Modulating Antimicrobial Activity by Synthesis: Dendritic Copolymers

Jan 31, 2012 - School of Chemistry, Cardiff University, Main Building, Park Place, ... in both polymer architecture and film morphology compared with ...
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Modulating Antimicrobial Activity by Synthesis: Dendritic Copolymers Based on Nonquaternized 2-(Dimethylamino)ethyl Methacrylate by Cu-Mediated ATRP Giovanni Vigliotta,† Massimo Mella,§,|| Damiano Rega,† and Lorella Izzo*,†,‡ †

Dipartimento di Chimica e Biologia, Università degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano Fisciano Salerno, Italy NANOMATES, Research Centre for NANOMAterials and nanoTEchnology, via Ponte Don Melillo, 84084 Fisciano Salerno, Italy § Dipartimento di Scienze Chimiche ed Ambientali, Università degli Studi dell’Insubria, via Lucini 3, I-22100 Como, Italy || School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom ‡

S Supporting Information *

ABSTRACT: The synthesis of novel star-like heteroarms polymers A(BC)n containing m-PEG (block A), methylmethacrylate (MMA), and nonquaternized 2-(dimethylamino)ethyl methacrylate (DMAEMA) (blocks BC) is here reported. We demonstrated that copolymer films with comparable amounts of DMAEMA have antimicrobial properties strongly depending on the topological structure (i.e., the number of arms) of the composing copolymers. We interpret the highest antimicrobial activity of A(BC)2 with respect to A(BC)4 and linear copolymers (respectively, A(BC)2 ≥ A(BC)4 > A(BC)) as probably due to the formation of strong hydrogen bonds between close amino-ammonium groups in the A(BC)2 film. Strong hydrogen bonds seem to be somewhat disfavored in the case of the linear species by the difference in both polymer architecture and film morphology compared with the A(BC)2 and A(BC)4 architectures.

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INTRODUCTION The last two decades have seen increasing interest in the development of antimicrobial polymers to prevent contamination and colonization of materials used in food packaging, textiles, water treatment, and for biomedical application (e.g., surface coatings of medical devices).1−3 Unfortunately, polymers able to release antimicrobial agents into the local environment naturally loose their capacity to kill bacteria over time.4−6 For this reason, nonleaching antimicrobial materials, that is, with the antimicrobial agent permanently fixed to the surface through covalent bonding, are undoubtedly an attractive alternative strategy. Poly(hexamethylene) biguanides have been known to possess antimicrobial activity since the 1980s,7 and many other polycations display antimicrobial activity in solution.1 However, antimicrobial properties of polycations are strongly reduced when they are cross-linked or insolubilized, probably due to the diminished accessibility of the cationic groups.8,9 Knowing that a star-like architecture is capable of substantially modifying the physical properties even of a homogeneous polymer,10−13 we wondered if a branched polymer structure may be able to overcome the problems connected with cross-linking by modulating inter- and intrachain interactions and thus improving the accessibility of cationic pedant groups. In this idea, we were supported by theoretical results indicating that topologically distinct heteroarms polymers in good and selective solvents14 may © 2012 American Chemical Society

change their aggregation mechanism (and thus the chain organization in a film) upon varying the number and length of the arms, a fact that could be exploited to modulate the exposition of cationic groups (vide supra) with respect to the local environment, thus conserving the antibacterial properties of the pendants. As for the latter, properly quaternized 2(dimethylamino)ethyl methacrylate (DMAEMA) is often used to introduce positive charges in the polymer backbone.15−19 However, we reckoned that quaternization may not indeed be necessary because a locally humid or wet environment may suffice in inducing the formation of charges thanks to the basicity of pendant amino-groups.20,21 Notice that the close proximity of DMAEMA monomers may strengthen the basicity of an amino pendant as it may allow the formation of a salt bridge with vicinal ones. To test these hypotheses, we obtained branched heteroarms polymers insoluble in water and containing nonquaternized DMAEMA, from which films were prepared. The antimicrobial properties were then evaluated using Escherichia coli, Pseudomonas sp., Dechlorosoma sp., and Staphylococcus aureus bacteria and rationalized on the basis of the polymeric architecture. Received: December 5, 2011 Revised: January 27, 2012 Published: January 31, 2012 833

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(−N(CH3)2), 52.0 (−OCH3, MMA), 54.5 (CH2 main chain), 57.2 (−O−CH2−CH2−N(CH3)2), 63.2 (−O−CH2−CH2−N(CH3)2), 70.7 (−OCH2CH2−), 176.3−178.2 (−CO). The molar mass of copolymers has been determined by the degree of polymerization (DP) as evaluated from 13C NMR, by the molar mass of monomers MM, and by the signal intensities I: Mn = DPm‑PEG (MMEO) + DPMMA(MMMMA) + DPDMAEMA(MMDMAEMA), where DPMMA = DPm‑PEG (2Ic/Ia) and DPDMAEMA = DPm‑PEG (Ih/Ia). The polydispersity index (Mw/Mn) has been evaluated by GPC. Synthesis of Low-Molecular-Weight m-PEG[G-1]-(PMMA-ranPDMAEMA)2 Copolymer. The synthesis was performed in toluene at 90 °C as reported above. The reaction was carried out in a 100 mL glass flask charged, under nitrogen atmosphere, with 0.1 g of m-PEG[G1]-Br2 macroinitiator in 15 mL of dry toluene. After the dissolution of the macroinitiator, CuBr, bpy, 2.5 mL of MMA, and 1.2 mL of DMAEMA were added (molar ratio m-PEG-[G-1]-Br2/CuBr/bpy = 1/4/8). The mixture was thermostatted at 90 °C and magnetically stirred. The reaction was stopped with n-hexane after 4 h. The copolymer was recovered, dissolved in the minimum amount of chloroform, and passed over a column of activated Al2O3 to remove the catalyst. The solution was dried in vacuum, and the polymer was washed with cold methanol and then dried. 1H NMR (400 MHz, CDCl3): δ 0.83−1.10 (CH3 main chain), 1.79−1.87(CH2 main chain), 2.27 (−N(CH 3 ) 2 ), 2.56 (−O−CH 2 −CH 2 −N(CH 3 ) 2 ), 3.57 (−OCH3), 3.61(−OCH2CH2−), 4.06 (−O−CH2−CH2−N(CH3)2). 13 C NMR (400 MHz, CDCl3): δ 16.6−18.9 CH3 main chain), 29.9 (−COC(CH3)2−), 44.9 (quaternary carbon in the main chain), 46.0 (−N(CH3)2), 52.0 (−OCH3, MMA), 54.5 (CH2 main chain), 57.2 (−O−CH2−CH2−N(CH3)2), 59.3 (CH3O−, m-PEG chain), 63.2 (−O−CH2−CH2−N(CH3)2), 70.7 (−OCH2CH2−), 176.3−178.2 (−CO). The molar mass and the polydispersity index were evaluated as reported above. Mn = 4500 Da. Mw/Mn = 1.4. NMR Analysis. Spectra were recorded on a Bruker Avance 400 MHz spectrometer at 25 °C with D1 = 3 s. The samples were prepared by introducing 20 mg of sample in 0.5 mL of CDCl3 into a tube (0.5 mm outer diameter). TMS was used as internal reference. Differential Scanning Calorimetry (DSC). DSC measurements were carried out on powders samples with a mass ranging between 5 and 7 mg. The tests were carried out by means of a DTA Mettler Toledo (DSC 30) under a nitrogen atmosphere. The range of temperature investigated was −50 to +200 °C with a heating rate of 10 °C/min. Gel Permeation Chromatography (GPC). GPC chromatograms were recorded on a system equipped with a Waters 1525 binary pump, a Waters 2414 RI detector and four styragel columns (range 103 to 106 Å). The measurements were carried out at 25 °C, using THF as eluent (1.0 mL min−1) and narrow dispersity polystyrene standards as references. UV−vis Measurements. To evaluate the amount of sorbed and released benzoate from the polymeric films, UV−vis measurements of the aqueous solution were recorded on a Perkin-Elmer Lambda EZ201 instrument, using PESSW 1.2 Revision E software, in the range 800− 200 nm with 1 cm quartz cells. Microbiological Assays. To evaluate the antimicrobial effects of different polymers, we preincubated E. coli, Pseudomonas sp., and Dechlorosoma sp. in Luria−Bertani (LB) medium (10 g L−1 trypton, 5 g L−1 yeast extract, 10 g L−1 NaCl) and grown for 12 h in aerobiosis at 250 rpm. E. coli were incubated at 37 °C, whereas the other strains were incubated at 32 °C. Subsequently, bacteria were collected by centrifugation for 10 min at 3500g, resuspended at concentration of 0.005 OD600 mL−1 in distilled, sterile water in presence of 1 cm2 of polymeric films, each cut into four equal parts, and incubated in aerobiosis to the corresponding growth temperature at 250 rpm. A control with bacteria without film was also carried out. For cell survival determination at different time, 100 μL of suspension were spread on LB agar dishes (15 g L−1 agar) and incubated for 24 h, and colony forming units (CFUs) were calculated. Microbiological assays for polymer diffusion were performed as already reported.23 E. coli or Staphyococcus aureus were preincubated

EXPERIMENTAL SECTION

Materials. Poly(ethylene glycol) monomethylether (m-PEG) (Mn = 2000 Da, PDI = 1.16), benzaldehyde dimethyl acetal, 2,2bis(hydroxymethyl)propionic acid, p-toluenesulfonic acid monohydrate (TsOH), acetone, N,N′-dicyclohexylcarbodiimide (DCC), 4(dimethylamino)pyridine (DMAP), methanol, Pd/C 10%, 2-bromoisobutyryl bromide (BMPB), triethylamine (TEA), diethyl ether, ethanol, CuBr, 2,2′-bipyridine (bpy), chloroform, and Al2O3 were purchased from Aldrich and used without any further purification. All manipulations involving air-sensitive compounds were carried out under nitrogen atmosphere using Schlenk or drybox techniques. Toluene (Aldrich) was dried over sodium and distilled before use. CH 2 Cl 2 (Carlo Erba), methylmethacrylate (MMA) and 2(dimethylamino)ethyl methacrylate (DMAEMA) (Aldrich) were dried over CaH2 and then distilled, the latter under a reduced pressure of nitrogen. Escherichia coli (strain JM109) was purchased from Promega (http://www.promega.com/products; cat. no. P 9751). Pseudomonas sp., Dechlorosoma sp. and Staphylococcus aureus strains were derived from the collection deposited in the microbiology laboratory directed by G. Vigliotta. Preparation of m-PEG-Br Linear, m-PEG-[G-1]-Br2, and mPEG-[G-2]-Br4 Macroinitiators. m-PEG-Br linear macroinitiator was synthesized according to the literature procedure.10 For the preparation of m-PEG-[G-1]-Br2 and m-PEG-[G-2]-Br4 macroinitiators, first m-PEG-[G-1]−OH2 and m-PEG-[G-2]−OH4 were synthesized according to the literature procedure and characterized by 1H NMR.22 In a typical procedure, m-PEG-[G-1]− OH2 (1.25 g, 0.59 mmol; 1 equiv) was dissolved, under nitrogen atmosphere, in 15 mL of dry CH2Cl2 into a 100 mL two neck roundbottomed flask equipped with condenser, dropping funnel, and magnetic stirrer. Then, 0.22 g of DMAP (1.77 mmol) and 0.12 mL of TEA (0.88 mmol) were added, and the reactor was thermostatted at 0 °C. After cooling, 0.36 mL of BMPB (2.95 mmol; 5 equiv) in 5 mL of dry CH2Cl2 was added dropwise during 1 h. Subsequently the temperature was allowed to raise room temperature, and the reaction was continued under stirring for further 24 h. The solution was filtered, and the product was precipitated in cold diethyl ether, filtered, washed with cold ethanol, and dried in vacuum.10 m-PEG-[G-1]Br2(Yield: 66%). 1H NMR (400 MHz, CDCl3): δ 1.91 (s, CO(CH3)2Br); 3.70−3.91 (bs, −CH2−, m-PEG). m-PEG-[G-2]-Br4 (Yield: 63%). 1H NMR (400 MHz, CDCl3): δ 1.91 (s, CO(CH3)2Br); 3.70−3.91 (bs, −CH2−, m-PEG). Preparation of m-PEG-(PMMA-ran-PDMAEMA) Linear, mPEG[G-1]-(PMMA-ran-PDMAEMA)2, and m-PEG-[G-2]-(PMMAran-PDMAEMA)4 Copolymers by ATRP. m-PEG-(PMMA-ranPDMAEMA) linear copolymer was synthesized in toluene at 90 °C. The reaction was carried out in a 100 mL glass flask charged, under nitrogen atmosphere, with 0.1 g of m-PEG-Br linear macroinitiator in 15 mL of dry toluene. After the dissolution of the macroinitiator, 0.013 g of CuBr, 0.03 g of bpy, 5 mL of MMA, and 2.5 mL of DMAEMA were added. The mixture was thermostatted at 90 °C and magnetically stirred. The reaction was stopped with n-hexane after 18 h. The copolymer was recovered, dissolved in the minimum amount of chloroform, and passed over a column of activated Al2O3 to remove the catalyst. The solution was dried in vacuum and the polymer was washed with cold methanol and then dried. m-PEG[G-1]-(PMMA-ran-PDMAEMA) 2 and m-PEG-[G-2](PMMA-ran-PDMAEMA)4 copolymers were synthesized using a molar ratio m-PEG-[G-1]-Br2/CuBr/bpy = 1/4/8 and m-PEG-[G1]-Br4/CuBr/bpy = 1/8/16, respectively. m-PEG[G-1]-(PMMA-ran-PDMAEMA)2 with 8 and 20% of DMAEMA have been synthesized following the above-reported procedure using a molar ratio m-PEG-[G-1]-Br2/CuBr/bpy = 1/4/8 and 0.5 and 1.0 mL of DMAEMA, respectively. 1 H NMR (400 MHz, CDCl3): δ 0.83−1.10(CH3 main chain), 1.79−1.87 (CH2 main chain), 2.27 (−N(CH3)2), 2.56 (−O−CH2− CH2−N(CH3)2), 3.57 (−OCH3), 3.61(−OCH2CH2−), 4.06 (−O− CH2−CH2−N(CH3)2). 13C NMR (400 MHz, CDCl3): δ 16.6−18.9 (CH3 main chain), 44.9 (quaternary carbon in the main chain), 46.0 834

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for 12 h as above-described in LB medium at 37 °C. We put 0.1 OD600 of each strain in 4 mL of autoclaved LB agar precooled to 42 °C and poured in 90 mm diameter Petri dishes. Films of A(BC)2 were lain down the agar surface, avoiding the formation of air bubbles, and plates were incubated at 37 °C for 24 h. Pieces of LB agar containing 70 μg mL−1 ampicillin were used as control. To evaluate the role of bacterial proteins in polymer interaction, the same procedure reported above and employed for polymers diffusion analysis was used, except that films were presaturated in the presence of an excess of aspecific proteins by incubating for 4 h at room temperature in TTBS, 5% milk (10 mM Tris, pH 7.4, 0.9% NaCl, 0.01% Tween-20, 5% non fat dry milk from Biorad) with constant shaking. After incubation, films were washed in TTBS without milk before they were laid in the agar surface. A control to evaluate toxicity of milk solution was also introduced by stratifying on the agar surface 10 μL of TBS-tween. For experiments with conditioned supernatant, A(BC)2 film was incubated in distilled water with 0.005 OD600 mL−1 E. coli for 24 h. Successively, residual bacteria were removed from water by centrifuging at 3000g 5 min at 4 °C. To verify the presence of soluble molecules released from previous incubation, we inoculated conditioned supernatant with 0.005 OD600 mL−1 E. coli from a fresh actively growing culture in the absence of film and incubated for 24 h at 37 °C. Antimicrobial effect was evaluated by CFUs determination as reported above. Theoretical Modeling. The free-energy change associated with the formation of a strong hydrogen bond between protonated and neutral DMAEMA pendants is estimated using ab initio calculations at the MP2/6-311++G(d,p)//MP2/6-31+G(d) level with the self-consistent reaction field/polarizable continuum model approach in Gaussian 09.24 To limit costs and the bias due to the implicit solvent, we used in our model protonated and unprotonated trimethyl amine, representing DMAEMA pendants, and two water molecules. The latter partially accounts for possible hydrogen bonds between the solutes and the aqueous solvent (Supporting Information) and allows one to estimate the dimerization Gibbs energy change using: ΔGdim = G(protonated amine dimer) + G(water dimer) − G(ammonium-water) − G(aminewater). To model the effects due to copolymer composition (i.e., molar fraction X of DMAEMA vs MMA), length of the BC branches, and polymer topology, we considered as a first approximation the BC branches to be linearly stretched and running parallel to each other in A(BC)n species. Chains are thus represented as ordered sequences of monomers. The probability for two DMAEMA monomers to be found as first and second intrachain neighbors was estimated for a chosen BC composition and length (i.e., number of monomers N) by randomly sampling with no repetition (i.e., overlap between DMAEMA monomers) the positions of NX DMAEMA/(X DMAEMA + XMMA) DMAEMA monomers (C) in a chain containing N monomers. We generated 10 000 chains, and the number of first and second neighbors was accumulated as an indication of the number of different ways a strong hydrogen bond could be formed along a chain. To compare such results with the chances of interchain contacts in multibranched species, we sampled two chains of length N/2 using a similar approach as the single chain one. In this case, possible interchain contacts are represented by zeroth (monomers occupying the same ordered position), first, and second neighbors belonging to the other chain. (See Figure 6B for a schematic representation.) In the calculation discussed in the main text, we employed N = 800, which is roughly the total amount of DMAEMA and MMA monomers present in the discussed polymers. Further details of the theoretical modeling are provided in the Supporting Information.

DMAEMA and MMA. The latter reduces hydrophilicity and provides a spacer between the DMAEMA monomers along the polymer backbone. (Figure 1).

Figure 1. Architecture of A(BC)n copolymers [m-PEG(PMMA-ranPDMAEMA)n].

The architectures were synthesized by methods we previously described for analogous star-like structures,10 using the ATRP of MMA and DMAEMA in the presence of Cu(I)Br/bipyridine and a macroinitiator based on PEGmonomethylether (m-PEG, Mn = 2000 Da, polydispersity index Mw/Mn = 1.16) properly modified to have different degree of branching at the terminal chain. 22 A(BC) n copolymers with n = 2 and 4, a molar amount of DMAEMA = 34%, and molecular masses in the range 80−90 kDa were synthesized. An analogous linear copolymer with a similar composition and molecular mass was obtained for comparison purpose. The characteristics of copolymers are reported in Table 1. The chemical composition was determined by NMR characterization (1H, 13C, and HSQC-NMR), and the molar fraction of DMAEMA was calculated by the signal intensities I of full-assigned 13C NMR spectra (Figure 2A) using the following equation:

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XDMAEMA = (Ih /2) ·[(Ia /2) + Ic + (Ih /2)]−1

RESULTS AND DISCUSSION We designed A(BC)n architectures where “A” is a PEG chain used to inhibit protein adhesion (thus avoiding possible bacterial proliferation) and to produce a hydrophilic environment, and “BC” is a random chain copolymer based on

(1)

The presence of copolymeric structures was confirmed by GPC of the final products that gave monomodal curves (Mw/ Mn = 1.4 to 1.5) with molar masses higher than the ones of the starting macroinitiators (Figure 2B). 835

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Table 1

a

architecture

Mn (kDa)a

Mw/Mnb

XDMAEMAc

XMMA

XPEG

Tg (°C) (dry sample)

Tg (°C) (wet sample)d

H2O(l) uptake (%)

A(BC) A(BC)2 A(BC)4

80 82 90

1.3 1.4 1.6

0.30 0.34 0.34

0.62 0.58 0.60

0.08 0.08 0.06

84 89 84

101 90 106

1.9 9.6 2.9

Calculated from 13C NMR spectra. (See the Experimental Section.) bEvaluated from GPC. cCalculated from 13C NMR spectra using eq 1. Evaluated after immersion of films in liquid water for 24 h using the equation: [(wtwet sample − wtdry sample)/wtdry sample]·100.

d

Figure 2. 1H, 13C, and HSQC NMR spectra (CDCl3, 25 °C) (A) and GPC (B) of the A(BC)2 structure.

It was found that the number of colonies decreased with respect to the control over the time, indicating that all polymers were acting as bactericidal agents. In fact, all bacteria were completely killed by the three morphologically different polymers over time (e.g., 24−48 h). However, a different effectiveness in the antimicrobial effects is clearly evidenced within the first 5 h (Figure 4). In particular, the A(BC)2 architecture displayed the most important antimicrobial activity after 5 h, with 99.98% ± 0.01 of bacteria killed over total population. At this time, also the most branched structure exhibited important antimicrobial properties, only very slightly in defect with respect to the A(BC)2 architecture. In fact, 99.76% ± 0.015 of bacteria were killed by A(BC)4, which means a number of survived bacteria of 22.0 × 103 ± 2.0 × 103 CFUs/mL for A(BC)4 versus 1.8 × 103 ± 1.0 × 103 CFUs/mL for A(BC)2. The linear copolymer was much less effective, killing only 45.15% ± 2.15 of bacteria after 5 h. The same experiments were repeated using a Pseudomonas strain, and comparable results were obtained; in fact after 5 h the A(BC)2

To prove the control of the synthetic approach over the architecture and so the formation of copolymers with branched structures, a low-molecular-mass copolymer A(BC)2 (Mn = 4500 Da, Mw/Mn = 1.4) was synthesized to make visible the termini chain by 13C NMR. As matter of fact, in addition to the resonances of the carbons main chain, the NMR spectrum showed minor resonances attributable to the end groups and to the carbon on the junctions (Figure 3). The intensity of the signals of the methoxyl end group on the m-PEG portion (δ = 59.2, CH3O−) and the methyl groups on the junctions (δ = 29.9, −COC(CH3)2−) were 1:4 molar ratio, hence definitely confirming the presence of two PMMA-ran-PDMAEMA blocks on the m-PEG chain. To determine antimicrobial activity, we prepared films of thickness ∼120 μm from linear, A(BC) 2 and A(BC) 4 copolymers by casting from chloroform (Table 1). The films, all with surface area of ∼1 cm2, were incubated with E. coli in distilled water; bacteria were harvested at different times (0 min, 15 min, 5 h, and 24 h), plated on a rich medium, and incubated for 24 h. Subsequently, the CFUs were determined. 836

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Dechlorosoma (syn. Azospira) genus.25 In this case, the A(BC)2 film displayed a slightly lower bactericidal activity (95.30% ± 0.4 after 5 h), whereas the relative activity relationship with the architectures was similar to that observed for E. coli and Pseudomonas, albeit less marked. The total absence of antimicrobial activity for the A(B)2 copolymer with no DMAEMA in the backbone clearly suggests the bactericidal properties to be due to the dimethyl-amino groups introduced via DMAEMA, which can naturally form an ammonium cation when moist (pKb∼3.1). Furthermore, considering that the amount of DMAEMA in the three films was comparable, one can state that the different antimicrobial properties presented by the films must be strictly related to the polymeric architecture. Having evidenced a stronger antimicrobial power for A(BC)2 (albeit only slightly compared to A(BC)4), it is reasonable to presume that the differences here reported are determined by the amount of formed cationic groups accessible to bacteria and that this feature is modulated by the copolymer architecture. Such hypothesis was studied indirectly by loading the polymeric films with the benzoate anion from a 2 ppm aqueous solution (Figure 5). From Figure 5 clearly emerges the fact that A(BC)2 is both more efficient and effective (1.2 ± 0.06 ppm) in sorbing the anion than A(BC)4 and the linear copolymer (0.4 ± 0.04 ppm and 0.3 ppm respectively) so as to justify the similar behavior of the two copolymers as bactericidal materials. Furthermore, the amount quickly desorbed by A(BC)4 seems to be compatible with the amount sorbed only on the surface; the remaining benzoate is extracted quantitatively only after doubling the ionic force of the surrounding solution. A different attitude of the materials to form inter- or intrachains strong (−N(CH3)2−H+N(CH3)2−) hydrogen bonds20,21 could explain the different amount of positive charges of the three copolymers, as highlighted by the experiments with benzoate and the different bactericidal power (particularly concerning the linear architecture). Notice that it is indeed possible to provide a theoretical estimate for the Gibbs energy change associated with the formation of such a strong hydrogen bond, and this is roughly −6 kcal mol−1(Figure 6A); such a datum clearly supports the hypothesis of the stable dimeric nature for the charge bearing moieties (Figure 6) when vicinal (i.e., sufficiently close so that conformations bringing two dimethyl amino groups at the appropriate distance are available) DMAEMA groups are indeed present. In reality, the ΔG we provide is likely to be somewhat underestimated due to the fact that we considered both the ammonium and amine as free species (Supporting Information), thus overestimating the decrease in entropy upon dimerization. In this respect, we notice that it is also possible to calculate roughly how many DMAEMA monomers may be sitting at a distance sufficiently short to allow them forming protonated diamino groups. Assuming initially that BC arms are fully extended (see the Theoretical Modeling section for a more complete discussion), one notices that, in a linear species, only DMAEMA groups that are first or second neighbors can form (−N(CH3)2−H+N(CH3)2−) due to the somewhat limited conformational freedom afforded by the two-carbon length of the pendant groups. When it comes to form intrachain protonated diamino groups, the same applies also to the arms of a branched species. The latter, however, has also the possibility of forming interchain protonated dimers within the same macromolecules due to the vicinity of the BC arms in the region close to the junction. For linearly stretched parallel chains in A(BC)2 containing an identical number of MMA (B) and DMAEMA (C) monomers as a reference linear copolymer, we estimated that the possible interchain “contacts” (Figure 6B) between two amino groups is roughly 2.5 times higher than

Figure 6. (A) Schematic representation of the dimerization process employed to estimate the stabilization of a protonated trialkyl ammonium ion due to a neutral trialkyl amine pendant in the MMA-DMAEMA arms. SCRF/PCM refers to the implicit solvent model employed in the calculations. The process represented is the desolvation of a protonated amino group and a vicinal free one to form a protonated diamino species with a much lower free energy, a fact that may increase the local basicity constant of the dimethyls amino pendants. (B) Definition of intra- and interchain contacts between DMAEMA (C) groups for randomly sample model linear chains. Geometrically accessible contacts in linearly stretched chains are first and second intrachain neighbors and zeroth, first, and second interchain neighbors.

the intrachain ones when a relative population C/(B + C) = 1/ 3 (i.e., roughly the fractional amount of DMAEMA in the polymers reported in Table 1) is used and the location of the two monomer along each chain is randomly sampled as to assume random insertion frequency. Albeit our estimate of vicinal DMAEMA groups may be modified when the flexibility of BC arms is taken into account, our results nevertheless provide a robust indication of the role played by the polymer topology. Besides, we would expect chain flexibility to play similar roles in both branched and linear copolymers, thus supporting that our simplified analysis indeed gives indications about the existence of an actual topological effect. Given the data discussed above, we thought that it could be of interest to investigate the way the amount of DMAEMA, and consequently of positive charges, affect the antimicrobial properties of films. In this respect, we synthesized the A(BC)2 structure with different percentages of DMAEMA (Table 2) and incubated films of ∼1 cm2 in the presence of E. coli for 5 h. We opted to run this test on the A(BC)2 architecture due to both its high bactericidal activity and sorption characteristics, two features that appear to be promising for practical applications. The copolymer with 34% in mol of DMAEMA showed the highest antimicrobial activity, meaning 99.98% ± 0.01 of bacteria killed with respect to the control in 5 h. However it was only slightly higher than copolymer characterized by 20% of DMAEMA, which was able to kill the 99.51% ± 0.18 of bacteria (that means a number of 11.0 × 103 ± 4.0 × 103 838

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Table 2

a

architecture

Mn (kDa)a

Mw/Mnb

XDMAEMAc

XMMAc

XPEGc

Tg (°C) (dry sample)

Tg (°C) (wet sample)d

H2O(l) uptake (%)

A(BC)2 A(BC)2 A(BC)2

71 78 82

1.6 1.6 1.4

0.08 0.20 0.34

0.84 0.72 0.58

0.08 0.08 0.08

85 80 89

83 79 90

1.6 3.7 9.6

Calculated from 13C NMR spectra. (See the Experimental Section.) bEvaluated from GPC. cCalculated from 13C NMR spectra using eq 1. Evaluated after immersion of films in liquid water for 24 h using the equation: [(wtwet sample − wtdry sample)/wtdry sample]·100.

d

CFUs/mL of survived bacteria versus 1.8 × 103 ± 1.0 × 103 CFUs/mL of the copolymer with 34% of DMAEMA). On the contrary, the copolymer containing just 8% of DMAEMA showed a marked lower activity (Figure 7) (75.6% ± 2.7); it

from the film surface to the environment. Having established that the highest activity was shown by the two arms copolymer with 34% of DMAEMA, to investigate the presence of such a phenomenon, films of A(BC)2 and an agar disk with ampicillin as control for leaching were placed in a nutrient broth containing E. coli. After 24 h of incubation at 37 °C, the agar region underneath the surface of the films did not show any bacterial growth (Figure 8.2A). More interesting, any area of

Figure 7. Antimicrobial activity of A(BC)2 copolymers with variable amount of DMAEMA.

nevertheless remained far more effective than the linear copolymer despite the roughly four-fold decrease in DMAEMA. It thus seems that within the same architecture the antimicrobial action is strongly influenced by the amount of DMAEMA in the range 8−20%, whereas for higher amounts it become less sensitive. Despite this dependency, it also appears that the copolymer topology plays a key role in defining the bactericidal power, even more important than the total amount of ionizable groups introduced during the synthesis. In this respect, our simplified model assuming linear and parallel chains seems to provide an accurate account for the limited decrease in antimicrobial power on going from 34 to 20% of DMAEMA and the sudden drop evidenced at 8%. In fact, the number of possible “interchain contacts” decreases by a factor of only 2 on going from 34 to 20%, whereas it is one order of magnitude smaller in the A(BC)2 species containing only 8% DMAEMA. It is also worth noting that we found differences in the water uptake of each sample (Table 2), which is clearly modulated by the percentage of DMAEMA. Whether this is due to the number of ionized groups in water or to some change in film morphology as a function of the amount of diamino groups is not clear at the moment. With respect to the mechanism of action, it is often reported that soluble cationic antimicrobials act on the bacterial cells in two steps: first, they are adsorbed onto the cell surface and diffuse through the cell wall successively binding to the cytoplasmic membrane; finally, once bonded to the membrane, they disrupt the latter, causing cell death.17,26−28 Considering that the cationic pedant groups of DMAEMA are not long enough to completely penetrate the cell wall (5−20 nm), one could argue that the antimicrobial activity of copolymers reported here is determined by leaching of polymeric blocks

Figure 8. Microbiological assay of polymer diffusion. A piece of agarized LB medium containing ampicillin used as positive control (indicated with C) and A(BC)2 films with 34% of DMAEMA (indicated with f) were tested with E. coli (A) and Staphylococcus aureus (B). In all of the pictures, the diffusion of ampicillin in the environment is clear from the circular halo indicating growth inhibition. The absence of growth inhibition in the immediate proximity of the films (f) and, on the contrary, the presence of growth inhibition in the agar surface evidenced by the removal of films (* in panels 2A and 4B), indicate, respectively, the absence of diffusion and the antimicrobial effect by contact.

growth inhibition in the immediate proximity of the films was not visible, this area being evident in the case of agar containing ampicillin (Figure 8.1A). To verify that the absence of bacterial growth in the areas of inhibition evidenced by the removal of the film (areas indicated in Figure 8 with the symbol *) was not due to a slower growth induced by anoxic conditions (the latter may occur with facoltative anaerobic microorganism such as the bacteria tested here), the Petri dishes in Figure 8, panel 2A, were further incubated aerobically at 37 °C for 3 days to facilitate the growth in those areas. Despite the additional incubation time, no growth was evidenced; the latter finding thus indicates that most of bacteria were killed by contact with films. It is also important to point out that the same film has been used several times (at least 20 times), showing an 839

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Pseudomonas sp.), copolymers like m-PEG(PMMA-ranPDMAEMA)2 displayed very high antimicrobial activity after just 5 h of exposure, a property that was only very weakly reduced for the corresponding four arm species, whereas it became substantially less for the linear copolymer. The A(BC)2 structure was also found to be bactericidal for Gram positive bacteria (i.e., Staphylococcus aureus). The data of water uptake by the three polymer topologies suggested that the latter plays a role in defining not only the bactericidal properties but also somewhat the film morphologies (e.g., number and volumes of cavities or channels). This conclusion, as well as the idea that more active polymers have a higher density of surface charges, was indirectly confirmed by the measurement of interaction between polymeric films and benzoate and from biological results performed on A(BC)2 films containing a different amount of cationic groups. A semiquantitative explanation of the dependency of the antimicrobial properties on the topology and DMAEMA fraction has also been proposed on the basis of simplified polymer models that accounts for the change in the number of possible protonated diamino groups as a function of the number of branches and monomer composition. Finally, it is worth noting that all films have been used several times, showing an unchanged antimicrobial activity and potential application as bactericidal, recyclable materials. More in general, this study highlights the way the organization and topology of polymeric chains play a fundamental role in the design of novel antimicrobial material acting just by contact. Further work is in progress in our laboratories to obtain a more detailed understanding of the dependency of material properties on the synthetic variables and film preparation as well as a better picture of their mode of action.

unchanged antimicrobial power and consequently the absence of surface wear or passivation. In addition to this evidence, we also incubated an A(BC)2 film in distilled water with E. coli for 24 h, collected the water after having removed the bacteria by means of centrifugation, and inoculated the conditional supernatant with E. coli collected from a living culture grew in absence of films. After incubation at 37 °C for 24 h, the latter bacteria were harvested for the CFUs determination, which, indicating no significant variations compared with the control, thus confirmed the absence of diffusible molecules involved in the antimicrobial action of polymers. All of these results clearly indicate that bacteria are killed by contact. The observation thus implies that the cationic groups exert their action by local interaction on the superficial layers of the bacterial envelope (e.g., by destroying the outer membrane of Gram negative). Such interactions are probably sufficient for a lethal action. It is, for instance, reported that the electrostatic interactions between positively charged polyelectrolytes and the negative charges of the bacterial cell wall can destabilize the outer membrane by allowing the leaching of divalent cations (Mg2+ and Ca2+) that neutralize the phosphate groups.29,30 This phenomenon implies the “adsorption” of bacteria on the charged matrices. Alternatively, the cationic groups might be responsible for interfering with the total charge of cell surface or with the ionic cellular transport. It cannot, however, be discounted a priori that polymer molecules on the film surface may be able to “stretch out” entire BC arms, whose length (roughly 35−130 nm depending on the polymer architecture) may be sufficient to reach the cytoplasmatic membrane and interfere with it, modifying its structure due to strong Coulomb interaction. With the goal of understanding if the charge-driven bactericidal effect may be modulated by the presence of different charge-compensating species such as proteins, we also investigated the possibility that the contact between films and bacteria could be hampered by host proteins. For this purpose, we measured toxic effects for films presaturated with an excess of aspecific proteins deriving from not fat milk by carrying out assays similar to those described in Figure 8. The results indicated that bactericidal effect was not modified by the presence of proteins and was always dependent on contact. So, these preliminary data evidenced a minor role of host proteins in the adhesion to a film; however, further tests need to be carried to shed some light on such possibility and to clarify the action mechanism of the polymers here reported. Finally, preliminary tests on antimicrobial properties of A(BC)2 copolymer with 34% of DMAEMA were conducted also on a group of Gram positive bacteria. In particular, because of its importance in medical and food contamination, we used Staphylococcus aureus. As for Gram negative bacteria reported above, bactericidal activity of A(BC)2 was very high, and once again the lack of diffusion of antimicrobial species (Figures 8, 3B, and 4B) suggested the need for contact between the polymeric surface and bacteria.

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ASSOCIATED CONTENT

S Supporting Information *

Details of the theoretical calculations and modeling. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Ruth Duncan and Dr. Niklaas J. Buurma for useful discussions and suggestions. This work was financially supported by MIUR (FARB 2010, 2011) and by the scheme “Rientro dei Cervelli”.

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REFERENCES

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CONCLUSIONS In conclusion, we have demonstrated the synergic role played in generating intrinsically active antimicrobial films by the copolymerization of nonquaternized DMAEMA into purposely tailored polymer architecture. Antimicrobial activity of these films can be explained by the direct interaction of films with bacteria. For Gram negative bacteria (i.e., E. coli and 840

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