Antibacterial Cellulose Fiber via RAFT Surface Graft Polymerization

Dec 8, 2007 - ... School of Chemistry, Eastern Ave., University of Sydney, NSW 2006, ... Emma Watson , Alexander M. Tatara , Dimitrios P. Kontoyiannis...
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Biomacromolecules 2008, 9, 91–99

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Antibacterial Cellulose Fiber via RAFT Surface Graft Polymerization Debashish Roy,† Jeremy S. Knapp,‡ James T. Guthrie,† and Sébastien Perrier*,†,§ Department of Colour and Polymer Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, U.K., and Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, U.K. Received August 2, 2007; Revised Manuscript Received October 18, 2007

2-(Dimethylamino)ethyl methacrylate (DMAEMA) was polymerized from cellulosic filter paper via reversible addition-fragmentation chain transfer (RAFT) polymerization. The tertiary amino groups of the grafted PDMAEMA chains were subsequently quaternized with alkyl bromides of different chain lengths (C8-C16) to provide a large concentration of quaternary ammonium groups on the cellulose surface. The antibacterial activity of the quaternized and nonquaternized PDMAEMA-grafted cellulosic fibers was tested against Escherichia coli. The antibacterial activity was found to depend on the alkyl chain length and on the degree of quaternization, i.e., the amount of quaternary amino groups present in the cellulose graft copolymers. The PDMAEMA-grafted cellulose fiber with the highest degree of quaternization and quaternized with the shortest alkyl chains was found to exhibit particularly high activity against E. coli.

Introduction In recent years, interest has grown in the preparation of materials with antibacterial properties, for use in a wide range of fields such as food packaging, sanitary materials, and household, medical, and military items.1,2 Antimicrobial surfaces can be obtained by incorporating antimicrobial species either through covalent bonding or via noncovalent (via leaching) interaction to the surface. One of the major disadvantages of the leaching approach is that the substrate progressively becomes ineffective and less durable. This is because the antimicrobial species are free to leave the surface and, thus, their effective concentration decreases. Several techniques have been reported that claim to have created a surface with permanent antimicrobial properties, including immobilizing antimicrobial polymers onto the surface via chemical reactions, polymerizing directly from the surface using monomers containing antimicrobial groups, or incorporating antimicrobial groups onto monomers after polymerization.1,3–7 The antimicrobial properties of these substrates are based on the contact between the biocidal active surface and microorganisms, without the release of active molecules.3 This lack of release of biocides also prevents contamination of the environment; indeed, surface-bonded antibacterial agents might be expected to cause fewer problems due to residual toxicity. Another advantage may be the longer lifetime of the antimicrobial substrate, as its activity has been shown, in some cases, to be regenerated by washing with appropriate solvents.4 These advantages have made the nonleaching approach the technique of choice to produce permanent, sterile-surface materials.5–7 The commonly used low molecular weight antibacterial agents are based on phenols, halogens (e.g., iodine), biguanides, heavy metals (e.g., silver, tin, and mercury), phosphonium salts, * Corresponding author: e-mail, [email protected]; tel, +61 2 9351 3366; fax, +61 2 9351 3329. † Department of Colour and Polymer Chemistry. ‡ Integrative and Comparative Biology. § Current address: Key Centre for Polymer Colloids, School of Chemistry, Eastern Ave., University of Sydney, NSW 2006, Australia.

and quaternary ammonium salts.2,8,9 Among these antibacterial agents, quaternary ammonium compounds (QACs) are the most widely used agents due to their good antibacterial properties, their low toxicity, their lack of skin irritation, their low corrositivity, their good environmental stability, and their excellent cell membrane penetration properties.10–17 The antibacterial activity of the QACs is strongly dependent on their overall molecular structure and the length of their alkyl chains.18,19 The general structure of QACs comprises a nitrogen atom of valency five. Four of the substituent radicals (R1-R4) are alkyl or heterocyclic radicals and the fifth (X-) is a small anion.8 The complex cations of the quaternary ammonium compounds form salts with chloride ions, bromide ions, and iodide ions. These associated anions do not play any part in the antibacterial action.20 For antibacterial activity, at least one of the R groups must have a chain length in the range C8-C18.21 This long alkyl chain provides a hydrophobic segment that is compatible with the lipid bilayer of the bacterial cytoplasmic membrane.22,23 The generally accepted mechanism for the antimicrobial action of such quaternary ammonium compounds can be described as (i) adsorption of positively charged QACs on the negatively charged cell surfaces of the microorganisms, by electrostatic interaction, (ii) diffusion though the cell wall, promoted by the long lipophilic alkyl chain, (iii) binding to the cytoplasmic membrane, (iv) disruption of the cytoplasmic membrane, and (v) the loss of cytoplasmic constituents, resulting in the death of the microorganisms.18,24 In comparison with low molecular weight QACs, cationic polymers with quaternary ammonium groups have some advantages. These include enhanced antibacterial activity, reduced residual toxicity, low tissue irritation tendencies to mammals, increased efficiency and selectivity, and prolonged lifetime.24,25 Since the polymeric cationic antibacterial compound carries a much higher charge density than that of the monomeric cations, the adsorption of polycations on to the negatively charged bacterial cell surfaces would be expected to take place to a greater extent. The binding of polymers to the cytoplasmic membranes is also expected to be facilitated by the polycations.

10.1021/bm700849j CCC: $40.75  2008 American Chemical Society Published on Web 12/08/2007

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Scheme 1. Synthesis of Quaternized Cellulose-g-PDMAEMA for Antibacterial Assessment

This effect is due to the presence of a large number of negatively charged species in the membrane. Thus, the disruption of the cytoplasmic membrane, resulting in the loss of cytoplasmic constituents and the death of the microorganisms, is enhanced.24,26–28 Several studies have been conducted in which the major objective has been to impart antibacterial properties to a variety of substrates such as siliceous surfaces, cotton fabrics, filter papers, glasses, polystyrene fibers, high-density polyethylenes, low-density polyethylenes, polypropylenes, nylons, poly(ethylene terephthalate) films, expanded polytetrafluoroethylene, and poly(vinylidene fluoride) membranes.4,10,19,29–37 However, in such studies, the antimicrobial surfaces were prepared via either free radical polymerization or coupling reactions. These methods cannot ensure control over the molecular weight and the molecular weight distribution. Also, the controlled topology of polymers, which is necessary for increased antimicrobial activity, has been studied. It has been reported that the biocidal activity of the polymeric quaternary ammonium compounds depends on their molecular weights.24 It is also highly desirable to introduce polymeric quaternary ammonium compounds onto the surface, with good control over the molecular weight and the molecular weight distribution of the polymer chains. Living radical polymerization (LRP) is, therefore, the technique of choice for the synthesis of well-defined antibacterial surfaces. Indeed, the preparation of antibacterial glass surfaces, filter papers, polymeric microspheres, poly(vinylidiene fluoride) membranes, low-density polyethylene (LDPE), and stainless steel surfaces, via atom transfer radical polymerization, has been reported in recent publications.1,3,38–40 A stainless steel surface with antibacterial properties was prepared by nitroxide-mediated radical polymerization.41 The commercial interest in cellulose fibers as antimicrobial substrates has increased over the past few years.42 In most cases, low molecular weight antibacterial agents were impregnated into the fiber; i.e., the antibacterial agents are not strongly chemical bonded, just physically deposited on the surface, thus leading to a short lifetime of the attributes of the resultant substrate. In this study, a tertiary amino group containing monomer such as 2-(dimethylamino)ethyl methacrylate (DMAEMA) is directly polymerized from the cellulose surface via the RAFT method.

The pendant amino groups are subsequently quaternized using alkyl bromides to achieve cellulose fiber with surface antibacterial properties. Scheme 1 shows the synthesis pathway to antibacterial cellulosic fibers via the RAFT process.

Experimental Section Materials. All solvents and monomer were purchased from Aldrich (Gillingham, Dorset, U.K.) at the highest purity available unless otherwise stated. 2-(Dimethylamino)ethyl methacrylate (DMAEMA) (98%) was filtered before use through an activated basic alumina (Brockmann I) column. 2,2′-Azobis(isobutyronitrile) (AIBN) (99%, Fisher Scientific) was purified by recrystallization from ethanol and dried at room temperature in a vacuum oven and stored in a freezer. The S-methoxycarbonylphenylmethyl dithiobenzoate (MCPDB) RAFT agent was synthesized following a procedure available in the literature.43 The synthesis procedure for the cellulose-s-methoxycarbonylphenylmethyl dithiobenzoate RAFT agent (cellulose-CTA) is described elsewhere.44 1-Bromooctane (99%), 1-bromododecane (97%), and 1-bromohexadecane (97%) were purchased from Aldrich (Gillingham, Dorset, U.K.) and used as received. Cultures of Escherichia coli were obtained from the Departmental Culture Collection, Faculty of Biological Science, University of Leeds. Ringer solution tablets were purchased from OXOID (Basingstock, U.K.). To prepare quarter-strength Ringer solution, one tablet was dissolved in 500 mL of distilled water and then sterilized by autoclaving at 121 °C for 15 min. Lecithin, egg (90%) (Fisher Scientific), was purchased from Aldrich. Tween 80 (DIFCO Laboratories, USA) was used as received. For the antibacterial tests, all of the glassware was sterilized in an autoclave at 121 °C for 20 min. Unmodified cellulose filter paper, cellulose-g-poly(2-dimethylamino)ethyl methacrylate) and quaternized cellulose-g-PDMAEMA samples were sterilized with 70% ethanol solution and dried under vacuum at 60 °C, overnight, before the antibacterial assessment, in order to remove ethanol. Elemental Analysis (EA). The cellulose samples were analyzed for C, H, N, and Br content in the Microanalysis Laboratory of the Chemistry Department at the University of Leeds, U.K. The C, H, and N contents were determined by combustion followed by chromatographic separation and thermal conductivity detection using a Carlo Erba EA 1108 elemental analyzer or a FLASH EA 1112 series CHNS-O analyzer (Thermo Electron Corp.). Halogen (Br) was determined as follows.

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Bromine Analysis. The completely dried test samples (5–10 mg) were combusted by the Schoniger oxygen flask combustion method.45 The combusted products were washed into flask with 5–10 mL of deionized water. To this solution, 1 drop of bromophenol blue (0.1% in ethanol) was added. Nitric acid solution was added dropwise until yellow, and a 0.5 mL excess of nitric acid was then added. Fifty milliliters of ethanol and diphenyl carbazone indicator (0.1% in ethanol) were added. Titration was carried out using 0.05 M Hg(II) nitrate (in water), to a purple end point. A blank determination was also carried out, and the value subtracted from the sample titration. A standard determination was carried out on a standard of known percentage of chlorine (p-chlorobenzoic acid). The Hg(II) nitrate solution was standardized against a known amount of sodium chloride (X mg) as sodium chloride solution (250 mL). Ten milliliters of this solution was taken for titration to give the factor, as defined in eq 2.

% bromine )

(titer - blank) × Factor 80 × 100 wt of sample 35.5

(1)

The “Factor” in eq 1 was calculated using eq 2.

Factor )

60.68 10X 250 titer × 100

(2)

Here, X is the known weight of sodium chloride that was used in the standardization of Hg(II) nitrate. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). ATR-FTIR spectra of the quaternized and nonquaternized samples were obtained on a Perkin-Elmer Spectrum One FTIR spectrometer using a single reflection, horizontal ATR accessory. The accessory has a diamond ATR crystal fixed at incident angle of 45°. For the measurement of ATR-FTIR spectra, each sample piece was mounted on the top of the ATR crystal and a premounted sample clamp was then lightly pressed on the sample. Each spectrum was collected in the range of 4000–400 cm-1 by cumulating 100 scans at a resolution of 4 cm-1. The scan speed was set at 0.5 cm/s. Baseline correction was applied for all spectra using Perkin-Elmer Spectrum software. Thermogravimetric Analysis (TGA). Thermogravimetric analyses on quaternized and nonquaternized samples were carried out using a TA Instrument TGA 2050 thermogravimetric analyzer under a nitrogen atmosphere. The samples (5–10 mg) were heated from room temperature to 500 °C at a rate of 10 °C min-1. The TA Instruments Thermal Advantage Universal Analysis software was used for calculating the onset, end decomposition temperature, and residual mass. All of the samples were dried under vacuum at 40 °C for 24 h prior to TGA measurements. Scanning Electron Microscopy (SEM). The surface morphology of the unmodified cellulosic samples and the modified cellulosic samples was observed by SEM using a JEOL JSM-820 scanning microscope, operated at an accelerating voltage of 5–10 keV. The dried cellulose samples were coated with a 30 nm gold layer using a Bio-Rad diode sputter coating unit. Electron micrographs of the sample were recorded at different magnifications, ranging from 2000× to 2500×. Tetrahydrofuran Size Exclusion Chromatography (SEC). The number-average molecular weight (Mn) and polydispersity index (Mw/ Mn)) of homo poly(2-(dimethylamino)ethyl methacrylate) formed in the graft polymerization solution were performed using an SEC system equipped with a LC 1120 HPLC pump (Polymer Laboratories, U.K.), a MIDAS (type 830) autosampler (Spark Holland, Netherlands), a differential refractive index (DRI) detector (Shodex, RI-101), a 5.0 µm bead-size guard column (50 × 7.5 mm), and two PLgel 5.0 µm MIXED-C columns (300 × 7.5 mm) in series (Polymer Laboratories, U.K.). Tetrahydrofuran (THF) with triethylamine (TEA) (5%) and 2,6di-tert-butyl-4-methylphenol (0.2 g) was used as the eluent at a flow rate of 1.0 mL min-1 at ambient temperature. Toluene (0.5%) was used as a flow rate marker. The SEC system was calibrated with poly(methyl methacrylate) (PMMA) EasiVial (Polymer Laboratories, U.K.) standards with molecular weights ranging from 690 to 1944000 g mol-1.

Nuclear Magnetic Resonance Spectroscopy (1H NMR). 1H (400 MHz)) NMR spectra were recorded on a Bruker 400 UltraShield spectrometer at 25 °C. Methylene chloride-d2 was used as a solvent during the assessing of DMAEMA monomer conversion while methanold4 was used as the solvent to obtain the spectra of quaternized PDMAEMA polymer. TMS was used as reference. Grafting Ratio. The grafting ratio (wt %) of each PDMAEMAgrafted filter paper was calculated using the following formula.

grafting ratio (wt %) )

weightgraft - weightcell-CTA × 100 (3) weightcell-CTA

Here, “weightgraft” is the dry weight of each cellulose-CTA sample after grafting with PDMAEMA and “weightcell-CTA” is the initial weight of each cellulose-CTA sample. Synthesis of Cellulose-g-poly(2-(dimethylamino)ethyl methacrylate) for the Antibacterial Test. In a typical reaction, the cellulose-CTA sample (0.95 g, RAFT agent loading ) 1.33 × 10-3 mol) was mixed with monomer (DMAEMA) (21.3 g, 1.33 × 10-1 mol), free chain transfer agent, s-methoxycarbonylphenyl dithiobenzoate (MCPDB) (0.4 g, 1.33 × 10-3 mol), initiator, and azobisisobutyronitrile (AIBN) (0.022 g, 1.33 × 10-4 mol) in ethanol (37.7 g). The molar ratio of cellulose CTA to free CTA (MCPDB) ([cell-CTA]0/[free CTA]0) was 1:1. The initial monomer to cellulose-CTA ratio was 100:1, and initial monomer concentration was 2.0 M. The molar ratio of cellulose CTA to initiator ([cell-CTA]0/[AIBN]0) was 1:0.1. The reaction was stopped after 60 h by cooling the flask in an ice–water bath, and the contents were exposed to air. The percentage monomer conversion in solution was measured by 1H NMR spectroscopy The molecular weight and polydispersity index of free PDMAEMA were determined by SEC after evaporating the excess monomer and solvent (Mn ) 16400 g/mol, PDI ) 1.3). The grafting ratio was calculated using eq 3. Grafting ratio, 27 wt %; monomer conversion, 90%; FTIR (cm-1) 2945 (C-H stretching, -CH3 and -CH2-), 2821, 2770 (C-H stretching, -N(CH3)2), 1724 (CdO), 1454 (-CH3 and -CH2bending), 1144 (C-N stretching). Anal. Calcd: C, 59.15; H, 5.15; N, 1.6, S, 12.1; Br, 2.5. Quaternization of Homo Poly(DMAEMA). PDMAEMA (0.82 g) was placed in 12 mL of ethyl alcohol containing an excess amount (3.0 g) of 1-bromohexadecane. The reaction mixture was stirred at 60 °C, for 40 h. The excess ethanol was removed from the reaction mixture under reduced pressure. The quaternized polymer was then precipitated in cold ethyl acetate and dried at 40 °C, under vacuum for 48 h. The weight gain of PDMAEMA, due to the quaternization reaction, was measured via gravimetry. The weight of quaternized PDMAEMA was 1.4 g. The weight increase due to quaternization ) (1.4 – 0.82) ) 0.58 g. FTIR (cm-1): 2921 (-CH2- stretching), 2852 (C-H stretching, -N(CH3)2), 1722 (CdO), 1465 (-CH3 and -CH2- bending), 1144 (C-N stretching). Anal. Calcd: C, 57.6; H, 9.9; N, 3.35; Br, 17.3. Quaternization of Cellulose-g-PDMAEMA Copolymer. Celluloseg-PDMAEMA copolymer samples were quaternized using three different alkyl bromides, 1-bromooctane, 1-bromododecane, and 1-bromohexadecane. In a typical quaternization reaction using 1-bromooctane, cellulose filter paper with 27 wt % graft ratio (0.254 g) was placed in 10 mL of ethyl alcohol containing an excess amount (3.0 g) of 1-bromooctane. The samples were stirred at 60 °C for 36 h. Each sample was thoroughly washed with ethanol (5 × 300 mL), THF (5 × 300 mL), THF: water (1:1) (5 × 300 mL), and water (5 × 300 mL). Further more, each sample was Soxhlet extracted with ethanol for 24 h. Finally, the sample was dried at 40 °C, under vacuum for 72 h. The actual weight gain (W1) of the copolymer sample, due to the quaternization reaction, was measured. FTIR (cm-1): 2923 (-CH2- stretching), 2853 (C-H stretching, -N(CH3)2), 1722 (CdO), 1465 (-CH3 and -CH2- bending), 1140 (C-N stretching). Anal. Calcd: C, 51.95; H, 6.25; N, 2.4; Br, 11.95 (quaternized with 1-bromooctane). The quaternization reaction completion was calculated using the following equation as described by Ramadan et al.46

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W1 × 100 W2

Roy et al. (4)

Here, W1 is the weight increase due to quaternization and W2 is the theoretical weight of alkyl bromide required for complete quaternization. The theoretical weight of alkyl bromide, W2 was calculated by the route shown in eq 5.

W2 )

Mw of alkyl bromide × wt of DMAEMA Mw of DMAEMA (157.22)

weight of DMAEMA )

graft ratio × sample weight 100

(5) (6)

Antibacterial Assessment of Quaternized Cellulose-g-PDMAEMA Copolymers. Gram-negative bacteria, E. coli, were inoculated into liquid culture of Nutrient Broth (peptone (15 g/L), yeast extract (3 g/L), NaCl (6 g/L), and dextrose (1 g/L)) and incubated overnight at 37 °C with shaking in an orbital incubator. This culture was then centrifuged for 10 min at ca.14000g (g ) relative centrifugal force). The supernatant was discarded and the pellet resuspended in Ringer quarter strength saline. The resulting bacterial culture was then centrifuged again for 10 min at ca.14000g. The supernatant was discarded again and the pellet resuspended in Ringer saline. After mixing properly, the resulting bacterial suspension was held in a sterile medical flat bottle as the stock E. coli suspension. The stock solution was further diluted (1 in 100) to give a working suspension of approximately 106-107 CFU/mL. Five milliliters of E. colisuspension was then added to the each Universal bottle containing virgin cellulose filter paper, cellulose-g-PDMAEMA or quaternized cellulose-g-PDMAEMA. The ungrafted paper, grafted paper and quaternized grafted paper plus E. coli were incubated for 1 h with gentle shaking at 37 °C. A control culture without filter paper was also treated in the similar way. After incubation period, 1 mL of the bacteria culture was taken from each bottle and then added to 9 mL of sterile neutralizer (2% lecithin and 3% Tween 80 in distilled water, heated to 80 °C with stirring in order to solubilize the lecithin)20 or Ringer saline and left for 5 min to provide a 10-1 dilution. This 10-1 dilution was further serially diluted down to 10-7. Aliquots (0.1 mL) of diluted samples were then spread, in triplicate, onto plates of nutrient Agar. After incubation at 37 °C for 24 h, the plates were examined, the number of colony forming units (CFU) was counted manually, and the results, after multiplication by the dilution factor, were expressed as mean colony forming units per milliliter (CFU/mL). Counts performed with or without a neutralizing agent showed that the neutralizing agent had no deleterious effect on E. coli.

Results and Discussions Synthesis of Cellulose-g-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and Its Subsequent Quaternization. In this study, the synthesis of cellulose-g-PDMAEMA was performed in the presence of ethanol and a nonattached (free) chain transfer agent to control the molecular weight (Mn) and molecular weight distribution (PDI) of the grafted PDMAEMA side chains. The subsequent quaternization of the grafted PDMAEMA chains was performed using alkyl bromides such as 1-bromooctane, 1-bromododecane, and 1-bromohexadecane. Long alkyl chain bromides were used to produce quaternary nitrogen atoms attached to appropriate lipophilic groups, providing a high charge density, for biocidal activity.24 The quaternization reactions were initially optimized for homo PDMAEMA, prior to reaction on the cellulose-grafted PDMAEMA chains. The quaternization reaction was confirmed by 1 H NMR, by elemental analysis, by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and by thermogravimetric analysis (TGA).

Figure 1. 1H NMR of homo PDMAEMA (a) before quaternization and (b) after quaternization with 1-bromohexadecane.

1

H NMR Analysis. The 1H NMR analysis of PDMAEMA, before and after quaternization (Figure 1), confirmed that the pendant amino groups were successfully quaternized. The -CH2-N- multiplet and the -N-(CH3)2 multiplet of PDMAEMA at 2.65 and 2.35 ppm, respectively, disappear after quaternization. New signals are observed at 3.1 ppm (singlet), corresponding to the methyl groups [-N+-(CH3)2], and at 3.4 ppm (multiplet), corresponding to the methylene protons (-CH2-N+-CH2) of the quaternized compound. The signal of the methylene protons (-C(O)-O-CH2-) is also shifted from 4.1 to 4.54 ppm. In addition, peaks at 1.3 ppm, characteristic of the (-CH2-) of the long alkyl chains, and the peaks of the terminal methyl of the alkyl chain at 0.93 ppm, confirm the presence of the long alkyl chains on the pendant amino groups of the PDMAEMA. Elemental Analysis. The elemental analysis results for the bromine content of quaternized homo PDMAEMA and cellulose-g-PDMAEMA copolymers are given in Table 1. The percentage of bromine (% Br )17.3%), found in the quaternized homo PDMAEMA, suggested the successful quaternization of pendant tertiary amino group of homo PDMAEMA. The % Br in the PDMAEMA-grafted cellulosic copolymer was in good agreement with the calculated degree of quaternization obtained using eq 4. The cellulose-g-PDMAEMA copolymer, quaternized with the shorter alkyl chain bromide (1-bromooctane) possessed a higher % Br (11.95%, Table 1) and degree of quaternization (33.6%) compared with the samples quaternized with longer alkyl chain bromides. The quaternization reaction completions (degree of quaternization) were 21.7% and 22.2% for the samples quaternized with 1-bromododecane and 1-bromohexadecane, respectively (Table 1). ATR-FTIR Spectroscopy. ATR-FTIR spectroscopy was used to confirm the presence of PDMAEMA on the cellulosic copolymer. The ATR-FTIR spectra (Figure 2b) of the cellulose-

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Table 1. Elemental Analysis Results and Degree of Quaternization

samples

C

H

homo PDMAEMA quaternized homo PDMAEMAa cellulose-g-PDMAEMA (C8)b cellulose-g-PDMAEMA (C12)c cellulose-g-PDMAEMA (C16)d

60.1 57.6

9.5 9.9

N

Br

quaternization reaction completione (%)

8.4 0.75 3.35 17.3

51.95 6.25 2.4

11.95

33.6

53.55 5.9

1.95

8.35

21.7

55.55 5.5

1.65

7.7

22.2

a Homo PDMAEMA quaternized with 1-bromohexadecane. b Celluloseg-PDMAEMA quaternized with 1-bromooctane. c Cellulose-g-PDMAEMA with quaternized 1-bromododecane. d Cellulose-g-PDMAEMA quaternized with 1-bromohexadecane. e Determined using the eq 4.

Figure 2. ATR-FTIR spectra of (a) ungrafted cellulose chain transfer agent (cellulose-CTA) and (b) cellulose-g-PDMAEMA copolymer.

Figure 3. ATR-FTIR spectra of homo PDMAEMA before quaternization (a) and after quaternization with 1-bromohexadecane (b).

g-PDMAEMA copolymer show the appearance of new bands at 2945 cm-1(C-H stretching of methyl and -CH2- groups), 2821, 2770 cm-1 (C-H stretching of -N(CH3)2 group), 1454 cm-1 (-CH2- bending (scissors) vibration and CH3 antisymmetric deformation), and 1144 cm-1 (C-C-N bending) in comparison with that of ungrafted cellulose chain transfer agent (cellulose-CTA, Figure 2a). These bands are characteristic peaks of PDMAEMA (Figure 3a). The absorption peak for ester group at 1747 cm-1 (CdO stretching) in the cellulose-CTA sample (Figure 2 a) is moved to about 1723 cm-1, characteristic of the CdO stretching of PDMAEMA. ATR-FTIR spectroscopy was also used to follow the quaternization reaction. Figure 3 shows the ATR-FTIR spectra of the homo PDMAEMA and of quaternized PDMAEMA. Figure

Figure 4. ATR-FTIR spectra of cellulose-g-PDMAEMA copolymer before quaternization (a) and after quaternization with 1-bromooctane (b).

Figure 5. TGA thermograms of homo PDMAEMA before and after quaternization with 1-bromohexadecane.

4 shows the ATR-FTIR spectra of cellulose-g-PDMAEMA and (cellulose-g-PDMAEMA)-C8samples. Since the long alkyl chain compounds were used in quaternization, peaks in the range of 2921–2853 cm-1(C-H antisymmetric and symmetric stretching of -CH3 and -CH2- group) suggested the successful addition of alkyl chains to the polymer. The same peaks were also observed for (cellulose-g-PDMAEMA)-C12 and (cellulose-gPDMAEMA)-C16 samples. Thermogravimetric Analysis. Thermogravimetric analysis (TGA) was used in an investigation of the thermal properties of quaternized homo PDMAEMA and of grafted PDMAEMA. TGA thermograms of homo PDMAEMA, quaternized with a C16 alkyl chain and cellulose-g-PDMAEMA fibers, quaternized with alkyl chains of C8, C12, and C16, are shown in Figures 5 and 6. It was observed that the quaternized homo PDMAEMA polymer chains decomposed at lower temperature than the nonquaternized PDMAEMA chains, as shown in Figure 5. Thus, the quaternized homo PDMAEMA polymer chains decomposed at lower temperature than the nonquaternized PDMAEMA chains, as shown in Figure 5. In a similar way, the decomposition temperatures of cellulose-g-PDMAEMA fibers decreased with increasing amounts of quaternary ammonium groups, due to an increase in the bromine content of the polymer.47 Quaternized cellulose-g-PDMAEMA copolymer, with a shorter alkyl chain (C8), decomposes at lower temperatures than samples containing longer alkyl chains (C12 and C16), as it has a higher bromine content (Table 1). This effect is due to those of the high degree of quaternization and, hence, the higher bromine content of the cellulose-g-PDMAEMA (C8) sample (Table 1). A similar thermal degradation profile of the quaternized polymers has also been observed by other groups.17,47,48

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Figure 6. TGA thermograms of cellulose-grafted PDMAEMA samples, before and after quaternization (Q-C8, Q-C12, and Q-C16 ) celluloseg-PDMAEMA quaternized with alkyl chain lengths of 8, 12, and 16, respectively).

Figure 7. A typical size exclusion chromatogram showing the molecular weight distribution of homo PDMAEMA chains (Mn ) 16500 g/mol, PDI ) 1.30).

Size Exclusion Chromatography (SEC). The homo PDMAEMA polymer chains formed in the graft polymerization were isolated and analyzed by SEC to estimate the molecular weights (Mn) and polydispersity index of chains attached to the cellulose surface. Since a free chain transfer agent (free CTA) was used in the graft polymerization, the formation of homo polymers and graft copolymers was expected to be controlled. The SEC trace of the free polymer chains was monomodal and had a reasonably narrow molecular weight distributions (PDI ) 1.3), as illustrated in Figure 7. An attempt has been made to analyze the quaternized homo PDMAEMA (C16) sample by size exclusion chromatography (SEC), to establish the effect of quaternization on the molecular weight (Mn) and polydispersity of the polymer chains. Since the quaternized PDMAEMA (C16) sample was not soluble in THF or in water due to its long alkyl chain, it was not possible to analyze the sample via THF SEC analysis or aqueous SEC analysis. Antimicrobial Effects of Quaternized Cellulose-g-PDMAMEA Copolymer. The ability of the quaternized celluloseg-PDMAEMA substrates to kill bacteria was tested following the method described in the Experimental Section. Two control experiments were run in which either no cellulose fiber was present or an unmodified cellulose was used. Results show a slight variation in bacteria number, possibly due to inhibited bacteria growth in salt solution, but the variations remain a lot lower than those observed for samples with modified cellulose. Initially, the antibacterial activity of cellulose-g-PDMAEMA sample (grafting ratio 9 wt %) quaternized with a longer alkyl

Roy et al.

chain (C16) bromide was evaluated (Table 2a). We observed that the cellulose-g-PDMAEMA substrate had a strong hydrophobic character, due to the long alkyl chains on the quaternary ammonium groups. This hydrophobicity resulted in poor wetting of the quaternized cellulose-g-PDMAEMA copolymer, thus preventing close contact with the bacteria (Figure 8). As a result, the surface did not reduce the number of bacteria significantly. In order to improve on the wetting of the cellulose sample, a cellulose-g-PDMAEMA substrate was synthesized with a greater proportion of hydrophilic polymer (27 wt % grafting ratio) and shorter alkyl chain (C8). This sample showed good wetting and was used as the basis of this study. Cellulose-g-PDMAEMA copolymers were also quaternized with longer alkyl chain lengths (C12 and C16) to investigate their effect. The antibacterial assessment results are shown in Table 2b. The results of the antibacterial tests show a greater reduction in bacterial counts in the presence of cellulose-g-PDMAEMA copolymers substituted with pendant quaternary ammonium groups compared with unmodified cellulose. The decreasing numbers of E. coli when in the presence of quaternized cellulose-g-PDMAEMA copolymers suggest antibacterial activity of the subtrate, which is influenced by the variation in the alkyl chain lengths of the quaternary ammonium groups. The viable cell-counting method confirmed that the activity against E. coli is greater when cellulose-g-PDMAEMA copolymer is quaternized by 1-bromooctane (C8) rather than with 1-bromododecane (C12) and 1-bromohexadecane (C16). The difference in antibacterial activities of cellulose-g-PDMAEMA quaternized with 1-bromooctane (C8) compared to that seen for cellulose-g-PDMAEMA quaternized with 1-bromododecane (C12) and 1-bromohexadecane (C16) can be due to three factors: (a) the greater hydrophobicity seen for the cellulose-g-PDMAEMA samples with C12and C16alkyl groups compared to the sample with C8, (b) the greater degree of quaternization seen for the cellulose-g-PDMAEMA sample with C8alkyl groups, and (c) the length of the alkyl chain. The decrease in antibacterial activity with an increase in alkyl chain length has also been observed by others.41,49–52 Indeed, the origin of the binding of polycationic antibacterial agents to the cytoplasmic membrane of bacteria and their diffusion through the cell walls may be linked to both electrostatic interactions and hydrophobic interactions, in relation to the molecular weight/alkyl chain length of the cationic polymer.19 Thus, an optimal range of alkyl chain lengths and molecular weights exists for the antibacterial action of the polymers.41 Most of these studies suggested that, ideally, the alkyl chain should contain eight carbon atoms to help in establishing an optimal balance between the hydrophobicity of the cationic antimicrobial and cytoplasmic membrane of the targeted bacteria.41,49,53 The major constituents of the cytoplasmic membrane are proteins and phosphoglyceride lipids which consist of both a hydrophilic end (phosphate with other polar residues) and a hydrophobic end (two long-chain fatty acid tails with 12–20 carbon atoms).18,54 Cationic polymers with an octyl chain are therefore assumed to strongly interact with the cytoplasmic membrane of the bacteria due to an affinity of the molecular structure, leading to the highest activity.53 Our results confirm these observations for a cellulose-g-PDMAEMA substrate, as we observe a dramatic change in antibacterial activity when the alkyl length is dropped from 16 or 12 carbons to 8 carbons. An increase in the alkyl chain lengths of the quaternary ammonium groups of the cellulose-g-PDMAEMA samples results in an increase in the hydrophobic character of the

Antibacterial Cellulose Fiber

Biomacromolecules, Vol. 9, No. 1, 2008 97

Table 2. Antimicrobial Activity of Unmodified Filter Paper and Modified Filter Paper

samples a

b

control (no filter paper) unmodified filter paper cellulose-g-PDMAEMA quaternized cellulose-g-PDMAEMA (C16) control (no filter paper) unmodified filter paper cellulose-g-PDMAEMA quaternized cellulose-g-PDMAEMA (C8) quaternized cellulose-g-PDMAEMA (C12) quaternized cellulose-g-PDMAEMA (C16)

polymer, or the polymer bound substrates, as observed in the initial test. This increase in hydrophobicity may therefore limit interactions between polymer and bacteria. Indeed, it is more difficult to reach the target cytoplasmic membrane of bacteria for surface-immobilized polymeric antibacterial agents than in the case of free low molecular weight cationic antimicrobial agents.50 It has been observed that unmodified filter paper, cellulose-g-PDMAEMA and quaternized cellulose-g-PDMAEMA (C8) could wet easily. However, samples quaternized with longer alkyl chains (C12 and C16) bromides could not wet easily in the aqueous medium of the antibacterial test. Thus, the interaction between the bacteria and cellulose surfaces with longer alkyl chain lengths is expected to be weaker than interactions with short alkyl chain lengths. In a recent study, Dizman et al.52 reported that the antibacterial activity of polymers with pendant quaternary ammonium groups against Gram-negative bacteria such as E. coli decreased with an increase in the length of the alkyl chain. The structure of the cell wall of E. coli is much more complicated than that of the Gram-positive species. There is an outer membrane mainly consisting of phospholipids, proteins, and lipopolysaccharides (LPS).18 One of the key functions of the outer membrane is to protect the bacteria from attack by foreign substances such as antibacterial agents. In addition, the hydrophobicity of an antibacterial polymer agent containing long alkyl chains makes it difficult to kill E. coli because of the fewer interactions between the outer membrane and the bacteria. This complicated structure of Gram-negative bacteria may explain why quaternized cellulose-g-PDMAEMA (C12) and cellulose-g-PDMAEMA (C16) samples are not very active against E. coli. It has been observed that the grafting ratio of PDMAEMA onto cellulose can affect antibacterial activity. Samples bearing

Figure 8. (a) Unmodified filter paper, (b) cellulose-g-PDMAEMA, and (c) quaternized cellulose-g-PDMAEMA (C16) in the antibacterial test system showing good (a and b) and poor (c) wetting behavior.

no. of bacteria remaining afterincubation (CFU/mL)

grafting ratio (%)

no. of bacteria added to sample (CFU/mL)

9.0 9.0

1 × 107 1 × 107 1 × 107 1 × 107

4.8 × 106 6.7 × 106 4.8 × 106 2.5 × 106

27.0 27.0 27.0 27.0

1 × 106 1 × 106 1 × 106 1 × 106 1 × 106 1 × 106

3.4 × 105 2.6 × 105 3.5 × 102