figure 13 shows the trends of growth inhibition by PEn composites within 48 h, compared to a negative (PE0, unloaded) and a positive control (PEAgNO3). The mean CFU/mL values for all experiments performed in triplicate, with their standard deviations, are reported in Tables S2−S4 (Supporting Information). As shown in Figure 13, PEn composites show different behaviors in terms of time and effectiveness of bacterial reduction. The “wavy” growth behavior found for some PEn in the experiments against S. aureus (Figure 13a) is related to its typical metabolism. In details, S. aureus displays a much lower generation time with respect to E. coli or P. aeruginosa. At the beginning, the antibacterial action of PEn seems more efficient than after an incubation period of 12−16 h, when the growth of S. aureus reaches a maximum and the growth curves are wavy-like. However, within 24 h, also such an apparent “second growth” of S. aureus is depressed by the antibacterial action of PEn composites. The number of viable cells was then employed to calculate the killing percentage (Tables 2, 3, and 4), useful to define if the polymer presents bactericidal or bacteriostatic action. The bactericidal activity is defined as the reduction of the number of viable bacterial cells ≥99.9% inoculum within 18−24 h. Whereas, the bacteriostatic activity is defined as the reduction of between 90 and 99% inoculum, always within 18 and 24 h.60 In particular, PE1, PE2, PE3, and PE4 exceeded 99.0% reduction

study. In any case, a significant increase in the nanometric scale of the surface roughness of pristine PE0 has been observed by increasing the amount of the silver additive 1 on going through composites PE1c, PE1b, and finally PE1a (Table 1 and Figure 4).59 Representative PEn composites have been also investigated by SEM spectroscopy. The EDX analyses confirmed, by a semi qualitative analysis, the chemical composition of composites and also determined the level of additive microscopic dispersion in the samples analyzed. High resolution SEM images and EDX spectra show that the silver-containing aggregates are as a matter of fact randomly distributed and well isolated conglomerates on the surface (Figure 8 and Figure S2 in Supporting Information),

Figure 8. SEM image with a large view of the surface of PE7 composite.

with two-dimensional diameters ranging from 10 to 100 μm. A magnification of SEM and EDX for PE5 is shown in Figure 9,

Figure 9. Magnification of SEM image and EDX spectrum of a silver additive conglomerate on the surface of PE5 composite.

where dimensions within 100 μm of diameter are well visible. For such 1:1000 w/w concentrations of the silver additives, the conglomerates are well separated from each other. By changing the additive/PE ratio, also the dimensions of conglomerates change accordingly. In Figures 10 and 11 the SEM images of PE1 composites, prepared with different additive/PE ratios, are reported. In detail, Figure 10 displays the PE1a composite (with a 1:500 w/w ratio), and the dimensions of conglomerates E


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Figure 10. High-resolution SEM image and EDX spectra of silver additive conglomerates on the surface of PE1a composite prepared with 1:500 w/w ratio.

Figure 11. High-resolution SEM image and EDX spectra of silver additive conglomerates on the surface of PE1c composite prepared with 1:2000 w/w ratio.

within 16 h, PE5, PE6, and PE7 exceed 90% reduction, showing than a constant activity up to 48 h. In particular, PE6 reaches 99.9% reduction already at 16 h, while maintaining almost constant its activity until the end of the test. Finally, PE8 and PE9 have the lowest activity, reaching the 98% reduction only at the end of the test. It is worth of mention the behavior of PE5 and PE6, which showed the best activity against P. aeruginosa: both composites have reached 98% reduction within 16h, and 99.9% within 24h, then maintaining constant bactericidal activity until the end of the test (Table 4). The composites PE4 and PE8 have instead reached 99.9% reduction within 48 h. Finally, concerning E. coli growth inhibition tests (Table 3), PE5 proved to be the best composite, reaching 99.9% reduction within 12 h of exposure and maintaining this activity constant for the whole duration of the test and comparable to that of the positive control PEAgNO3. PE6 also showed a good performance, reaching the bactericidal activity within 16 h and keeping it constant up to 48 h. PE1 reached 99.9% within 24 h, whereas PE2, PE3, and PE9 are by far the composites with the lowest activity against E. coli. PE7 and PE8 exceed both 90% reduction within 16h, although not reaching reduction percentages deemed as bactericidal. All these results are in line with previous findings on the intrinsic antibacterial activity of the silver additives 1−9.46 2.5. Silver Ion Release from the Composite Materials. The ability of PE1−PE9 composites to release Ag+ ions in aqueous environment was investigated through release tests, with the aim to confirm the mechanism of their antibacterial action. The migration of Ag ions was tested by using three simulants (distilled water, acetic acid 3% v/v, and ethanol 10% v/v) under two assay conditions (40 °C for 10 days in Table 5 and 80 °C for 2 h in Table 6), according to EU Legislation on the migration of chemicals from plastic materials.52 The test conditions correspond to the more severe (worst foreseeable) conditions of contact. PE0 and PEAgNO3 squares were tested as negative

Figure 12. Number of viable cells (CFU/mL) versus time of PE1a− PE1c composites, loaded with different amounts of compound 1, compared to blank (PE0: unloaded) against (a) S. aureus, (b) E. coli, and (c) P. aeruginosa.

of S. aureus growth within 4 h, thus showing a good bacteriostatic activity almost immediately after the first exposure times (Table 2). Albeit showing a slight decrease between 12h and 20 h, during which S. aureus shows its maximum growth capacity, PE1, PE2, PE3, and PE4 reached 99.9% reduction within 24h, thus showing also bactericidal activity. By contrast, PE5−PE9 displayed a lower antibacterial activity within 12h. However, F


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the results are similar, only PE1 and PE6 exceed 1% release, with values that remain, however, within 2%. silver release is even less under contact condition at 80 °C for 2 h (Table 6). Another observation is that the silver release ability of PEn is dependent on the typology of the silver additive. In particular, PE1−PE6 always release silver ions, in low amounts even in drastic conditions, and very less than PEAgNO3. This release seems anyway sufficient to ensure a good or very good bactericidal and bacteriostatic action. Instead, PE7−PE9, loaded with phosphine-containing silver additives, showed the lowest release, in both normal and drastic conditions, in accordance with the lower antibacterial activity of silver complexes 7−9 with respect to 1−6.46 More in details, the percentage release of PE7 and PE9 after 2 h heating at 80 °C, in all conditions, was almost zero (Table 6). Similarly, PE7−PE9 in simulant A and PE7 in simulant C, after heating at 40 °C for 10 days, displayed an almost zero percentage release (Table 5). However, the mean release values in mg/dm2 (Tables S5 and S6 in Supporting Information), from which such ∼0.00 percentage releases were calculated, are not negligible: a very low Ag+ release is again present and it is sufficient to ensure the antibacterial activity of PE7−PE9, which is lower than that of PE1−PE6 but not completely absent. It is worth of mention that the actual composites show reduced silver release compared to previously reported composites with other silver acylpyrazolonate additives.40 This result can be related to the different structure of acylpyrazolone ligands. In fact, the HQ ligands in the previous work contain aromatic phenyl and pyridine rings,40 while the HQ here employed include also aliphatic hydrocarbon and fluorocarbon substituents, which likely improve the adhesive forces with the polyethylene matrix affording more homogeneous composites. With the aim to give better insight on the intrinsic mechanism of Ag+ release from PEn composites, we have investigated the aqueous solutions after contact with representative composites PE1 and PE3 by heating at 80 °C for 2 h, in accordance with the release tests conditions, previously discussed. The ESI-MS spectra, performed in the negative mode, display peaks due to the (QR)− ligands (see Figure S3 in Supporting Information), which demonstrates that the release of Ag+ occurs together with that of the acylpyrazolonato from the agglomerates of the additive, placed on the surface of PEn composites. 2.6. Test by Contact on the Composite Materials. The test by contact was performed out on PE1, PE4 and PE7 composites. PEAgNO3 and PE0 were also investigated in the test, as positive and negative controls, respectively. Each composite was tested in triplicate. Apart from the non additioned PE0 disk, all the other composites demonstrated inhibition of E. coli growth on the contact surface (Figure 14). Notably, an inhibition zone was found not only below but also around the disks of PE1, PE4, and PE7, similarly to PEAgNO3. This evidence underlines that the activity of PEn composites is through release of Ag+ from the disk surface, further confirming the ability of PEn to break down bacterial growth through a very small amount of additive. It is well-known that Ag+ ions are able to bind to the carboxyl, phosphate and hydroxyl groups of the surface of lipoproteins on the negatively charged cell walls, thus destroying bacterial cell membrane integrity and provoking oxidative damage.20 The membrane permeability and the formation of reactive oxygen species (ROS) were in fact previously observed with composites loaded with similar silver acylpyrazolonate additives.40 2.7. Toxicity Test on the Composite Materials. We have investigated the eventual toxicity of PEn against higher organisms

Figure 13. Reduction of CFU/mL after exposition to PEn composites (n = 1−9), PE0 (unloaded) and PEAgNO3 (loaded with AgNO3): (a) PEn vs S. aureus, (b) PEn vs E. coli, and (c) PEn vs P. aeruginosa.

and positive controls, respectively. In details, the Tables 5 and 6 show the percentage release of Ag+ with respect to the Ag content of PEn samples. Instead, the absolute values of Ag+ release and their standard deviations, in units mg/dm2, are reported in Tables S5 and S6 (Supporting Information). The most evident observation is that, under contact condition at 40 °C for 10 day (Table 5), all PEn displayed a minimal release (less than 1%) in distilled water with respect to PEAgNO3. Even in more drastic conditions (acetic acid 3% or ethanol 10% v/v) G


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ACS Applied Materials & Interfaces Table 2. Killing Percentage of PEn vs. S. aureus time

PE1

PE2

PE3

PE4

PE5

PE6

PE7

PE8

PE9

PEAgNO3

0 4h 8h 12 h 16 h 20 h 24 h 48 h

0.0 99.2 99.6 94.3 94.4 96.3 99.9 100.0

0.0 99.3 99.6 94.3 94.4 96.1 99.9 100.0

0.0 99.1 99.5 95.3 94.4 96.1 99.9 100.0

0.0 99.3 99.7 95.0 95.0 96.2 100.0 100.0

0.0 39.0 62.8 68.8 98.8 99.8 94.8 99.4

0.0 58.7 69.5 70.5 100.0 99.0 99.8 100.00

0.0 48.2 69.1 70.1 96.5 96.6 90.3 99.4

0.0 43.4 66.8 72.2 84.3 65.4 66.5 97.5

0.0 49.0 62.4 64.2 72.1 72.2 69.4 98.1

0.0 94.7 99.3 99.8 99.4 99.4 99.9 99.9

Table 3. Killing Percentage of PEn vs. E. coli time

PE1

PE2

PE3

PE4

PE5

PE6

PE7

PE8

PE9

PEAgNO3

0 4h 8h 12 h 16 h 20 h 24 h 48 h

0.0 3.4 42.8 42.8 82.4 95.1 99.9 99.9

0.0 14.8 33.0 33.0 46.1 45.5 80.3 98.7

0.0 23.6 41.8 41.8 51.8 74.1 82.5 94.7

0.0 45.5 53.2 53.2 99.3 99.5 99.5 99.9

0.0 90.0 99.8 99.9 99.9 99.9 99.9 99.9

0.0 58.7 69.5 70.5 100.0 99.0 99.9 100.0

0.0 48.2 69.1 70.1 96.5 96.6 90.3 99.4

0.0 43.4 66.8 72.2 84.3 65.4 66.5 97.5

0.0 49.0 62.4 64.2 72.1 72.2 69.4 98.1

0.0 94.7 99.3 99.8 99.4 99.4 99.9 99.9

Table 4. Killing Percentage of PEn vs. P. aeruginosa time

PE1

PE2

PE3

PE4

PE5

PE6

PE7

PE8

PE9

PEAgNO3

0 4h 8h 12 h 16 h 20 h 24 h 48 h

0.0 16.5 34.7 46.5 77.8 78.8 95.4 99.8

0.0 13.5 19.4 34.1 98.3 95.7 99.1 99.4

0.0 20.0 26.1 46.5 72.5 75.0 92.3 98.1

0.0 32.4 47.2 48.5 49.2 57.4 97.8 100.0

0.0 45.3 58.5 56.5 98.8 99.8 100.0 100.0

0.0 76.7 73.9 88.7 98.1 98.3 100.0 100.0

0.0 73.0 78.9 80.0 98.9 99.5 95.4 97.2

0.0 59.5 78.7 77.0 76.7 67.1 98.8 100.0

0.0 43.3 50.1 60.6 86.1 74.0 70.7 94.6

0.0 71.7 100.0 60.6 86.1 74.0 99.3 99.8

Table 5. Specific Ag+ migration from PEn square composites with embedded Ag(I) complexes 1−9, expressed in terms of percentage release in several simulants by heating at 40°C for 10 days

Table 6. Specific Ag+ Migration from PEn Square Composites with Embedded Ag(I) Complexes 1−9, Expressed in Terms of Percentage Release in Several Simulants by Heating at 80°C for 2 h

simulant A

simulant B

simulant C

simulant A

simulant B

simulant C

sample

distilled water

acetic acid 3% v/v

ethanol 10% v/v

sample

distilled water

acetic acid 3% v/v

ethanol 10% v/v

distilled water acetic acid 3% v/v ethanol 10% v/v PE0 PEAgNO3 PE1 PE2 PE3 PE4 PE5 PE6 PE7 PE8 PE9

0.00 0.00 0.00 0.00 100.00 0.15 0.35 0.32 0.20 0.15 0.85 ∼0.00 ∼0.00 ∼0.00

0.00 0.00 0.00 0.00 100.00 0.21 0.52 0.51 0.51 0.27 1.26 0.04 0.04 0.16

0.00 0.00 0.00 0.00 55.00 1.68 0.28 0.43 0.20 0.12 1.20 ∼0.00 0.04 0.12

distilled water acetic acid 3% v/v ethanol 10% v/v PE0 PEAgNO3 PE1 PE2 PE3 PE4 PE5 PE6 PE7 PE8 PE9

0.00 0.00 0.00 0.00 100.00 0.02 0.24 0.34 0.13 0.05 0.16 ∼0.00 0.08 ∼0.00

0.00 0.00 0.00 0.00 100.00 0.08 0.10 0.56 0.20 0.15 0.22 ∼0.00 0.08 ∼0.00

0.00 0.00 0.00 0.00 76.00 0.06 0.26 0.42 0.20 0.07 0.22 ∼0.00 0.04 ∼0.00

composites in sufficient amounts to affect the aqueous environment. In conclusion, composites PE1−PE6 are ideal candidates in several technological applications for their very good antibacterial activity and absence of toxicity toward higher organism, compared to AgNPs, which by contrast show a toxicological potential toward higher organisms and humans.64−66

through a well-known test of ecotoxicity, based on the use of Daphnia magna. While in previous studies AgNPs were found to display acute toxicity toward D. magna,61−63 our composites show no ability to induce deaths of the organisms tested, demonstrating the tolerance toward our composites by higher organisms. This finding is a proof that Ag+ ions are not released by PEn H


Research Article


Figure 14. Bactericidal effect promoted by contact with composites PE1, PE4, PE7, and PEAgNO3 compared with the effect of a non embedded PE0 disk for E. coli ATCC 25922 with MacConkey agar: (a) Petri plate with the blank PE0 and PEAgNO3 disks: bacterial growth can be appreciated only below the non embedded PE0 disk, while a zone of inhibition around the PEAgNO3 disk can be observed; (b) Petri plate with three PE1 disks; (c) Petri plate with three PE4 disks; (d) Petri plate with three PE7 disks. In (b), (c), and (d) no bacterial growth was found below and around the disks. The picture was taken after lifting the disks: the growth below the PE disk is clearly visible, whereas below the PE1, PE4, PE7, and PEAgNO3 disks there is absence of bacterial growth. (3-methyl-1-phenyl-4-heptafluorobutanoil-pyrazol-5-one) were synthesized as previously reported.41,42 All reactions and manipulations were performed in the air. Solvent evaporations were always carried out under vacuum conditions using a rotary evaporator. IR spectra were recorded from 4000 to 600 cm−1 with a PerkinElmer Spectrum 100 FT-IR instrument by total reflectance on a CdSe crystal. Thermal gravimetric analyses (TGA) were carried out in a N2 stream with a PerkinElmer STA 6000 simultaneous thermal analyzer (heating rate: 7 °C/min). The negative electrospray ionization mass spectra (ESI-MS) were obtained on a Series 1100 MSI detector HP spectrometer instrument, using a water mobile phase. Images of powder granules of Ag(I) complex 1 and their dimensions were taken by an electronic microscope MOTICAM 1000 (1.3 M Pixel, USB 2.0). ICP analyses for specific silver ions migration were carried out with a 7500 cx Agilent Technologies ICP-MS Spectrometer. The AFM analyses were performed on an AFM microscope (AFM Veeco 5000 Dimension). Dried samples were fixed on a holder double-side tape and 10 × 10 μm2 areas were scanned by semicontact mode in the air. Three different locations were tested and an average value of RMS was reported. AFM pictures were taken by non contact mode AFM55 with a cantilever from Bruker made by SiN and Si tip at the 300 kHz working frequency. The measurements were done by using a constant driving voltage of 200 mV. Acquisition times were in the range of 20 ms each point of a 300 × 300 matrix. SEM spectra and EDX analyses were carried out with a field emission scanning electron microscope AURIGA (FIB-FESEM) from Carl Zeiss SMT, equipped with a back scattered detector (BSD) to obtain high quality microphotographs and a system of microanalysis by EDX (Oxford Instruments) for the determination of local or mapping distribution of chemical elements. 4.2. Synthesis of the Silver Complexes. Silver complexes 1−9 have been prepared and characterized as previously reported.46 4.3. Preparation of the PEn Composites. Polyethylene composite PEn disks, with embedded Ag(I) complexes 1−9, were prepared in the following manner: the silver complex, in the form of powder, was mixed in a glass capsule with PE granular powder (1.00 g) in a 1:1000 weight ratio. The capsule was heated up to the melting point of PE (98 °C) until to 150 °C, while its content was stirred half hour to give a homogeneous dispersion. The dispersion was then left to cool at room temperature; after solidification, the loaded polymer matrix was removed from the capsule and placed in contact with a hot quartz surface (130 °C): within a few minutes, the matrix melted and distributed homogeneously onto the quartz surface to give a thin liquid layer. After reduction of the quartz surface temperature down to 80 °C, the polymeric matrix layer turned into a soft solid film, suitable to be cut into small disks of 6 mm diameter and of thickness in the range 0.8−1.0 mm. 4.4. Microbiological Studies on the PEn Composites. The antibacterial activity of PE1a−c and PEn composite materials (n = 1−9, corresponding to PE containing Ag(I) complexes 1−9) was tested against the two Gram-negative bacteria E. coli ATCC 25922 (PBI International) and P. aeruginosa ATCC 27853 (OXOID-remel) and the Gram-positive bacteria S. aureus ATCC 25923 (BPI International). The experiments were performed in triplicate and the graphs shown in

3. CONCLUSIONS Novel antibacterial polyethylene composites have been prepared through an easily up-scalable embedding procedure of Ag(I) acylpyrazolonato complexes, by using an advantageous 1:1000 weight ratio of silver additive to PE. Their relevant spectral and thermal properties are essentially unchanged with respect to unloaded PE0, even though the surface of composite PEn disks displays a sufficiently homogeneous dispersion of the additive, which appears as small granules with an average diameter in the range 10−100 μm. The antibacterial efficiency of the composites is a function of the structure of silver additive: PE1−PE3 composites, loaded with silver coordination polymers 1−3, and PE4−PE6 loaded with monomeric silver complexes 4−6 containing ancillary imidazole ligands, display from good to excellent antibacterial activity against three bacterial strains (S. aureus, E. coli, and P. aeruginosa). This broad-spectrum antibacterial activity of PEn against both Gram-positive and Gram-negative bacteria is a relevant result, being well-known that the killing of Gram-negative bacteria is much more difficult compared to Gram-positive bacteria. By contrast, PE7−PE9 loaded with monomeric silver complexes 7−9 bearing ancillary phosphine ligands, show a somewhat reduced activity against all bacterial strains. In any case, PEn composites can be reused several times, displaying the same antibacterial activity. Contact and release tests confirm that the long-standing activity is related to a slow and controlled release of silver ions by the active surface of the materials. These findings highlight that Ag(I) complexes, with some unsaturation on the silver coordination environment, combined with main features such as insolubility in water, stability to heat and light and intrinsic antibacterial activity, can be employed as efficient additives to polyethylene. Moreover, this study has proved that a minimal amount of silver additive (even in a 1:1000 weight ratio), located at the outermost surface of a biomaterial, can confer significant antibacterial activity, even in vivo, while avoiding any toxicity issues. Our research shows possible improvements toward the embedding of our silver additives in different polymeric matrixes, such as polyurethane, polyvinyl chloride and poly(methyl methacrylate), thus providing a new avenue of explorations in fighting the ever increasing antibacterial resistance in pathogenic microorganisms. 4. EXPERIMENTAL SECTION 4.1. Materials and Methods. All chemicals were purchased from Aldrich (Milwaukee) and used as received. 4-Acyl-5-pyrazolone ligands HQcy (3-methyl-1-phenyl-4-cyclohexanecarbonyl-pyrazol-5-one), HQbe (4-(3-methylbut-2-enoyl)-3-methyl-1-phenyl-pyrazol-5-one) and HQfb I


Research Article

ACS Applied Materials & Interfaces Figures 11 and 12 are based on the mean values. Tables S1−S4 in Supporting Information give the mean CFU/mL values and the corresponding standard deviations. Bacteria were grown aerobically at 37 °C for 18 h using Tryptone Soya Broth (OXOID) as the growth medium. Bacterial cultures in the range of (1−3) × 106 CFU/mL were added to sterilized test tubes containing 4 mL of autoclaved physiological solution. For sterilization of the tubes, an Alfa-10-plus autoclave (PBI International) was used, operating at 121 °C for 15 min. A 40 mg amount of loaded PEn disk, previously reduced to granules, was added to the test tubes containing bacterial suspensions. All tubes were kept on an IKA KS 130 BASIC agitator for 24 h at slow speed. To study the growth inhibitory effect of the PEn composites on the bacterial cultures, 100 μL of supernatant fraction were withdrawn from the tubes at time intervals of 4 h (t0, 4, 8, 12, 16, 20, 24, 48 h). To obtain the bacterial colony count, the supernatant fraction was diluted and included uniformly into Petri dishes containing Plate Count Agar (OXOID). Adopting the same procedure, an unloaded PE disk was used as negative control (indicated as PE0). In addition, by using the same method, other tests were conducted on composite samples PE1a−c prepared with different additive/PE ratios to evaluate the antimicrobial activity at 3 different concentrations of silver complex 1 as additive (1:500, 1:1000, 1:2000). The average of CFU/mL, obtained from tests conducted in triplicate, was used to calculate the percentage of reduction applying the following formula:

% of reduction =

Guideline 202 of the Organisation for Economic Co-operation and Development.67 D. magna were purchased from Ecotox LDS Conaredo (MI), Italy and reared in the culture room. The embryonic development of D. magna eggs takes about 3 days under optimal conditions (6.000 lx light intensity at the top of the Petri dish and at 20−22 °C) the first neonates may even appear before 72 h incubation, but the largest hatching will occur between 72 and 80 h of incubation. The Toxkit microbiotests were carried out on multiwall test plates composed of six rinsing wells and 24 wells for the toxicant dilutions. Each compartment of the pot tests was filled with 10 mL of standard Freshwater (IsoMedium, formula according to ISO 634168) kit DAPHTOXKIT F MAGNA Ecotox LDS Conaredo (MI), Italy. To also provide the neonates hatched from the ephippia with food prior to the test, a 2 h “pre-feeding” was applied with a suspension of Spirulina microalgae. The transfer of the Daphnia neonates into the test wells was performed with a micropipette. The small size of the young born daphnids meant that this transfer was usually carried out by using a light table provided with a dark light strip and a transparent stage to increase the contrast. Transfer of the daphnids to the multiwell plate was accomplished in two steps: (1) Transfer of the 20 neonates from the Petri dish into the rinsing wells of the multiwell plate; (2) Transfer of the neonates from the rinsing wells to the four test wells of the same rows. Then, 10 mL of the solutions coming from the previous release tests (carried out at 40 °C for 10 days in simulant A; that is, distilled water) of the composites PE1−PE9 were added to the compartments. Finally, a parafilm strip was placed over the multiwall plate and the cover was put on tightly. The plate was left in an incubator at 20 °C in the dark. After 24 and 48 h incubation, the multiwall plate was placed on the lamp holder and the number of dead and immobilized daphnids was counted. The presence of toxic compounds is related in direct proportion to the number of D. magna death. In the case of PE1−PE9, no deaths of the daphnids were observed, indicating the tolerance of higher organisms toward the compounds.

B − AB × 100 B

where B = CFU/mL average recovered from unloaded PE0 and A = CFU/mL average recovered from PEn composites. 4.5. Release of Silver Ions From the PEn Composites. Polyethylene squares of dimensions 10 × 10 cm with embedded the complexes 1−9 were prepared in the following manner: the silver additive, in the form of powder, was mixed in a Teflon mold, with an area of 1 dm2, with PE granular powder (7.00 g) in a 3:1000 weight ratio. The Teflon mold was heated up to the melting point of PE (98 °C), while its content was stirred to give a homogeneous dispersion. The dispersion was then left to cool at room temperature; after solidification, the loaded polymer matrix was removed from the mold and was reduced into pieces suitable for migration testing. The migration tests were performed under different contact conditions using distilled water (simulant A), 3% acetic acid (simulant B), 10% ethanol v/v (simulant C).52 Samples were immersed in 100 mL of simulant in a conical flask with ground glass stopper. In this manner both faces of the sample were in contact with the simulant. All conic flasks, covered with an aluminum foil, were kept in controlled atmosphere at two assay conditions: 40 °C for 10 days and 80 °C for 2 h. After the incubation period, pieces were removed and the simulant was treated to analyze the Ag+ released in the simulants by inductive coupled plasma spectroscopy with mass spectrometry detection (ICP-MS). Adopting the same procedure, an unloaded PE square was used as negative control and a loaded PE square with AgNO3 as positive control. A solution containing In (10 μg/L) was used as internal standard for ICP-MS measurements. Calibration curves for investigated element were obtained by using aqueous (3% nitric acid) standard solutions prepared by appropriate dilution of stock standards (Ag standard solution 10 mg/L for ICP-MS). The experiments were performed in triplicate to ensure the repetitiveness. The absolute values with their standard deviations in mg/dm2 are reported in Tables S5 and S6 (Supporting Information). 4.6. Contact Tests. Bacterial aqueous suspensions of E. coli (106 CFU/mL, 0.5 mL) was streaked over a plate containing MacConkey Agar, differential medium for the isolation of coliforms and intestinal pathogens in water, dairy products, and biological specimens (OXOID S.p.A), and were spread uniformly. Disks of PE1, PE4, PE7, the blank PE0 and the positive control PEAgNO3 were gently placed over contaminated MacConkey Agar in Petri dishes. Petri dishes were incubated overnight at 37 °C for 24 h. After incubation, growth inhibition was evaluated by visual inspection, observing the dish, inverted, on a light table (Precision Illuminator, model B95, Northern Light). 4.7. Acute Toxicity Test on the PEn Composites. The acute toxicity test for Daphnia magna was carried out in accordance with Test

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b09742. Electronic Microscope image of granulometry of silver additives (Figure S1), SEM images of selected polyethylene composites (Figure S2), ESI-MS spectra of the aqueous solutions after contact with PE1 and PE3 at 80 °C for 2 hours (Figure S3) and Tables S1−S6 (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*(F.M.) Phone: +39 0737402217; e-mail: [email protected]. *(J.P.) Phone: +39 0737402234; e-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the University of Camerino and University of Granada.

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REFERENCES

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Preparation of Polyethylene Composites Containing Silver(I

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