Novel composite plastics containing silver(I) acylpyrazolonato additives display potent antimicrobial activity by contact.

DOI: 10.1002/chem.201404812

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& Antibiotic Polymers

Novel Composite Plastics Containing Silver(I) Acylpyrazolonato Additives Display Potent Antimicrobial Activity by Contact Fabio Marchetti,*[a] Jessica Palmucci,[a] Claudio Pettinari,*[b] Riccardo Pettinari,[b] Francesca Condello,[b] Stefano Ferraro,[a] Mirko Marangoni,[c] Alessandra Crispini,[d] Stefania Scuri,[b] Iolanda Grappasonni,[b] Mario Cocchioni,[b] Massimo Nabissi,[b] Michele R. Chierotti,[e] and Roberto Gobetto*[e]

Abstract: New silver(I) acylpyrazolonato derivatives displaying a mononuclear, polynuclear, or ionic nature, as a function of the ancillary azole ligands used in the synthesis, have been fully characterized by thermal analysis, solution NMR spectroscopy, solid-state IR and NMR spectroscopies, and Xray diffraction techniques. These derivatives have been embedded in polyethylene (PE) matrix, and the antimicrobial activity of the composite materials has been tested against three bacterial strains (E. coli, P. aeruginosa, and S. aureus): Most of the composites show antimicrobial action comparable to PE embedded with AgNO3. Tests by contact and release tests for specific migration of silver from PE composites clearly indicate that, at least in the case of the PE, for composites containing polynuclear silver(I) additives, the antimi-

crobial action is exerted by contact, without release of silver ions. Moreover, PE composites can be re-used several times, displaying the same antimicrobial activity. Membrane permeabilization studies and induced reactive oxygen species (ROS) generation tests confirm the disorganization of bacterial cell membranes. The cytotoxic effect, evaluated in CD34 + cells by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide) and CFU (colony forming units) assays, indicates that the PE composites do not induce cytotoxicity in human cells. Studies of ecotoxicity, based on the test of Daphnia magna, confirm tolerability of the PE composites by higher organisms and exclude the release of Ag + ions in sufficient amounts to affect water environment.

Introduction In recent years the need for control of pathogenic microorganisms in contaminated environments has led to the development of antimicrobial materials[1] that can protect humans from infectious disease. Among the wide range of antimicrobial plastics, metal-polymer nanocomposites and, particularly, silver-polymers are the subject of increased interest.[2] Silver compounds are known to exhibit strong antimicrobial activity towards a broad spectrum of bacteria.[3] In low concentrations, silver is not toxic for human cells,[4] and it has been found to be effective in killing numerous types of infectious bacteria.[5] Silver ions are proposed to react with electron donor groups (N, O, or S atoms), which are present in bacteria as, for example, amino, imidazole, and phosphate.[6] Although the exact antibacterial mechanism of colloidal silver nanoparticles remains unclear, it has been proposed that silver nanoparticles themselves are active.[7] However, more recent studies seem to indicate that, to be active, the silver nanoparticles (AgNPs) must first be converted to ionic silver, which is the effective antimicrobial species, through oxidation of the zero-valent silver, which limits the concentration of ionic silver available.[8] Syntheses of AgNPs in commercially available polymers such as poly(vinyl alcohol) (PVA) or poly(vinylpyrrolidone) (PVP) have been carried out because of their excellent surface capping ability.[9] However, extensive release of the AgNPs from the ma-

[a] Prof. F. Marchetti, Dr. J. Palmucci, Dr. S. Ferraro School of Science and Technology Chemistry Section, University of Camerino Via S. Agostino 1, Camerino (MC) (Italy) E-mail: [email protected] [b] Prof. C. Pettinari, Dr. R. Pettinari, Dr. F. Condello, Dr. S. Scuri, Prof. I. Grappasonni, Prof. M. Cocchioni, Dr. M. Nabissi School of Pharmacy, Chemistry Section University of Camerino Via S. Agostino 1, Camerino (MC) (Italy) E-mail: [email protected] [c] Dr. M. Marangoni Analisi Control S.r.l. Via San Claudio, 5, 62014 Corridonia (MC) (Italy) [d] Prof. A. Crispini Centro di Eccellenza CEMIF.CAL-LASCAMM CR-INSTM Unit della Calabria Dipartimento di Chimica e Tecnologie Chimiche University of Calabria 87030 Arcavacata di Rende (CS) (Italy) [e] Dr. M. R. Chierotti, Prof. R. Gobetto Dipartimento di Chimica, University of Torino Via P. Giuria 7, and Centro di Eccellenza NIS 10125 Torino (Italy) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404812. Chem. Eur. J. 2014, 20, 1 – 16

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Full Paper terials could lead to environmental hazards,[10] and promote the development of resistant microbial strains.[11] More recent attempts to circumvent such limitations are based on microorganism-triggered release of AgNPs from biodegradable calcium phosphate carriers. In this approach, the growing bacteria dissolves the carrier containing nutrients and thereby releases the AgNPS, thus enabling a significant reduction of silver use.[12] Our interest in silver derivatives and their potential application as antimicrobial agents recently led us to investigate different classes of ligands.[13, 14] We have previously shown that 4acyl-5-pyrazolones[15] can be used to form silver(I) complexes that are mono-, di-, and even polynuclear.[16] Here, we have extended the previous studies to the synthesis of novel silver(I) complexes containing 4-acyl-5-pyrazolonate (Q) ligands with different electronic and steric features, and several imidazoles L as ancillary ligands (Scheme 1), with the aim of obtaining

Results and Discussion Synthetic procedures Derivative Ag(QPh) (1) was obtained as a colorless precipitate by reaction between AgNO3 and HQPh in methanol using NaOMe as deprotonating agent (Scheme 2). Derivatives 2–5 were subsequently obtained by mixing an acetonitrile suspen-

Scheme 2. Synthesis of derivatives 1–5.

sion of 1 with an excess or large excess of the corresponding imidazole, to afford Ag/QPh/imidazole derivatives with 1:1:1 composition (derivatives 2 and 5) or 1:1:2 (derivatives 3 and 4), respectively (Scheme 2). They are all air-stable and quite highmelting derivatives that are soluble and stable in dimethyl sulfoxide (DMSO) and acetonitrile and, in the case of 4 and 5, also in alcohols and chlorinated solvents. Conductivity measurements carried out on solutions of 1–3 in DMSO and on solutions of 4–5 in acetonitrile seem to indicate partial dissociation and formation of ionic species. This is in accordance, at least for 3, with the ionic structure in the solid state (see X-ray data below), whereas in the case of 4 we can hypothesize a partial dissociation according to Equation (1):

Scheme 1. Pro-ligands HQ and ligands L used.

a number of AgI derivatives with different nuclearity as potential antimicrobial agents to embed in plastics. Moreover, because polyethylene (PE) is one of the most common plastics, and is extensively used in packaging and containers for safe drinking water including bottles and taps, specific tests were carried out on PE disks with embedded AgI derivatives to assess the antimicrobial activity of composites against suspensions of E. coli, P. aeruginosa, and S. aureus, and to give insight on their mechanism of action. Our goal is to demonstrate that, in place of plastic composites of AgNPs with their associated side effects in terms of silver release and environmental impact, the embedding of insoluble silver(I) coordination polymers in a polymeric matrix may give rise to a new concept in the field of plastics with permanent antimicrobial activity: “contact action by polymer/polymer composites”.

½AgðQPh ÞðMeimÞ2 Ð ½AgðMeimÞ2 þ þðQPh Þ

ð1Þ

IR data of 1–5 show two strong absorptions in the range 1580–1653 cm1 assigned to n(C=O) stretching modes, likely indicating asymmetric chelation of QPh through the carbonyl groups or even an ionic formulation of QPh in the solid state,[15] as confirmed by X-ray data in the case of derivative 3. In the 1H NMR spectra of derivatives 2–5, the integration ratio of H resonances of QPh and imidazole ligand is in accordance with the anticipated 1:1 (derivatives 2 and 5) or 2:1 for-

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Full Paper imidazole (imH), with the formation of a monomeric specie of general formula [Ag(Qpy,CF3)(imH)]. Solid-state 15N NMR characterization Solid-state NMR spectroscopy has provided more in-depth information on the coordination shift experienced by the Ndonor atoms interacting with the silver atom.[17] Earlier studies reported the NMR spectroscopic analysis of AgI complexes with substituted pyrazole ligands.[18] All 15N data are listed in Tables 1 and 2, and the 15N CPMAS spectra are reported in Figures 1 (complexes 1–5) and 2 (6–12). For nitrogen atom label-

Table 1. 15N NMR chemical shifts (ppm) with assignment for the complexes 1–5 and the corresponding ligands HQPh, imH, Meim, and Bzim. Peak multiplicity and JAgN coupling constants (Hz) are reported in parentheses.

HQ

Ph

N1

N2

165.6

253.5 251.9

imH Meim[19] Bzim 1 2 3

167.1 161.9 167.3

207.6 (m, br) 198.3 (m, br) 237.6

4

163.7

217.2 (br)

5

167.1

204.8 (m, br)

N3

N4

220.5 215 234.3 177.2 (m, br)

149.4 119 153.7 147.6

181.9 (m, br)

149.4 148.2 141.9 138.7 155.0 (br)

201.9 (d, 32) 198.6 (d, 46) 188.0 (m, br)

Scheme 3. Synthesis of derivatives 6–12.

mulation (derivatives 3 and 4). Derivative 6 was obtained Table 2. 15N NMR chemical shifts with assignment for the complexes 6–12 and as a colorless precipitate by reaction of AgNO3 with HQCF3,py the corresponding ligands HQpy,CF3, Bzim, 2Mebzim, 2EtimH, COdim, 2tfbim, in methanol using NaOMe as deprotonating agent and Me3py. Peak multiplicity and JAgN coupling constants (Hz) are reported in parentheses. (Scheme 3). Derivatives 7–12 were subsequently obtained by mixing an solution of 6 in acetonitrile with equivalent N3 N4 N5 N1 N2 NPy amounts of the corresponding N-donor, to afford Ag/ py,CF3 HQ 163.3 239.7 137.2 – – – Qpy,CF3/N-donor derivatives with 1:1:1 composition, apart for Bzim – – – 234.3 153.7 – di(imidazol-1-yl)methanone, which afforded derivative 10 2Mebzim – – – 234.9 143.8 – with 2:2:1 composition (Scheme 3). Derivatives 7–9 and 2EtimH – – – 218.3 147.0 – COdim – – – 220.6 149.1 – 11–12 are monomeric substances (in the case of 9 it is fur2tfbim – – – 210.3 127.9 – ther confirmed by X-ray data, see below), whereas 10 is – – – – – 260.7 Me3py[21] likely composed of binuclear units, with the ditopic di6 168.7 236.3 208.6 (br) – – – (imidazol-1-yl)methanone ligand bridging two {Ag(Qpy,CF3)} 232.0 7 168.3 229.7 182.6 (d, 86) 201.8 (m, br) 156.2 – moieties. In the IR spectra of 7–12, the n(C=O) absorptions 8 165.4 (br) 218.6 (br) 183.1 (br) 200.8 (br) 147.7 – are found to be essentially unchanged with respect to 9 166.9 227.6 182.6 (d, 85) 201.1 (m, br) 140.7 – those in free, neutral HQpy,CF3, in accordance with the coor10 165.4 (br) 232.1 181.9 (m, br) 198.3 (br) 144.7 – dination of Qpy,CF3 primarily through the N atoms of the pyr11 166.6 215.2 174.2 (m, br) 200.9 (br) 125.1 – 12 168.6 218.8 207.5 – – 236.9 (d, 81) azole and pyridine rings. TGA analyses show that 1–12 are thermally stable species, with decomposition not starting before 100 8C for 4 and 7–9, at approximately 150 8C for 3 ing, we refer to the Scheme 1. 15N CPMAS spectra of the liand 5, at approximately 180 8C for 12, at approximately 200 8C for 2 and well beyond for all the others. It is worth mentioning gands and 13C CPMAS spectra of all compounds with chemical that derivative 10 decomposes in acetone/chloroform solution, shifts are reported in the Supporting Information (Figures S1, according to the pathway described in the Scheme 3. The deS2 and S3, respectively). In principle, the existence of two isocomposition product 10’ has been structurally analyzed (see topes 107Ag and 109Ag (natural abundance 51.8 and 48.2 %, rebelow), proving the transformation of the COdim ligand into spectively) should give rise to two doublets with correspondChem. Eur. J. 2014, 20, 1 – 16

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Figure 2. 15N (40.56 MHz) CPMAS spectra of the ligand HQpy,CF3 and complexes 6–12 recorded with a spinning speed of 9 kHz. Figure 1. 15N (40.56 MHz) CPMAS spectra of the ligand HQPh and complexes 1–5 recorded with a spinning speed of 9 kHz.

by the 15N peak at 137.2 ppm (compared with the pyridyl signal, which resonates at ca. 280 ppm). For complexes 1, 2, 4, and 5 (Figure 1, Table 1) the N2 chemical shift suggests nitrogen coordination to the silver atom according to Scheme 2. Indeed, all signals experience coordination shifts toward lower frequencies around 45.5 ppm. For sample 4, the small value of the measured JAgN couplings with respect to those reported previously[22] suggests weaker AgN bonds (as confirmed by the X-ray structure, see below). The 15N CPMAS spectrum of 3 is consistent with the X-ray structure (see below): the absence for the N2 signal of either splitting or broadening due to JAgN coupling confirms the lack of coordination to the metal, whereas the small shift toward lower frequencies (ca. 16 ppm) with respect to the free ligand is consistent with the formation of a cocrystal between the free HQPh ligand and the Ag(imH) dimer. Interestingly, in the series of compounds 6–12 (Figure 2, Table 2), the shift upon coordination of the N2 signal is smaller than in the previous series (ca. 13 ppm, max. 24.5 ppm). This can be related to the different coordination type of the silver atom, which leads to longer AgN distances (see X-ray structure below). The NPy signal undergoes a high frequency shift with respect to the pyridinium position of the free ligand. However, it resonates at lower frequency compared with that of the pyridine, indicating that NPy is coordinated to the metal. The splitting of some resonance in the 13C and 15N CPMAS spectra of 6 suggests the

ing silver-nitrogen coupling constants. However, owing to very similar g (magnetogyric ratio, 1.089·107 and 1.252·107 rad·T1 s1, respectively) the relation between J couplings is J109AgN/J107AgN = 1.15. Thus, the difference is often less than the spectral resolution. Indeed, for sp3 nitrogen atoms, for instance N2 in all complexes, the coupling is expected to be smaller and the two resulting doublets appear as a broad signal. For cases in which the doublet can be resolved, an averaged value between J109AgN and J107AgN is reported. Concerning the HQPh and HQpy,CF3 ligands, the X-ray structure of HQPh indicates the presence of two independent molecules in the unit cell[20] as confirmed by the splitting of the N2 resonance (253.5 and 251.9 ppm) in the 15N CPMAS spectrum (Figure 1). On the other hand, the single set of signals in the 13C CPMAS spectrum (see the Supporting Information) highlights the high similarity between the two molecules, both of which are characterized by the structure given in the Scheme 1. Concerning HQpy,CF3, the 13C and 15 N CPMAS spectra suggest the protonated structure reported in Scheme 4. This is characterized by Scheme 4. Structure of prolia pyridinium cation, as confirmed gands HQPh and HQpy,CF3. &

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Figure 3. a) Perspective view of the asymmetric unit content of [Ag(imH)2] [QPh] (3) with atomic numbering scheme (ellipsoids at the 40 % level) and b) crystal packing view of 3 showing the formation of columns and relative intermolecular interactions.

Figure 4. a) Perspective view of the asymmetric unit content of [Ag(QPh)(Meim)2] (4) with atomic numbering scheme (ellipsoids at the 40 % level) and b) the formation of dimers in the crystal packing with CH/p attractive intermolecular interactions.

presence of two independent molecules in the unit cell. The number of peaks in the 13C and 15N NMR data of complex 10 suggests a highly symmetric environment of the complex: indeed, for instance, only one signal is observed for N4 (d15N = 144.7 ppm) and for the methyl (d13C = 19.4 ppm). In 12, coordination of the Me3py ligand through the nitrogen atom is confirmed by the N5 shift from 260.7 (free ligand) to 236.9 ppm (complex).

silver cations are joined through a strong AgAg interaction of 3.202(1) (Figure 3 b). The so-formed silver dimer strongly interacts with the anions through hydrogen bonds of the N H···O type, between one imidazole coordinated ligand and the keto-oxygen atom of the [QPh] anion [N(6)O(1)i 2.691(3) , N(6)H(6)···O(1)i 173.58, i = x, y, z + 1]. In contrast to derivative 3, the single-crystal X-ray analysis of complex 4 demonstrated the neutral nature of the latter, at least in the solid state, confirming the general formula [Ag(QPh)(Meim)2]. The QPh ligand coordinates the silver(I) ion in a monodentate fashion, through the negatively charged nitrogen atom (Figure 4 a). Two Meim ligands complete the AgI coordination sphere, bounding the metal ion though their nitrogen atom, and generating an overall pseudo trigonal-planar distorted geometry. The maximum deviation from trigonality is due to the presence of the rotationally free phenyl ring of the QPh ligand, causing an enlargement of the N(1)-Ag-N(5) angle to 128.7(1)8. The central pyrazole ring of the QPh ligand is found to be nearly orthogonal with respect to the mean coordination plane, with a torsion angle of about 508. As a consequence, a CH/p attractive intermolecular interaction between the Northo-hydrogen atom of one coordinated Meim ligand and the

Crystallography The X-ray crystal structure analysis of derivative 3 confirmed the ionic nature of the isolated specie in the crystalline solid state (Figure 3 a). The silver cation is built up through coordination of the neutral nitrogen atoms of two imH ligands to the AgI ion, in a distorted linear geometry [N(3)-Ag-N(5) of 167.3(1)8]. The QPh ligand is found as counter ion in its unprotonated form (QPh) and therefore is not coordinated to the silver metal center. The C(3)O(1) bond distance of 1.248(3) proves that the deprotonation arises from the original keto form of the QPh molecule, as observed in similar HQR pyrazolones.[23] The [Ag(imH)2][QPh] unit, when repeated through an inversion center, gives rise to a binuclear structure in which the two Chem. Eur. J. 2014, 20, 1 – 16

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Figure 5. Perspective view of the asymmetric unit content of [Ag(Qpy,CF3)(2EtimH)] (9) with atomic numbering scheme (ellipsoids at the 40 % level).

rotationally free phenyl ring of QPh is the structural feature characterizing complex 4 crystal packing (Figure 4 b). Very short H–Ph plane distances characterize this interaction, all geometrical parameters being indicative of its presence.[24] Moving from HQph to HQpy,CF3, the ligand coordination mode with respect to the AgI ion changes drastically. Indeed, the crystal structure determination of complex 9 proves the monoanionic N2-chelating mode of the Qpy,CF3 ligand, with the AgI coordination sphere completed by the imidazole 2EtimH bound through its nitrogen atom (Figure 5). The bond distances and angles around the AgI ion are comparable to those found in the crystal structure of an analogous AgI complex of HQpy,CF3 ligand recently reported, containing the imidazole Meim similarly coordinated.[16a] However, in the case of complex 9, a greater asymmetry between the AgN bond distances within the NN-chelated ring is found compared with the previously reported derivative, with the distance to the pyridine nitrogen atom being longer than the other [AgN(1) and AgN(3) distances of 2.240(3) and 2.392(3) , respectively]. The geometry around the central metal ion could be defined as very distorted trigonal-planar, with a N(1)-Ag-N(3) “bite” angle of 71.4(1)8 and a N(1)-Ag-N(4) angle of 154.7(1)8. The overall planarity of the molecule is broken by both the high distortion from planarity of the NNchelated ring (internal torsion angle around the N(2)C(4) bond of 10.2(4)8) and the slight rotation of the 2EtimH ligand with respect to the NN-chelated ring, with the dihedral angle between their mean planes being 8.5(1)8. The packing mode of complex 9 is dominated by the formation of columns of molecules stacked on the top of each other slightly shifted with a repetitive Ag–Ag intermolecular distance of 3.9 (Figure S4). The interaction between columns is ensured by the presence of strong NH···O hydrogen bonds [N(5)–O(1)i 2.853(4) , N(5)H(5a)···O(1) 163.68, i = x, y + 2, z1/2] and intermolecular interactions involving the fluorine atoms of the CF3 groups and one of the methylene hydrogen atoms [C(15)– F(2)i 3.376(5) , C(15)H(15a)···F(2) 135.08]. As previously antici&

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Figure 6. a) Perspective view of the asymmetric unit content of [Ag(Qpy,CF3)(imH)] (10’) and b) [Ag(Qpy,CF3)(Me3py)] (12) with atomic numbering scheme (ellipsoids at the 40 % level).

pated, both the decomposition product 10’ and complex 12 have been structurally analyzed, and their characterization has confirmed the same type of N2-chelating mode of Qpy,CF3 seen in complex 9 (Figure 6 a, b). In both cases, the distorted trigonal-planar geometry around the silver ion is reached through its coordination to the imidazole ligand nitrogen atom (imH and Me3py in 10’ and 12, respectively), with N-Ag-N “bite” angles within the NN-chelated ring of 70.5(1)8 and 71.1(1)8 in the two molecules of the asymmetric unit in 10’ and 70.9(1)8 in 12. The same asymmetry of the two AgN bond distances with the coordinated Qpy,CF3 ligand seen in 9, is found in both 10’ and 12 complexes, albeit being less pronounced in the latter case (Table 3). Whereas the NN-chelated ring of both molecules in the asymmetric unit of 10’ is found to be closer to planarity, as shown by the torsion angles around the NC internal bond of 1.4(4)8 and 0.65(4)8, respectively, derivative 12 shows a distortion similar to that seen in 9, with a torsion angle of 7.2(4)8. In both cases, the rotationally free imidazole ligand is found to be slightly rotated with respect to the NN-chelated ring, with dihedral angles between their mean planes of 10.1(1)8 and 6



Full Paper Table 3. Relevant bond lengths [] and angles [8] for complexes 9, 10’, and 12. 9 Bond lengths AgN(1) AgN(3) AgN(4) Bond angles N(1)-Ag-N(3) N(1)-Ag-N(4) N(3)-Ag-N(4)

10’ 2.240(3) 2.392(3) 2.136(3)

71.4(1) 154.7(1) 133.9(1)

2.230(2), 2.233(3) 2.376(3), 2.358(3) 2.137(3), 2.127(3) 70.5(1), 71.1(1) 150.8(1), 150.5(1) 138.7(1), 138.4(1)

12 2.306(2) 2.368(2) 2.212(2) 70.9(1) 147.6(1) 140.6(1)

12.6(1)8 in 10’ and 5.9(1)8 in 12. The presence of the NH group on the imidazole ring means that the crystal packing of derivative 10’ is characterized by the formation of chains of molecules joined together through NH···O hydrogen bond interactions [N(5)–O(3)i 2.758(3) , N(5)H(5a)···O(3) 167.28, i = x1, y < 1, z; N(10)–O(1)ii 2.800(4) , N(10)H(10a)···O(1) 169.08, ii = x1, y1, z1]. Chains of coplanar molecules are linked between each other with CH···F interactions, forming layers mostly in the ab plane [C(6)F(4)iii 3.438(6) , C(6) H(6a)···F(4) 160.98, iii = 2x, 2y, z] (Figure S5). In contrast, in the absence of the N-H group on the imidazole ring in derivative 12, association of complementary molecules into dimers is reached through weak CH···O=C hydrogen bonds [C(6)–O(1)i 3.212(4) , C(6)H(6a)···O(1) 139.78, i = x, y, 1z], with the formation of a hydrogen bond ring described as R22(14) in the graph set notation (Figure S6).

Figure 7. SEM image of the PE composite containing derivative 1.

original inoculum, whereas bacteriostatic activity is defined as a maintenance of a reduction of less than 99.9 % of the total count of CFU mL1 in the original inoculum.[25] Unloaded and loaded PE granules (see experimental section) with AgNO3 (indicated as PE0 and PEAgNO3 respectively) were tested as negative and positive controls, respectively. As shown, the growth inhibitory test carried out with PE1–PE12 granules showed different performances in terms of time and rate of action (Figure 8). In fact, in S. aureus, all PEn composites reached and passed a 90 % reduction within 8 h of exposure and almost all PEn composites led to 99 % reduction within 24 h of exposure. In particular, PE4, PE7, PE8, and PE9 reached 99.9 % reduction (Figure 8 a, b). All composites showed a homogeneous trend during the period of exposure. A similar result was found for all tested PEn composites against P. aeruginosa, although some PEn showed small variations compared with PEAgNO3 during 24 h of exposure. In particular, PE10 and PE12 showed a decrease of activity between 12 and 16 h, but this reduction was recovered starting from 16 to 24 h (Figure 8 c, d). Notably, the activity of PE10 and PE12 was comparable to that of PEAgNO3 and was strongly effective in the first period of exposure, with a 90 % of reduction obtained in just 4 h. All PEn reached a good bacteriostatic activity (99 % reduction) within 24 h, although PE1, PE6, and PE9 showed slower activity than other PEn, and PE4 and PE5 exhibited a more homogeneous trend. The same composites showed bactericidal activity, reaching 99.9 % reduction. In E. coli a markedly different behavior was observed in the first period of exposure, compared with that against P. aeruginosa and S. aureus. Almost all PEn passed 90 % of reduction within 24 h of exposure and, in particular, PE4, PE5, and PE10 achieved 99.9 % reduction within 16 h (Figure 8 e, f). The different behavior of PEn composites against the three bacteria strains could be explained by the structure of the cell wall in different bacteria strains. To be effective, a biocide needs to bind to the bacterial cell wall. However, the cell wall structure is different in Gram-negative and Gram-positive bacteria. The Gram-negative bacteria (such as E. coli) have an outer membrane barrier outside the cell wall.[26] This membrane is formed by lipopolysaccharides (LPS) and proteins, and represent an additional barrier for foreign macromolecules.[27]

Preparation and characterization of composite plastics Novel polyethylene (PE) composite materials PEn (n = 1–12, PE composites containing AgI derivatives 1–12, respectively) were prepared by embedding the AgI derivatives in a PE matrix in a 1:1000 weight ratio. The ratio was chosen on the basis of preliminary antimicrobial tests carried out on 1:5000, 1:1000, and 1:500 weight ratio composites, with the 1:1000 ratio being the most suitable for killing bacteria within 24 h. The PE composites, characterized by IR and TGA analyses, did not display any detectable change of their infrared (Figure S7) or thermal properties (Figure S8) with respect to unloaded PE, apart a slight shift of decomposition temperature. The surface characterization of PE composites was carried out by EDX and SEM analyses. As an example, the SEM spectrum of composite PE1 is shown in Figure 7, with the black area being PE matrix and the white parts being the AgI derivative 1: the AgI additive is well dispersed in the polymeric matrix with a wide size distribution (between 1 and 10 mm). Antibacterial activity of PEn composites The bactericidal and bacteriostatic activity of the PE composites on two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and one Gram-positive (Staphylococcus aureus) bacterial strains was evaluated. Bactericidal activity is defined as a reduction of 99.9 % of the total count of CFU mL1 in the Chem. Eur. J. 2014, 20, 1 – 16

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Full Paper and the component of the cell membrane carrying negative charge density, so impairing cell respiration by blocking its energy system and resulting in cell death.[31] We therefore also used E. coli (Gram-negative) and S. aureus (Gram-positive) as models to study in detail the mechanism of action of PEn composites (n = 1, 3, 4, 6 and 7) on membrane permeabilization, reactive oxygen species (ROS) production and protein release.

Test by contact on PEn composites The test by contact was carried out on PE1, PE3 and PE6 composites, to include different structural types of additives (polymeric and ionic). PEAgNO3 and PE0 were used in the test as positive and negative controls, respectively. Apart from the simple PE disk (PE0) all the other composites demonstrated inhibition of E. coli growth on the contact surface (Figure 9).

Antibacterial activity according to ISO standard Figure 8. Reduction of CFU mL1 after exposure to polymers PEn (n = 1–12); PE0 (unloaded) and PEAgNO3 (loaded with AgNO3): a) PEn (n = 1–6) versus S. aureus; b) PEn (n = 7–12) versus S. aureus; c) PEn (n = 1–6) versus P. aeruginosa; d) PEn (n = 7–12) versus P. aeruginosa; e) PEn (n = 1–6) versus E. coli; f) PEn (n = 7–12) versus E. coli.

Gram-positive bacteria (such as S. aureus) have a cell wall that is characterized by a less complex structure.[28] Therefore, the lower performance observed against E. coli than S. aureus and the different activity could be explained by considering the cell structure of the bacteria. As observed above, the different cellular structure could also explain the lower activity against E. coli than against P. aeruginosa (both Gram-negative bacteria). To disorganize the outer membrane of Gram-negative bacteria, the PEn composites probably bind to negatively charged LPS membrane, affecting the membrane potential with a consequent disorganization of the outer membrane and cell death.[29] Studies on the negatively charged density on the cell surface showed that different Gram-negative bacteria possess different negative charges. The diverse results in activity of PEn composites against different Gram-negative bacteria (PEn are less active against E. coli than against P. aeruginosa) could be explained by the higher negative charge density on the cell surface of P. aeruginosa compared with that in E. coli.[30] This should increase the binding strength between silver centers &

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To further verify the antibacterial activity by contact of the loaded PEn composites, a standardized ISO test was carried out.[32] The

Figure 9. Bactericidal effect promoted by contact with composites PE1, PE3, PE6, and PEAgNO3 (indicated in the photos as AgNO3) compared with the effect of a nonembedded PE disk (indicated in the photos as PE) for Escherichia coli ATCC 25 922 with MacConkey agar: a) Bacterial growth can be appreciated only below the nonembedded PE disk; PEAgNO3 shows a zone of inhibition around the disk. b) The picture was taken after lifting the disks: the growth below the PE disk is clearly visible, whereas below the PE1, PE3, PE6, and PEAgNO3 disks there is absence of bacterial growth.

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Full Paper results were elaborated according to the Japanese industrial standard (JIS).[33] This test clearly demonstrated inhibition of bacteria growth on the contact surface between the tested PEn square composites and the bacterial inoculum, not only after 24 h incubation but even after 72 h, to assess the ability of the PEn composites (n = 1, 3, 4, 6, and 7) over time. Each composite was analyzed in triplicate. Compared to E. coli, composites PE4 and PE7 show a value of antibacterial activity R > 3 log microbial growth reduction, corresponding to a percentage of reduction of 99.9 % (Table 4). In particular, PE4 had an

sites (n = 1, 3, 4, 6, and 7) and PEAgNO3 under both contact conditions (Tables 5 and 6). In fact, all PEn samples showed less release than that of PEAgNO3.

Table 5. Specific Ag + migration from PEn square composites with embedded silver(I) derivatives 1, 3, 4, 6, and 7, expressed in terms of percentage release in several simulants by heating at 80 8C for 2 h.

Table 4. Antibacterial activity (R values) according to ISO standard.[32] Sample

R (24 h) E. coli

R (72 h)

R (24 h) S. aureus

R (72 h)

PE0 PEAgNPs PE1 PE3 PE4 PE6 PE7

0.0 6.7 2.3 2.2 6.9 1.9 4.0

0.0 0.6 5.4 3.9 5.2 2.1 5.5

0.0 2.0 0.1 6.3 7.0 0.8 0.1

0.0 0.0 1.0 1.1 5.6 1.6 1.9

Sample

Simulant A Simulant B Simulant C 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 PE3 PE4 PE6 PE7

0.00 – – 0.00 100.00 0.51 14.51 5.71 1.84 0.48

– 0.00 – 0.00 100.00 1.19 9.52 18.57 2.51 6.19

– – 0.00 0.00 76.00 0.68 4.08 4.05 1.51 2.86

Table 6. Specific Ag + migration from PEn square composites with embedded silver(I) derivatives 1, 3, 4, 6, and 7, expressed in terms of percentage release in several simulants by heating at 40 8C for ten days.

antibacterial activity equal to that achieved with PEAgNPs. It is interesting to note, however, that the activity of composites was maintained over time, whereas that of PEAgNPs in the third exposure tended to lose its activity. The log value of PEAgNPs after the third exposure is in fact R = 0.6 log microbial growth reduction, whereas composites PE1, PE3, PE4 and PE7 maintain a log value > 3 even after the third exposure. These data further confirm that, in contrast to PEAgNPs, PEn composites do not release Ag + ions and their antibacterial activity persists over time, especially in the case of composites PE1 and PE3. Compared to S. aureus, composites PE3 and PE4 reach very high reduction values after 24 h, largely exceeding a log value of 3, corresponding to a percentage of reduction of 99.9% (Table 4). Compared with the PEAgNPs composite with embedded Ag nanoparticles, most PEn composites show a significant antibacterial action, particularly PE4, which maintains a log microbial growth reduction value of more than 3, whereas PEAgNPs display a total loss of antibacterial activity after the third exposure (Table 4).

Release tests from PE composites were carried out for composites PE1, PE3, PE4, PE6, and PE7 to further confirm the mechanism of antimicrobial action by contact. The migration was tested by using three simulants (distilled water, acetic acid 3 % v/v, and ethanol 10 % v/v) under two assay conditions (40 8C for ten days and 80 8C for 2 h), according to EU Legislation on the migration of chemicals from plastic materials.[34] The test conditions correspond to the more severe (worst foreseeable) conditions of contact. PE0 and PEAgNO3 squares were tested as negative and positive controls, respectively. A significantly different release profile was observed between PEn compowww.chemeurj.org


Simulant A Simulant B Simulant C 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 PE3 PE4 PE6 PE7

0.00 – – 0.00 100.00 0.68 9.90 8.10 0.67 2.14

– 0.00 – 0.00 100.00 2.33 7.09 12.40 4.16 19.27

– – 0.00 0.00 55.00 0.60 2.87 8.4 1.27 2.56

Such difference could be explained by the insolubility of derivatives 1, 3, 4, 6, and 7 in water with respect to water-soluble silver nitrate, which facilitates the migration of Ag + ions in simulants. Furthermore, PE1 and PE6 contain polynuclear silver(I) derivatives and, likely for this reason, they show lower levels of release among the tested samples. In contrast, no significant difference was observed between PE4 and PE7 composites, both loaded with monomeric silver(I) derivatives 4 and 7. PE3, loaded with the ionic silver(I) derivative 3, showed a slightly higher level of release then the other two composites. As expected, tests in acetic acid as acidic simulant give the highest values of migration but, even in this case, PE1 and PE6 performances were very good in terms of limited levels of silver release. In summary, we can conclude that PE1 and PE6 do not release silver and likely act by a contact antimicrobial mechanism, whereas for the other PEn composites containing monomeric neutral or ionic silver species, we cannot exclude the conclusion that antimicrobial activity is connected to silver ion release. In particular, this should explain the high R values

Release test on PEn composites

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Figure 10. a) Percentage of PI fluorescent emission for E. coli cells exposed to PEn composites (n = 1, 3, 4, 6, and 7), PE0 (unloaded), PEAgNO3 (loaded with AgNO3), and PEAgNPs (loaded with AgNPs). The data are represented as the mean SD of at least three separate experiments. b) Representative phase-contrast (left) and fluorescence microscopy (right) images. Dead bacteria are stained red.

of antibacterial activity according to the ISO standard test detailed in Table 4 for PE3 and mainly PE4. Analysis of membrane permeabilization in treated E. coli and S. aureus cells Propidium iodide (PI) has been proposed as an appropriate probe with which to assess membrane permeabilization because it penetrates only when the bacterial membrane is permeabilized. Thus, E. coli and S. aureus bacteria cells were treated for 24 h with PEn composites (n = 1, 3, 4, 6, and 7) and then stained by PI. Red fluorescence emission was determined and represented as a percentage of fluorescent emission compared to control, in both bacterial strains. A similar effect towards cultured E. coli and S. aureus bacteria treated with PEn composites (n = 1, 3, 4, 6, and 7) was found, and, in particular, moderate levels of emission was observed for E. coli treated with PE1, PE4, and PE7 compared with cells treated with PE0 (Figure 10 a). Moreover, to assess the effective incorporation of PI, we examined bacterial cells by fluorescence microscopic analysis (Figure 10 b). We also evaluated whether PEn composites induced ROS generation in both strains. ROS production after treatment with PEn composites (n = 1, 3, 4, 6, and 7) was measured by using the DCFH-DA method, 1 and 2 h post-treatment in E. coli and S. aureus strains. As shown in Figure 11 a and b, after incubation with PEn composites, ROS formed in the samples were detected at 523 nm emission wavelength by using a fluorescence spectrophotometer. The results confirm the generation of free radicals in E. coli and S. aureus cells at 1 h and, in particular, at 2 h post-treatment with composites PE1, PE4, and PE7, whereas no effects were observed with composites PEAgNO3 and PE6. In particular, PE7 showed an higher effect at 2 h than both PEAgNO3 and PEAgNPs (a PE composite loaded with Ag nanoparticles) in S. aureus cells. These data clearly indicate an ability of PEn composites to break the bacterial cell membrane.[35] An increase in the ROS synthesis leads to the formation of highly reactive radicals associated with cellular destruction. Excess generation of reactive oxygen species can attack membrane lipids, &

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Figure 11. Formation of ROS in a) S. aureus and in b) E. coli cells exposed to PEn composites (n = 1, 3, 4, 6, and 7), PE0 (unloaded), PEAgNO3 (loaded with AgNO3), and PEAgNPs (loaded with AgNPs) for 1 and 2 h. The data are represented as the mean SD of at least three separate experiments.

which leads to a breakdown of membrane function. Certain transition metals might disrupt the cellular donor ligands that coordinate Fe. In particular, the main targets of these metals are the cellular donor ligands that coordinate Fe;[36] especially, the solvent-exposed [4Fe4S] clusters of proteins. The direct or indirect destruction of [4Fe4S] clusters by metals causes the release of additional Fenton-active Fe into the cytoplasm, resulting in increased ROS formation.[37] We also used Bradford’ pro10



Full Paper tein assay to verify possible protein leakage from bacterial cells. Protein leakage was analyzed by evaluating the ability of PEn composites (n = 1, 3, 4, 6, and 7) to induce protein leakage in E. coli and S. aureus membrane. Interestingly, different effects were found for the composites on two different bacteria (Figure 12 a, b). Protein leakage from E. coli cells was higher than that from S. aureus for all composites tested.

(n = 1, 3, 4, 6, and 7) for up to seven days, and cell viability was evaluated at the third and seventh day post-treatment. As shown in Figure 13 a, no inhibition of cell viability was found in any of the treated cells versus control cells. These results suggest that none of the compounds have an effect on CD34 + cell viability compared with the control, indicating a noncytotoxic effect of PEn composites in human cells. To strengthen the MTT data, the cytotoxic effect was evaluated for colony formation by CFU assay. As shown in Figure 13 b and c, no differences in colony formation were found in the PEn treated cells compared with the control cells, confirming that the PEn composites had no cytotoxic effects on CD34 + cells. CFU-GM assays are generally used to estimate the potential for myeloid toxicity,[40] and these data indicate that PEn composites do not produce an inhibition of myeloid colony formation. Acute toxicity test with D. magna A final objective of the present study was to understand the eventual toxicity of our composites against higher organisms. A classic test of ecotoxicity is based on the use of Daphnia magna. Previous release kinetics of Ag + ions from AgNPs into aqueous environment have shown that silver nanoparticles were acutely toxic to Daphnia magna.[41] In contrast, in the case of selected composites (PE1, PE3, PE4, PE6, and PE7) no deaths of the organisms tested were observed, indicating the tolerance towards the composites by higher organisms. This tolerance indicates that toxic substances are not released under the test conditions, or at least that Ag + is not released in sufficient amounts to affect the aqueous environment.

Conclusion The reaction between two acylpyrazolonato ligands and AgNO3 in the presence of base yielded two new coordination polymers (derivatives 1 and 6). By also using N-donor ancillary ligands it is possible to control the nuclearity, and neutral mono- or binuclear species have been isolated (2, 4, 5, and 7– 12); in one case an ionic compound was afforded (derivative 3). All derivatives 1–12 have been embedded in polyethylene to give PEn composite materials with a 1:1000 weight ratio of additive to PE. Their relevant spectral and thermal properties are essentially unchanged with respect to simple PE, even though the surface of composite PE disks displays a homogeneous dispersion of the additive, which appear as small granules with an average diameter in the range 1–10 mm. Such distribution leads to “large distances” on the surface of the composites without any silver additive; these distances are larger than the dimensions of a bacterium. Nevertheless, the PEn composites display excellent antimicrobial activity against three bacterial strains (Gram-positive S. aureus and Gram-negative E. coli and P. aeruginosa). The silver coordination polymers (derivatives 1 and 6) seem to be the most suitable for use as inexpensive antimicrobial additives to PE. As an average, they are in fact the most active additives; moreover, they are clearly easier to prepare and less expensive than others materials. Their synthe-

Figure 12. Leakage of protein from a) S. aureus and in b) E. coli cells exposed to PEn composites (n = 1, 3, 4, 6, and 7), PE0 (unloaded), PEAgNO3 (loaded with AgNO3), and PEAgNPs (loaded with AgNPs) for 1 and 2 h. The data are represented as the mean SD of at least three separate experiments.

Both PE4 and PE6 showed an effect that was comparable to that of PEAgNPs. This result suggests that the sensitivity of Gram-positive S. aureus was lower than that of Gram-negative E. coli. Other studies have attributed this difference to the different structure of the cell walls and their thickness. The wall thickness of Gram-positive bacteria is approximately 80 nm, whereas the wall thickness of Gram-negative bacteria is approximately 10 nm. Furthermore, Gram-positive cells have a thicker peptidoglycane layer than Gram-negative cells, and this layer forms a protective barrier against antibacterial agents such as silver.[38, 39] Cytotoxic effects of PE granules in human CD34 + cells The cytotoxic effect of PE composites was evaluated in CD34 + cells, by MTT assay. CD34 + were treated with PEn composites Chem. Eur. J. 2014, 20, 1 – 16

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Full Paper dried in vacuo to constant weight (20 8C, ca. 0.1 Torr). Elemental analyses (C, H, N) were performed inhouse with a Fisons Instruments 1108 CHNS-O Elemental Analyzer. IR spectra were recorded from 4000 to 400 cm1 with a PerkinElmer Spectrum 100 FTIR instrument by total reflectance on a CdSe crystal. 1H, 13C{1H}, and 19F{1H} NMR spectra were recorded with a 400 Mercury Plus Varian instrument operating at RT (400 MHz for 1H, 100 MHz for 13C, and 376.8 MHz for 19 F). H and C chemical shifts (d) are reported in parts per million (ppm) from SiMe4 (1H and 13C calibration by internal deuterium solvent lock), whereas F chemical shifts (d) are reported in ppm versus CFCl3. Peak multiplicities are abbreviated: singlet, s; doublet, d; triplet, t; quartet, q; and multiplet, m. Melting points are uncorrected and Figure 13. CD34 + cells were cultured for up to seven days in the presence of PE composites. a) Cell viability was were recorded with an STMP3 determined by MTT assay. The data are represented as the mean SD of at least three separate experiments. Stuart scientific instrument and b) Representative phase-contrast photomicrographs depicting colony formation assay for CD34 + cells. c) Total soft with a capillary apparatus. The agar colony counts were done by visualizing individual colonies in ten random fields. Data shown are representaelectrical conductivity measuretive of one of three separate experiments. reported as ments (LM, S cm2 mol1) of acetonitrile, methanol, and DMSO solutions of the sis does not require any additional reactant, whereas the other silver derivatives were recorded with a Crison CDTM 522 conderivatives also need the use of ancillary N-donor ligands, and ductimeter at RT. The positive and negative electrospray mass their isolation is straightforward—simple filtration from the respectra were obtained with a Series 1100 MSI detector HP specaction mixture. Contact and release tests confirm that, at least trometer, using acetonitrile as mobile phase. Solutions (3 mg mL1) for the composite materials containing the two silver(I) coordifor electrospray ionization mass spectrometry (ESI-MS) were prepared by using reagent-grade acetonitrile and methanol. For the nation polymers (derivatives 1 and 6), the antimicrobial activity ESI-MS data, mass and intensities were compared to those calculatessentially functions by simple contact of the active surface, ed by using IsoPro Isotopic Abundance Simulator, version 3.1. without any noticeable release of the biocide. PEn composites Peaks containing silver(I) ions were identified as the center of an are able to disrupt the bacterial cell membrane, thus provoking isotopic cluster. Thermal gravimetric analyses (TGA) were carried bacteria death. However, the composites are not toxic towards out in a N2 stream with a PerkinElmer STA 6000 simultaneous therhigher organisms and they are benign in aqueous environmal analyzer (heating rate: 7 8C min1). Energy dispersive X-ray ments. Moreover, PEn composites can be reused several times, analyses were carried out in a N2 stream with a 800 HS Shimadzu displaying the same antimicrobial activity. This feature can be and SEM spectra with a Cambridge Stereoscan 360 Scanning electron Microscope. ICP analyses for specific silver ions migration appropriate for the application of our composite plastics in were carried out with a 7500 cx Agilent Technologies ICP-MS Speca number of different situations, such as cases for mobile trometer. phones, power buttons in kitchens, and remote controls and

light switches in hotels, where accumulation of dirt is often overlooked and bacteria levels reach those of the toilet and the bathroom sink.[42]

Synthesis of [Ag(QPh)] (1) A solution of ligand HQPh (0.278 g, 1.0 mmol) and NaOMe (0.054 g, 1.0 mmol) in methanol (30 mL) was added to a solution of silver nitrate (0.170 g, 1.0 mmol) in water (10 mL). A colorless precipitate immediately resulted, which was filtered off, washed with Et2O (20 mL), and dried in vacuo to constant weight to give 1 (86 % yield), which was soluble in DMSO. M.p. 234–236 8C; 1H NMR ([D6]DMSO): d = 2.18 (s, 3 H; C3-CH3), 7.07 (t, 1 H) 7.27–7.36 (m, 5 H), 7.60 (d, J = 7 Hz, 2 H), 7.77 (d, J = 8 Hz, 2 H) ppm (10 H, Ar-H of QPh); 13 1 C{ H} NMR ([D6]DMSO): d = 17.8 (s, C3-CH3), 102.0 (s, C4), 120.8 (s), 123.9 (s), 127.0 (s), 128.4 (s), 128.5 (s), 129.6 (s), 139.8 (s), 141.5 (s; CAr of QPh), 152.6 (s, C3), 163.4 (s, C5), 188.4 (s) ppm (CO); IR: ﬃ 3055 (w, nCAr=H), 1612 (vs), 1592 (s, nC=O), 1575 (m), 1499 (vs), 1428 (s), 1354 (nC=C, nC=N, nC=N), 1222 (w), 1064 (w), 941 (s), 753

Experimental Section Materials and methods All chemicals were purchased from Aldrich (Milwaukee) and used as received. The acylpyrazolone ligands 3-methyl-1-phenyl-4-benzoyl-5-pyrazolone (HQPh) and 1-(2-pyridyl)-3-methyl-4-trifluoro acetyl-5-pyrazolone (HQpy,CF3) were synthesized as previously reported.[15, 16c] All of the reactions and manipulations were performed in air. Solvent evaporation was carried out under vacuum conditions by using a rotary evaporator. The samples for microanalyses were

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Full Paper (s) cm1; elemental analysis calcd (%) for C17H13AgN2O2 : C 53.01, H 3.40, N 7.27; found: C 52.69, H 3.29, N 7.21; LM in DMSO: 15.2 S cm2 mol1; TGA-DTA (mg % vs. 8C): heating from 30 to 600 8C with a speed of 8 8C min1; from 230 to 600 8C progressive decomposition, with a final black residual of 45 % weight.

27 853 (OXOID-remel), and the Gram-positive bacterium S. aureus ATCC 25 923 (BPI International). Bacteria were grown aerobically at 37 8C for 18 h using Tryptone Soya Broth (OXOID) as the growth medium. Bacterial cultures (106 CFU/mL) were added to sterile test tubes containing autoclaved physiological solution (4 mL). For sterilization of the tubes, an Alfa-10-plus autoclave (PBI International) was used, operating at 121 8C for 15 min. A PEn disk (40 mg), 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 disks on the bacterial cultures, 100 mL of supernatant fraction were withdrawn from the tubes at time intervals of 4, 8, 12, 16, 20, and 24 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.

All other analytical and spectroscopic data of derivatives 2–12 are available in the Supporting Information.

Solid-state NMR spectroscopy 15

N solid-state NMR spectra were recorded with a Bruker Avance II 400 instrument operating at 400.23 and 40.55 MHz for 1H and 15N nuclei, respectively. Cylindrical 4 mm o.d. zirconia rotors with a sample volume of 80 mL were employed and spun at 9 kHz. A ramp cross-polarization pulse sequence was used with a contact time of 4 ms, a 1H 908 pulse of 3.05 ms, recycle delays of 10–20 s, and 16 000–20 000 transients. A two-pulse phase modulation (TPPM) decoupling scheme was used with an rf field of 75 kHz. 15N chemical shifts were referenced with the resonance of (NH4)2SO4 (15N signal at d = 355.8 ppm with respect to CH3NO2).

Antibacterial activity according to ISO standard: The antibacterial activity by contact of all PEn composites was performed according to the ISO standard.[32] All tested square composites had dimensions of 50 50 mm (10 mm in thickness) and were tested, in triplicate, against two strains: (Gram-positive) Staphylococcus aureus ATCC 25 923 and (Gram-negative) Escherichia coli ATCC 25 922. Unloaded PE samples were used as negative control. PE loaded with AgNPs was used as positive control. The appropriate culture medium was inoculated with the test microbes and cultivated for 24 h at 37 8C under aerobic conditions, to achieve the concentration of 107 CFU/mL. Bacterial suspensions (0.4 mL) were inoculated onto the test surface and the inoculum was covered with a piece of polyethylene film (40 40 mm), gently pressed down to spread the inoculum to the edges. The Petri dishes containing the inoculated test specimens were incubated at (35 1) 8C and a relative humidity of not less than 90 % for 24 1 h. After the incubation time, the inoculum was processed by adding 10 mL SCDLP broth (Soybean casein digest broth with lecithin and polyoxyethylene sorbitan monooleate). From SCDLP broth, tenfold serial dilutions were made in phosphate-buffered physiological saline (PBS-saline) and aliquots of 1 mL for each dilution were placed in Petri dishes, and 15 mL of plate count agar (PCA) was poured to disperse the bacteria. The inverted Petri dishes were incubated at (35 1)8C for 48 h. After incubation, the numbers of colonies in the Petri dishes were counted. The numbers of bacteria surviving on the specimens tested were compared to the number of colonies present on the negative controls. The antibacterial activity (R) was determined according to the Japanese industrial standard[33] based on the following rating: no antimicrobial activity 0.5 log microbial growth reduction; slight antimicrobial activity = 0.5–1 log microbial growth reduction; significant antimicrobial activity > 1 to 3 log microbial growth reduction; strong antimicrobial activity > 3 log microbial growth reduction.

Crystal structure analyses Details of the crystal data collection are listed in Table S1. X-ray data for 3, 4, 7, 8, 10’, and 12 were collected with a Bruker-Nonius X8 Apex CCD area detector equipped with a graphite monochromator and Mo Ka radiation (l = 0.71073 ), and data reduction was performed by using the SAINT programs; absorption corrections based on multiscan were obtained by SADABS.[43] Both structures were solved by Patterson method (SHELXS/L program in the SHELXTL-NT software package) and refined by full-matrix leastsquares based on F2.[44] All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were included as idealized atoms riding on the respective carbon atoms with CH bond lengths appropriate to the carbon atom hybridization

Embedding procedure for silver(I) derivatives in polyethylene disks Polyethylene (PE) disks with embedded complexes 1–12 were prepared in the following manner: the coordination polymer, in the form of a powder, was mixed in a glass capsule with PE granular powder (1.00 g) in a 1:1000 weight ratio. The capsule was heated to the melting point of PE (98 8C), while its content was stirred to give a homogeneous dispersion. The dispersion was then allowed to cool to RT; after solidification, the loaded polymer matrix was removed from the capsule and placed in contact with a hot quartz surface (130 8C): within a few minutes, the matrix melted and distributed homogeneously onto the quartz surface to give a thin liquid layer. After cooling of the quartz surface to 80 8C, the polymeric matrix layer became a soft solid film, that could be cut into small disks of 6 mm diameter and a thickness in the range 0.8– 1.0 mm.

To verify the persistence of antibacterial activity over time, an additional test at 72 h was performed on the same square composites, previously washed with ethanol and irradiated by UV light for 15 min. Contact tests: Bacterial aqueous suspension (E. coli 106 CFU mL1, 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. Composite PEn disks and the blank disk were gently placed over contaminated MacConkey Agar in Petri dishes. Petri dishes were incubated overnight at 35 8C 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).

Microbiological studies Preparation of PE disks for cell culture analysis: The PEn composites were prepared as disks with a diameter corresponding to that of the well of the plates used. Before each treatment, the PE disk was inserted into the wells and sterilized by exposure to UV irradiation. Antibacterial activity studies: The antibacterial activity of all PEn composite disks was tested against the two Gram-negative bacteria, E. coli ATCC 25 922 (PBI International) and P. aeruginosa ATCC Chem. Eur. J. 2014, 20, 1 – 16

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Release tests for specific migration of silver from composites[34] PEn composite squares preparation: Polyethylene squares (Figure S9) with embedded complexes 1, 3, 4, 6, and 7 were prepared in the following manner: the coordination polymer, in the form of powder was mixed in a Teflon mould, with an area of 1 dm2, with PE granular powder (7.00 g) in a 3:1000 weight ratio. The Teflon mould was heated to the melting point of PE (98 8C), while its content was stirred to give a homogeneous dispersion. The dispersion was then cooled to RT; after solidification, the loaded polymer matrix was removed from the mould and was reduced into pieces suitable for migration testing.

Acute toxicity test using D. magna: The acute toxicity test for D. magna was carried out in accordance with Test Guideline 202 of the Organisation for Economic Co-operation and Development.[46] D. magna were purchased from Ecotox LDS Conaredo (MI), Italy and reared in the culture room in the Analisi Control S.r.l., Corridonia (MC), Italy. The embryonic development of Daphnia magna eggs takes about three days under optimal conditions (6.000 lux light intensity at the top of the petri dish and at 20–22 8C) 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 multiwell 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 6341[47]) 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 micro-algae. 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 solution coming from the previous release test (carried out at 40 8C for ten days in simulant A; i.e., distilled water) of the composites PE1, PE3, PE4, PE6, and PE7 were added to the compartments. Finally, a parafilm strip was placed over on the multiwell plate and the cover was put on tightly. The plate was left in an incubator at 20 8C in the dark. After 24 and 48 h incubation, the multiwell 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 Daphnia death. In the case of PE1, PE3, PE4, PE6, and PE7, no deaths of the Daphnids were observed, indicating the tolerance of higher organisms towards the compounds.

Migration tests: 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). 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 conical flasks, covered with a aluminum foil, were kept in a controlled atmosphere at two assay conditions: 40 8C for ten days and 80 8C for 2 h. After the incubation period, the pieces were removed and the simulant was extracted to analyze the Ag + released in the simulants by using inductively coupled plasma spectroscopy with mass spectrometry detection (ICPMS). By adopting the same procedure, an unloaded PE square was used as negative control and a loaded PE square with AgNO3 was used as positive control. A solution containing indium (10 mg L1) was used as internal standard for ICP-MS measurements. Calibration curves for the investigated element were obtained by using aqueous (3 % nitric acid) standard solutions prepared by appropriate dilution of stock standards (Ag standard solution 10 mg L1 for ICP-MS). Protein leakage from bacterial cells: Protein leakage from bacterial cells was measured in cell medium by Bradford’s protein assay. Briefly, cells of E. coli and S. aureus, with a concentration of 107 CFU mL1, were exposed for 24 h to PEn composites (n = 1, 3, 4, 6, and 7) in culture medium (1 mL). PEAgNPs, with Ag nanoparticles prepared according to literature reported procedure,[45] PEAgNO3 and PE0 were used as positive and negative controls, respectively. After treatment, culture medium was recovered, centrifuged for 5 min at 3000 rpm 4 8C, and supernatants were aliquoted and stored at 80 8C. Each sample (2 mL), in triplicate, was assayed for protein content.

Acknowledgements

PI uptake: E. coli and S. aureus cells, treated and untreated, were incubated with the PEn composites for 1 day, and then propidium iodide PI (2 mm) was added for 20 min. For microscopic analysis, an aliquot of each sample was cytospun onto glass slides and immediately analyzed by Cell imaging performed with an Olympus BX51 Microscope.

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This work was financially supported by the University of Camerino (Fondo di Ateneo per la Ricerca 2011–2012) and Nuova Simonelli Company.

Procedures used for fluorospectrophotometer analysis of ROS production, fluorospectrophotometer analysis of cell membrane damage and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay are available in the Supporting Information.

Keywords: antibiotics · antiproliferation · polymers · silver · solid-state structures

CFU-GM assay: The CFU-GM assay was used as a surrogate assay to assess cytotoxicity effects of PE compounds. For CFU-GM assay, 3 103/well human CD34 + cells (StemCell Technologies, Voden, IT) were incubated with Stem Span SFME II medium supplemented with StemSpa-ACF (StemCell Technologies) and treated with the appropriate PE composites for 7 days, in 96-well plate. The cells were then aspirated from the wells, diluted in MethoCult H4034 Optimum (StemCell Technologies) and dispensed in duplicate onto 35 mm culture plates, with a concentration of 2 103/well. Colonies were scored on day 14, counted microscopically and the percent-

[1] a) A. MuÇoz-Bonilla, M. Fernndez-Garca, Prog. Polym. Sci. 2012, 37, 281; b) M. Charnley, M. Textor, C. Acikgoz, React. Funct. Polym. 2011, 71, 329. [2] a) D. Crespy, K. Landfester, Polymer 2009, 50, 1616; b) A. Gautam, S. Ram, Mater. Chem. Phys. 2010, 119, 266. [3] S. L. Percival, P. G. Bowler, D. Russell, Hosp. Infect. 2005, 60, 1. [4] E. Amato, Y. A. Diaz-Fernandez, A. Taglietti, P. Pallavicini, L. Pasotti, L. Cucca, C. Milanese, P. Grisoli, C. Dacarro, J. M. Fernandez-Hechavarria, V. Necchi, Langmuir 2011, 27, 9165. [5] a) P. Gunawan, C. Guan, X. Song, Q. Zhang, S. S. J. Leong, C. Tang, Y. Chen, M. B. Chan-Park, M. W. Chang, K. Wang, R. Xu, ACS Nano 2011, 5,

Chem. Eur. J. 2014, 20, 1 – 16

www.chemeurj.org

14



Full Paper

[6]

[7] [8] [9]

[10] [11] [12] [13]

[14]

[15] [16]

[17]

[18]

[19] [20] [21] [22] [23] [24]

10033; b) S. Kar, M. Subramanian, A. K. Ghosh, R. C. Bindal, S. Prabhakar, J. Nuwad, C. G. S. Pillai, S. Chattopadhyay, P. K. Tewari, Desal. Water Treat. 2011, 27, 224; c) M. M. da Silva Paula, C. V. Franco, M. C. Baldin, L. Rodrigues, T. Barichello, G. D. Savi, L. F. Bellato, M. A. Fiori, L. da Silva, Mater. Sci. Eng. C 2009, 29, 647; d) M. J. Brady, C. M. Lisay, A. V. Yurkovetskiy, S. P. Sawan, Am. J. Infect. Control 2003, 31, 208. O. Gordon, T. V. Slenters, P. S. Brunetto, A. E. Villaruz, D. E. Sturdevant, M. Otto, R. Landmann, K. M. Fromm, Antimicrob. Agents Chemother. 2010, 54, 4208. O. Choi, K. K. Deng, N.-J. Kim, L. Ross, R. Y. Surampalli, Z. Hu, Water Res. 2008, 42, 3066. S. Chernousova, M. Epple, Angew. Chem. Int. Ed. 2013, 52, 1636; Angew. Chem. 2013, 125, 1678. a) M. Rai, A. Yadav, A. Gade, Biotechnol. Adv. 2009, 27, 76; b) K.-S. Chou, C.-Y. Ren, Mater. Chem. Phys. 2000, 64, 241; c) K. K. Y. Wong, X. Liu, MedChemComm 2010, 1, 125; d) J. Liu, D. A. Sonshine, S. Shervani, R. H. Hurt, ACS Nano 2010, 4, 6903. K. Helttunen, N. Moridi, P. Shahgaldian, M. Nissinen, Org. Biomol. Chem. 2012, 10, 2019. J. J. Bullen, H. J. Rogers, P. B. Spalding, C. G. Ward, FEMS Immunol. Med. Microbiol. 2005, 43, 325. S. Loher, O. D. Schneider, T. Maienfisch, S. Bokorny, W. J. Stark, Small 2008, 4, 824 – 832. C. Pettinari, F. Marchetti, G. Lupidi, L. Quassinti, M. Bramucci, D. Petrelli, L. A. Vitali, M. F. C. Guedes da Silva, L. M. D. R. S. Martins, P. Smolen´ski, A. J. L. Pombeiro, Inorg. Chem. 2011, 50, 11173. A. Ta˘ba˘caru, C. Pettinari, F. Marchetti, C. Di Nicola, K. V. Domasevitch, S. Galli, N. Masciocchi, S. Scuri, I. Grappasonni, M. Cocchioni, Inorg. Chem. 2012, 51, 9775. F. Marchetti, C. Pettinari, R. Pettinari, Coord. Chem. Rev. 2005, 249, 2909. a) A. Cingolani, Effendy, F. Marchetti, C. Pettinari, R. Pettinari, B. W. Skelton, A. H. White, Inorg. Chem. 2002, 41, 1151; b) A. Cingolani, Effendy, F. Marchetti, C. Pettinari, R. Pettinari, B. W. Skelton, A. H. White, Inorg. Chem. 2004, 43, 4387; c) A. Lorenzotti, F. Marchetti, C. Pettinari, R. Pettinari, B. W. Skelton, A. H. White, Inorg. Chim. Acta 2005, 358, 3190. a) E. Siebel, R. D.. Fischer, N. A. Davies, D. C. Apperley, R. K. Harris, J. Organomet. Chem. 2000, 604, 34; b) R. Pettinari, C. Pettinari, F. Marchetti, R. Gobetto, C. Nervi, M. R. Chierotti, E. J. Chan, B. W. Skelton, A. H. White, Inorg. Chem. 2010, 49, 11205; c) M. R. Chierotti, R. Gobetto, Eur. J. Inorg. Chem. 2009, 2581; d) D. M. Tçbbens, J. Glinneman, M. R. Chierotti, J. van de Streek, D. Sheptyakov, CrystEngComm 2012, 14, 3046; e) V. Bertolasi, R. Boaretto, M. R. Chierotti, R. Gobetto, S. Sostero, Dalton Trans. 2007, 5179. a) M. L. Gallego, P. Ovejero, M. Cano, J. V. Heras, J. A. Campo, E. Pinilla, M. R. Torres, Eur. J. Inorg. Chem. 2004, 3089; b) P. Cornago, R. M. Claramunt, M. Cano, J. V. Heras, M. L. Gallego, ARKIVOC, 2005, 21; c) M. L. Gallego, M. Cano, J. A. Campo, J. V. Heras, E. Pinilla, M. R. Torres, P. Cornago, R. M. Claramunt, Eur. J. Inorg. Chem. 2005, 4370. G. C. Levy, R. L. Lichter, Nitrogen-15 Nuclear Magnetic Resonance Spectroscopy, John Wiley & Sons, Inc., New York, 1979. Y. Akama, M. Shiro, T. Ueda, M. Kajitani, Acta Crystallogr. Sect. C 1995, 51, 1310. P. Lorente, I. G. Shenderovich, N. S. Golubev, G. S. Denisov, G. Buntkowsky, H.-H. Limbach, Magn. Reson. Chem. 2001, 39, S18. U. J. Scheele, M. Georgiou, M. John, S. Dechert, F. Meyer, Organometallics 2008, 27, 5146. A. Cingolani, Effendy, F. Marchetti, C. Pettinari, R. Pettinari, B. W. Skelton, A. H. White, Inorg. Chim. Acta 2002, 329, 100. M. Nishio, Y. Umezawa, K. Honda, S. Tsuboyama, H. Suezawa, CrystEngComm 2009, 11, 1757.

Chem. Eur. J. 2014, 20, 1 – 16

www.chemeurj.org


[25] a) P. J. Petersen, C. H. Jones, P. Bradford, Diag. Microbiol. Infect. Disease 2007, 59, 347; b) G. A. Pankey, L. D. Sabath, Clin. Infect. Dis. 2004, 38, 864. [26] E. R. Kenawy, S. D. Worley, R. Broughton, Biomacromolecules 2007, 8, 1359. [27] C. Z. Chen, N. C. Beck-Tan, P. Dhurjati, T. K. Dyk, R. A. La Rossa, S. L. Cooper, Biomacromolecules 2000, 1, 473. [28] H. Kong, J. Jang, Langmuir 2008, 24, 2051. [29] R. Kgler, O. Bouloussa, F. Rondelez, Microbiology 2005, 151, 1341; and references therein. [30] Y. C. Chung, Y. P. Su, C. C. Chen, G. Jia, H. L. Wang, J. C. Wu, J. G. Lin, Acta Pharmacol. Sin. 2004, 25, 932. [31] a) M. Nawaz, M. Y. Han, T. I. Kim, U. Manzoor, M. T. Amin, Sci. Total Environ. 2012, 431, 20; b) R. B. Thurman, C. P. Gerba, Crit. Rev. Environ. Contr. 1989, 18, 295. [32] ISO-22196: Plastics – Measurement of Antibacterial Activity on Plastics Surfaces. [33] JIS L-1902:2002, Testing Method for Antibacterial Activity of Textiles. [34] EU Food Contact Regulations for Plastics (food packaging and food Regulations 1935/2004, 79/112/EEC and 89/109/EEC) and EU Regulation 10/2011 (The Plastics Regulation), which indicates the rules for measuring overall and specific migration. (http://ec.europa.eu/food/food/ chemicalsafety/foodcontact/index_eu.htm). [35] M. J. Hajipour, K. M. Fromm, A. A. Ashkarran, D. J. de Aberasturi, I. R. de Larramendi, T. Rojo, V. Serpooshan, W. J. Parak, M. Mahmoudi, Trends Biotechnol. 2012, 30, 499. [36] S. Ninganagouda, V. Rathod, D. Singh, J. Hiremath, A. K. Singh, J. Mathew, M. ul-Haq, Biomed. Res. Int. 2014, 753419. [37] I. Bajpai, K. Balani, B. Bikramjit, J. Biomed. Mater. Res. Part B 2014, 102, 524. [38] K. Soo-Hwan, L. Hyeong-Seon, D.-S. Ryu, S.-J. Choi, L. Dong-seok, Korean J. Microbiol. Biotechnol. 2011, 39, 77. [39] W. K. Jung, H. C. Koo, K. W. Kim, S. Shin, S. H. Kim, Y. H. Park, Appl. Environ. Microbiol. 2008, 74, 2171. [40] R. E. Parchment, M. Gordon, C. K. Grieshaber, C. Sessa, D. Volpe, M. Ghielmini, Ann. Oncol. 1998, 9, 357. [41] Y.-J. Lee, J. Kim, J. Oh, S. Bae, S. Lee, I. S. Hong, S.-H. Kim, Environ. Toxicol. Chem. 2012, 31, 155. [42] K. R. Kirsch, S. Sirsat, J. Neal, S. Kline, B. Almanza, O. Stroia “A Microbial Analysis of Environmental Surfaces in Hotel Rooms” presented at 112th General Meeting of the American Society for Microbiology, San Francisco, California, June 16 – 19, 2012. [43] SMART, SAINT and SADABS, Bruker AXS, Inc., Madison, WI, 1997. [44] SHELXTL-NT Crystal Structure Analysis Package, Version 5.1, Bruker AXS Inc., Madison, WI, 1999. [45] A. Pal, S. Shah, S. Devi, Mater. Chem. Phys. 2009, 114, 530. [46] Organisation for Economic Co-operation and Development, 2004. Guideline for testing of chemicals 202. Daphnia sp., Acute Immobilisation Test. Paris, France. [47] ISO 6341:2012 Water quality “Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea)” Acute toxicity test.

Received: August 11, 2014 Published online on && &&, 0000 Please note: Minor changes have been made to this manuscript since its publication in Chemistry—A European Journal Early View. The Editor.

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Touch of (cell) death: Novel silver(I) acylpyrazolonato compounds were embedded on polyethylene matrix and the antimicrobial activity of the composite materials was tested against E. coli, P. Aeruginosa, and S. aureus (see figure). The composites show efficient inhibition of bacteria growth on the contact surface, exceeding 90 % reduction of bacteria within a few hours of exposure. The materials containing silver(I) coordination polymers as additives exert their antimicrobial action by simple contact, without release of the silver ions.

F. Marchetti,* J. Palmucci, C. Pettinari,* R. Pettinari, F. Condello, S. Ferraro, M. Marangoni, A. Crispini, S. Scuri, I. Grappasonni, M. Cocchioni, M. Nabissi, M. R. Chierotti, R. Gobetto* && – && Novel Composite Plastics Containing Silver(I) Acylpyrazolonato Additives Display Potent Antimicrobial Activity by Contact

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