Assessment of Antibacterial Properties of Polyvinylamine (PVAm) with

Apr 24, 2009 - Souvik Chakrabarty , Allison King , Pinar Kurt , Wei Zhang , Dennis E. Ohman , Lynn F. Wood , C. Lovelace , Raj Rao , and Kenneth J. Wy...
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Biomacromolecules 2009, 10, 1478–1483

Assessment of Antibacterial Properties of Polyvinylamine (PVAm) with Different Charge Densities and Hydrophobic Modifications Eva-Helena Westman, Monica Ek, Lars-Erik Enarsson, and Lars Wågberg* Department of Fiber and Polymer Technology, School of Chemical Science and Engineering, KTH, Royal Institute of Technology, SE-100 44 Stockholm, Sweden Received January 22, 2009; Revised Manuscript Received March 30, 2009

Hydrophobically modified and unmodified polyvinylamines (PVAm), including a total of five polymers, were tested against both gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacteria for antimicrobial activity. The assessment of PVAm in solution against bacteria is described, and the influence of the charge density and of the hydrophobic modification of the polyelectrolyte is discussed. The antimicrobial activity was found to depend upon the concentration of PVAm and also on the type of bacteria used. The results also indicated that no direct relationship exists between antimicrobial activity and charge density of the different PVAms. It was, however, observed that an alkyl chain length of six or eight alkane units had a substantial effect on the bacteria investigated. The best combined antibacterial activity for the two bacteria tested was achieved for PVAm with a C6 alkane substituent (PVAm C6). To evaluate the antimicrobial activity on a solid substrate, PVAm C6 was further studied after being deposited onto a glass slide and the results show a large reduction in bacterial infection.

Introduction Great interest surrounds materials with properties that limit the growth of unwanted microorganisms due to for example the alarming problem of increasing antibiotic resistance and to the development of molecular surface engineering. Various polymers have been intensively studied in terms of their antimicrobial activity.1-6 The use of high molecular weight substances as a functionalized layer on a surface provides one method to maintain valuable bulk properties while creating an antimicrobial barrier in environments where hygiene is of utmost importance. Areas of applications include, for example, water filtration, health care, and medical technology, where the protective surfaces can serve as a complement to standard hygienic procedures. Polycations modified with hydrophobic moieties to create an amphiphilic structure are known to exhibit antimicrobial properties, and they have been evaluated in the immobilized state on surfaces such as glass, carbohydrate-based materials and plastic.7-12 The effect of these polymers has been ascribed to the charge (e.g., quaternary ammonium functionality), but also to the degree of hydrophobicity, which depends on the length of the alkyl substituent of the polymer. The polymers, in contrast to their monomeric counterparts, are believed to be more efficient against bacteria due their greater size and charge density.4,6 Early work within the field showed the antimicrobial properties of glass slides functionalized with poly(4-vinyl-N-alkylpyridinium bromide).7 The work showed that an alkyl chain length of C6 was the most effective against Staphylococcus aureus when the polymers were covalently attached to the surface. To avoid the complexity of stepwise grafting reactions, polyethylene imine (PEI) was deposited onto a glass substrate and the antibacterial polymer, which was dissolved in an organic solvent, was allowed to solidify on the surface via evaporation.13 PEI was further evaluated in the immobilized state with respect to * Corresponding author. E-mail: [email protected].

its antimicrobial properties, and the hydrophobic/hydrophilic balance was studied to obtain a polymer that swells in water while preserving a strong interaction with lipid membranes.14 The bactericidal efficiency of immobilized polymers was reported to be optimal for a hydrophilic system, in which the internal hydrophobic interactions between the alkyl chains are low. The antibacterial activity of the polycationic agents has been ascribed to both physical and electrostatic interactions with bacterial membranes, which are in general negatively charged at a physiological pH.6,15 As electrostatic interaction is important, the charge-activity relationship of the polycations has been studied.16 It was observed that a charge density threshold must be exceeded to achieve a rapid decrease in bacterial population. It was suggested that the antibacterial effect was due to a release of counterions of the charged envelope of bacteria due to the interaction with the cationic surface and that, upon loss of Mg2+ and Ca2+, the stability of the outer membrane of the bacteria was lost, and the bacteria were killed. Today, no unified explanation exists for the antibacterial mode of action of the cationic polymers. It has, however, been fairly well-established that cationic polymers have a broad spectrum of efficiency, and the risk of bacteria developing resistance toward these substances is considered to be low because the mechanism of antimicrobial activity is believed to depend on more than one factor and to involve several functions and targets in/on the bacterial cell.17 The polyelectrolyte chosen in the present study, polyvinylamine, is a weak cationic polyelectrolyte, that is, the charge is pH-dependent, and it does not have any quaternary amine functionality. The charge density of polyvinylamine is also dependent upon the vinyl amine content and on the degree of hydrophobic modification, that is, substitution of the vinyl amine groups with alkyl chains. Modified polyvinylamines are interesting to study because they possess a high ratio of cationic groups/ alkyl substituents, are soluble in water, and can be immobilized to solid substrates by physical adsorption as an alternative to grafting techniques. Depending on the choice and degree of

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Table 1. Properties of the Different PVAm Used for the Evaluation of Antimicrobial Activity type of PVAm PVAm PVAm PVAm PVAm PVAm

B, unmodified D, unmodified C6, 6 carbon units C8, 8 carbon units C12, 12 carbon units

degree of alkylation (%)

degree of hydrolysis (%)

30 10 10

100 30 90.7 90.7 90.7

hydrophobic substituents, it is possible to control the solubility properties of modified polyvinylamines. Qiu et al. synthesized a nonionic amide derivative of polyvinylamine with side chains of hydrophilic oligosaccharides and hydrophobic hexanoyl groups.18 Modified polyvinylamines with a cationic charge have been prepared by partial substitution of the polyvinylamines with benzyl or alkane chains.19,20 Hydrophobic substituents on polyvinylamines can also be introduced in the polymerization step by synthesizing a copolymer of formamide and N-vinylcarboxamides.21 The authors have previously studied modified polyvinylamines with C6 alkyl substituents regarding their antibacterial effect on E. coli when immobilized on cellulose films.22,23 The main purpose of the present work was to assess the antibacterial effect of a set of polyvinylamines with alkyl substituents of different chain lengths. To test their potential antimicrobial effect, unmodified and hydrophobically modified polyvinylamines were screened against bacteria in aqueous solution. This was followed by an antibacterial assessment of polyvinylamines immobilized on glass surfaces, accomplished by adsorption of cationic polyelectrolytes onto the anionic solid substrates.

Experimental Section Materials and Bacterial Strains. Five types of PVAm, all with an average molecular weight (Mw) of 340000 Da, according to the supplier, were investigated for antimicrobial activity (see Table 1 for specifications). The polymers were synthesized by BASF, Ludwigshafen, Germany, according to a previously described method.23 The preparation is based on a radical polymerization of vinyl formamide, partial hydrolysis of the formamide units into vinylamines, and partial substitution of the amine units with alkyl chains of the desired length using epoxy-alkanes.23 Prior to use the polymers were dialyzed against deionized water for 72 h followed by freeze-drying. The polymers were subsequently stored in a desiccator and dissolved to the desired concentration in ultrapure water under stirring for 24 h before use. Figure 1 shows the general structure of the polymer. As expected the PVAm is prepared from polyvinylformamide via hydrolysis to different degrees, which refers to the conversion of amide groups to amines. The amines can then be substituted to different degrees with alkyl chains having different chain lengths. Table 1 shows that C6, C8, and C12 were the alkane substituents used, and the degree of substitution, DS, was 30, 10, and 10%, respectively, for these groups. E. coli, ATCC 11775 was obtained from SIK (The Swedish Institute for Food and Biotechnology), and B. subtilis was obtained from STFI-

Figure 1. Principal structure of the PVAm. The formamide units illustrate that the PVAm is prepared from polyvinylformamide that is hydrolyzed to a given extent. To control the hydrophobicity of the polymers, part of the primary amine substituents are further derivatized with alkyl groups, where R refers to a hexyl, octyl, or dodecyl chain.

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Packforsk AB (MERCK, 1.10649.0002). Ringer’s solution and tryptone glucose extract (TGE) were freshly prepared. The growth agar medium used for E. coli was Fluorocult E. coli 0157:H7 agar, from MERCK, and Petrifilm aerobic count plates (3M) were used for the enumeration of B. subtilis. For the in vitro experiments using glass slides (purchased from Menzel-Gla¨ser), Chromocult growth agar (VWR) was used. Ultrapure water with a resistivity of 18.2 MΩ · cm (Milli-Q plus system, Millipore) was used. Methods. Antibacterial Assessment. A 100 µL suspension of E. coli or B. subtilis was cultivated in 10 mL of TGE broth in a sterile flask, followed by shaking at 75 rpm at 37 °C (Stuart Scientific Orbital incubator SI 50). B. subtilis were cultivated for 16 h at 37 °C until they reached the vegetative state (108 cells/mL with no observable spores in a light microscope). The bacterial cell concentration was estimated by serial dilution in Ringers solution. Solutions of the five different polyvinylamines were prepared to give final concentrations of 100, 25, 10, 2.5, 1, and 0.25 µg/mL. Subsequently, the polymers were separately tested against 106 cells/mL of E. coli and B. subtilis. The polymers were dispersed in ultrapure water and bacteria were added to the solution of polymers to reach the desired amount of bacterial cells in the sample (106 cells/mL). The inoculated polymer solution was thoroughly stirred for 30 s, and samples of each inoculated polymer suspension were then immediately spread on an agar plate or Petrifilm. Bacteria in ultrapure water without the polymer were used as a reference. The plates were incubated at 37 °C for 20 h. The number of colonies, that is, viable bacteria on the agar plates, was counted on the following day. The E. coli used for the assessment were sorbitol-positive microorganisms, which resulted in yellow colonies on the Fluorocult agar medium. B. subtilis colonies were detected as red spots on the Petrifilm. Commercially available glass slides were rinsed in ultrapure water followed by rinsing with ethanol. To remove excess ethanol, the slides were again rinsed in ultrapure water. This procedure was conducted on all slides. Half the rinsed glass slides were left overnight in an aqueous polymer solution containing PVAm C6 (100 mg/L). On the following day, the treated glass slides were dried under a flow of nitrogen gas. To spread the bacteria evenly over the glass surfaces, one of two similar glass slides, that is, treated or untreated, was inoculated with 10 µL of bacteria in Ringer’s solution of 105 CFU/mL (CFU meaning colony forming unit). This gave a final concentration of 103 CFU on the glass surface. The second glass slide was then placed on top of the inoculated glass slide and the bacteria were consequently spread onto the two glass surfaces. Sterile gloves were used. The two glass slides were carefully separated and placed in separate Petri dishes, left to dry for two minutes, and subsequently covered by Chromocult growth agar kept at a temperature of 37 °C. The two treated surfaces were treated as a single sample. Untreated glass slides were used as controls. The Petri dish was sealed and incubated at 37 °C overnight. The experiments were duplicated. Charge Determination. To determine the charge of the polyvinylamines, the polyelectrolyte titration method was used.24,25 Polyvinylamines were titrated with potassium polyvinyl sulfate (KPVS) from Wako Pure Chemicals, Japan, using the cationic indicator orthotoluidine blue from Kebo AB (Sweden) for the end point detection. In the titration, KPVS forms polyelectrolyte complexes with cationic polyelectrolytes until the analyte is consumed. At the end point, excess KPVS forms complexes with the monovalent cationic indicator, inducing a color shift of KPVS. This response was detected with an equipment similar to that described by Horn.25 All the PVAm polymers were diluted to known concentrations in ultrapure water and the pH was adjusted by adding acid (HCl) or alkali (NaOH). A phosphate buffer was used to keep the pH of 7.5.

Results Antibacterial Assessment. The results of the antibacterial activity of PVAm are shown in Table 2 (E. coli) and Table 3

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Table 2. Evaluation of the Antimicrobial Activity of Different Types of PVAm against E. colia polymer concentration polymer PVAm PVAm PVAm PVAm PVAm

B D C6 C8 C12

blank ++++ ++++ ++++ ++++ ++++

0.25 1 2.5 10 25 100 µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL ++++ ++++ ++++ ++++ ++++

++++ ++++ ++ ++++ ++++

+++ +++ + ++ +

+++ +++ ++ +

++ + + +

++ + + +

a The difference in growth for the various concentration of polymer can be seen, where ++++ indicates full growth and - indicates no growth. Legend to the amount of growth: (++++) blank test or growth as blank test (106 CFU/mL sample); (+++) excessive growth but less than blank (not countable bacterial number); (++) limited growth (up to approximately 10000 CFU/mL sample, i.e., a 99% decrease); (+) little growth (up to approximately 1000 CFU/mL sample, i.e., a 99.9% decrease); (-) no growth (less than 10 CFU on agar plate).

Figure 2. Charge of different types of PVAm at pH ) 2, as determined with the polyelectrolyte titration method.

Table 3. Evaluation of the Antimicrobial Activity of Different Types of PVAm Polymers against B. subtilisa polymer concentration polymer PVAm PVAm PVAm PVAm PVAm

B D C6 C8 C12

blank ++++ ++++ ++++ ++++ ++++

0.25 1 2.5 10 25 100 µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL ++++ ++++ ++++ ++++ ++++

+++ +++ +++ + ++++

++ + ++ +

-

-

-

Figure 3. Charge of different types of PVAm at pH ) 7.5 as determined with the PET-method in the presence of a phosphate buffer.

a The difference in growth for the various concentration of polymer can be seen, where ++++ indicates full growth and - indicates no growth. Legend to the amount of growth: (++++) blank test or growth as blank test (106 CFU/mL sample); (+++) excessive growth but less than blank (not countable bacterial number); (++) limited growth (up to approximately 10000 CFU/mL sample, i.e., a 99% decrease); (+) little growth (up to approximately 1000 CFU/mL sample, i.e., a 99.9% decrease); (-) no growth (less than 100 CFU on Petrifilm).

(B. subtilis). The method used for evaluation is not standardized but it was used to determine the concentration intervals in which the polymers remained active. In turn, the difference in antimicrobial activity between the PVAm polymers can be assessed. The two tables show that the antimicrobial activity increased with the concentration of PVAm and that the effect was more pronounced for B. subtilis. This is in agreement with the findings of previous studies, where cationic polymers were observed to be more efficient against gram-positive bacteria.2,7 PVAm C8 was active against B. subtilis at concentrations in the range of 1-2.5 µg/mL, while the other PVAm samples were effective at concentrations of g10 µg/mL. For E. coli, on the other hand, PVAm C6 was the most efficient sample, showing an antibacterial effect at a concentration of 10 µg/mL, while the other samples were less efficient in their antimicrobial activity even at as high concentration as 100 µg/mL. PVAm C6, therefore, showed the best potential in the tests, with efficiency both against E. coli and B. subtilis. Charge of the Polyelectrolytes as a Function of pH. As mentioned earlier, the charge of the polyelectrolytes is believed to be critical for their antibacterial action and it was therefore considered vital to determine the charge of the different polyelectrolytes at different pH values. The charges of the PVAm polymers at pH ) 2 and pH ) 7.5 are shown in Figures 2 and 3, respectively. Like monoamines with a pKa of about 9, the polyvinylamines also show a decrease in charge when the pH is increased, but the dissociation function is, as expected, distributed over a broader pH interval as compared to the monoamines. At pH ) 2, the amine groups of the polymers are assumed to be fully protonated, whereas at pH 7.5, they will

Figure 4. Charge density of the PVAm C6 as determined with the polyelectrolyte titration as a function of pH, ranging from pH 2 to 10.

be only partially protonated. The charge is also determined by the degree of hydrolysis of the polyelectrolytes and the substitution of alkyl chains of the amine groups on the polymer. As evidenced by these figures, the charge is much lower at the higher pH and the chemical composition of the PVAm has a significant effect on the charge of the polymer. PVAm substituted with an alkyl chain with six carbon atoms (PVAm C6) showed the greatest impact against E. coli and had a good effect against B. subtilis and was therefore selected for experiments in which this polyelectrolyte was immobilized on solid substrates. To characterize this polymer in more detail, the charge of the polyelectrolyte was determined over a larger pH range. Figure 4 shows how the charge density of PVAm C6 changes between pH 2 and 10. The approximate charge at pH ) 10 is about 20% of the charge at pH ) 2. In these measurements, no buffers were used except for pH ) 7.5, where a phosphate buffer was used. These results indicate that the charge of PVAm C6 may vary when bacteria are deposited on the polyelectrolytetreated surfaces and covered with agar. However, nutritional solution and agar are usually buffered and kept at a neutral pH, since this is the pH region where most organisms have optimal

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Figure 6. Results from a “Leaching test” where agar solution was poured onto a glass slide containing deposited E. coli: left, reference (untreated) sample; right, sample with deposited PVAm C6.

Discussion

Figure 5. Antibacterial assessment of PVAm immobilized on glass slides. Left: reference (untreated) samples; right: treated (PVAm C6) samples inoculated with E. coli (∼103 CFU/sample), which results in dark colonies on Chromocult agar upon bacterial growth.

growth. For the systems tested, that is, in aqueous solution and with agar, it is safe to assume that the pH would not exceed pH ) 7.5, where the polymer maintains approximately 40% of its charge at pH ) 2. Effect of Immobilized Polyelectrolytes on Bacterial Growth. PVAm C6 was adsorbed onto glass slides according to the procedures described in the experimental section and was then exposed to E. coli. The results are shown in Figure 5. The number of colonies on the reference (untreated) slide was difficult to enumerate by manual counting after incubation; however, the number of colonies inoculated on the surface was known (103 CFU/ sample). The dark spots and patches indicate growth of E. coli on the surface of the glass slides, and a decrease in growth could be seen on the polymer-treated samples. To further evaluate surfaces with and without PVAm C6 as an antimicrobial coating, the glass slides were inoculated with bacteria (103 CFU) without a second glass slide to spread bacteria across the treated slides. A drop of bacteria suspension was placed on the slides but was shortly thereafter washed off by pouring agar into the Petri dish. This experiment was performed to test if the polymers were being washed off the surface as agar was poured on top and if the bacteria are killed on contact with the glass surface or by leaching of the polymer from the surface. If the polymer coating leached from the surface, this would be detected with a coated slide placed at the bottom of an inoculated agar plate. Figure 6 shows an untreated and treated slide from the previously described experiments where bacteria are spread in the surrounding agar with an untreated/treated glass slide placed at the bottom of the Petri dish. In comparison with the results shown in Figure 5, the agar with the treated sample exhibited infection, that is, bacteria grew in the agar media above the surface, but show limited growth on the actual surface. This indicates that bacteria were killed by contact with the surface. The observed edge effect (growth of bacteria) around the glass slides also indicates that no leaching of polymer occurred since otherwise, a zone of inhibition would have been observed around substrates that leach of antimicrobial agents.

The trials performed on unmodified/modified polyvinylamines show that these weak polyelectrolytes with a net positive charge possess antibacterial activity against both gram-positive and gram-negative bacteria. The results show differences in activity that can be related to differences in degree of hydrolysis, the charge and the hydrophobicity of the PVAm’s investigated. All of the tested polymers showed good results, with PVAm C8 producing the best results in terms of antibacterial activity when tested against the gram-positive bacterium B. subtilis. PVAm C6 was, however, more efficient against the gram-negative E. coli than the other PVAm samples, as these showed much worse performance over the studied concentration range. Thus, for the combination of the two species of bacteria, the PVAm C6 sample showed the best combined performance in terms of antimicrobial activity. As was mentioned earlier, a standardized MIC (minimum inhibitory concentration) procedure was not used in the present investigation, but if the definition that no bacterial growth should occur above the MIC value, the results in Tables 2 and 3 can be used to estimate the MIC values for the currently investigated polyelectrolytes for reason of future comparisons. Hence, by adopting this definition the MIC values would be between 2.5 and 10 µg/mL for PVAM C6 both for E. coli and B. subtilis. For PVAm C8 the MIC value would be 1-2.5 µg/ mL for B. subtilis and >100 µg/mL for E. coli. Differences in the results suggest that different bacteria are more or less susceptible to the charge and hydrophobicity of the PVAm. It has been previously demonstrated that gramnegative bacteria are less susceptible to amphiphilic polymers and are consequently more difficult to inhibit or kill.14 The present results are in agreement with this finding. The greater antimicrobial activity of PVAm C6 is also in accord with the findings of earlier work on grafted polyelectrolytes.7 B. subtilis was more sensitive to PVAm C8, which has longer hydrophobic moieties, even though the DS was 10% compared to that of PVAm C6, which was 30%. The charge of PVAm C8 is higher, and this may have influenced the results. This difference in activity among different PVAm may prove to be useful to enhance the overall antimicrobial effect if polymers are used together in, for example, a coating process where more than one polymer can be applied. The antibacterial effect of PVAm C6 immobilized on solid surfaces was also investigated. Absorbed amounts were not determined on the glass surfaces, but from previous experiments on silicon oxide the Sauerbrey mass determined with the quartz crystal microbalance, that is, the total mass of PVAm C6 and water trapped in the adsorbed layer, is about 2 mg/g.22 The results showed a decrease in bacterial colonization. The polymer

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Table 4. Comparison between the Experimental and Theoretical Charges of the Different Polymers under the Assumption that either only the NH2 Groups or all N-Groups have an Electrostatic Charge at pH ) 2a

polymer PVAm PVAm PVAm PVAm PVAm a

B D C6 C8 C12

charge if only charge if all exptl results NH2 groups are N-groups carry (mequiv/g) charged (mequiv/g) charges (mequiv/g) 13.1 ( 0.9 6.8 ( 0.5 10.6 ( 1.5 21.3 ( 0.9 7.98 ( 1.0

23 4.8 9.7 15 14

16 15 18 17

It is assumed that no counterions are present in the polyelectrolytes.

coating was efficient, as the decrease in CFU is 1000-fold. Additional glass surface experiments indicate that bacteria need to be in close contact with the surface to be affected. A leaching test was performed where bacteria were washed off the surface and grew freely in the surrounding agar. The results indicate that these surfaces do not readily release polymers into the agar if the agar is poured onto the modified PVAm slides (Figure 6). Furthermore, no zone of inhibition could be detected in close proximity to the glass slide. The result cannot exclude the possibility that the polymers are leached if the agar surface is left in contact with the polymer coating for a longer period of time, even though this is unlikely for charged polyelectrolytes adsorbed to an oppositely charged surface.26 Other studies have, however, shown that a possible leaching from the surface to agar may occur when polymers have been adsorbed onto glass substrates.13,14 When dealing with weak polyelectrolytes, it is important to control their charge and the pH of the medium. This is especially important in this case since the charge of cationic polymers is considered to influence antimicrobial activity.16 The results show that modified/unmodified PVAms are highly charged at pH ) 2 and that the charge drops significantly when the pH is increased. A significant charge does, however, remain at neutral pH on both unmodified and modified PVAms, the most prominent being PVAm C8. This can be related to antimicrobial activity, especially against B. subtilis. From the composition of the polyelectrolytes, it is possible to calculate a theoretical charge of the polyelectrolytes, assuming either that only the NH2 groups carry charges at pH ) 2 or that all amine groups are charged. It is furthermore assumed that no counterions are present in the pure polymers. These simple calculations are summarized in Table 4. In Table 4 it can be seen that, at pH ) 2, the experimental results correlate fairly well with the theoretical calculations, where only the NH2 groups are assumed to be charged, with the exception of pure PVAm and PVAm C8. The theoretical charges are also generally larger than the measured charges. This lower charge is probably due to the presence of counterions in the pure polymers, because they were only dialyzed against deionized water and not against water with a higher pH. This will no doubt lead to some protonation of the amino groups and a subsequent binding of counterions to the charged groups. This means that when weighing in samples for titration there will be fewer charged centers/unit mass. The higher charge of PVAm C8 is more difficult to explain, but it is suggested that the hydrophobic group of this polyelectrolyte can interfere with the titration dye,24,25 leading to an overconsumption of KPVS before the end-point in the titration is reached. It is not clear why this is not detected in the case of PVAm C12, but this polymer did show limited solubility in water, which might affect its titration behavior. The change of charge

with pH is more complicated to predict since some of these polyelectrolytes probably show complex solution behavior due to association phenomena when the charge is decreased. Naturally, these factors are important, but they were beyond the scope of the present investigation and will be considered in future research. It should also be stressed that the present results show that the charge does not seem to be a critical factor for the polyelectrolytes used in the present investigation. The molecular explanation of the observed antibacterial action remains to be identified. Previous research efforts have suggested that the charges of the polyelectrolytes will attract bacteria and strongly bind them to the surface. It has also been suggested that cationic polymer chains can further penetrate the phospholipid membrane of the bacteria to create cell death.7 Today, it is more or less clear that this is an oversimplification and that more complicated processes are probably involved in the bactericidal mechanism. The charges are definitely important in the physical attraction between the negatively charged bacteria and the positively charged polymer. In the present investigation, the charges on the PVAm polymers, even after interaction with the glass surface, are obviously sufficient to render the polymers bactericidal or bacteriostatic. It is also clear that the charge of the polyelectrolytes is not the most critical parameter for the polymers investigated in the present work, since PVAm C6 had the overall highest antibacterial efficiency despite being the polyelectrolyte with the lowest charge, as determined by PET. Regardless of the precise mechanism of action of the amphiphilic polymers, the combination of charge and hydrophobic substitution seems to be most important. The exact reason for this is not clear, but it has been shown27 previously that polyelectrolytes may induce a peeling of multilamellar vesicles and that the charge in combination with the hydrophobic substituents of the polymer will induce such strong interactions with the bacterial cell wall and its lipid bilayer that these components will preferably complex/interact with the polyelectrolyte and not with the other components of the cell wall. This will, hence, lead to a catastrophic decomposition of the cell membrane and to the final death of the bacterial cell. It must be stressed that this is only a hypothesis, and that it needs to be critically tested (potentially using model experiments with vesicles), though it is supported by earlier work.27 This followup work is currently in progress in the laboratory of the authors.

Conclusions The efficacy of a range of different PVAms, modified with hydrophobic side groups as well as unmodified, was assessed against gram-positive and gram-negative bacteria. All the PVAm types were proven to be active against the bacterial species, but at different concentrations. PVAm C6 was most potent against E. coli while PVAm C8 was more active against B. subtilis in its vegetative state. One reason for the antimicrobial activity of PVAm may be that these are sufficiently charged polyelectrolytes, even at the relatively high pH of 7.5. PVAm C8 showed the highest charge and was the most efficient polymer against B. subtilis, which suggests that the charge may affect some bacteria more than others. However, a combination of charge and hydrophobicity and probably other factors contribute to the antimicrobial activity of high molecular weight compounds, since PVAm C6 was more efficient against E. coli. PVAm C6 was also found active in the adsorbed state. The use of weak polyelectrolytes21 for antimicrobial surface treatment may be further enhanced since the adsorption behavior can be fine-tuned by controlling parameters such as pH and ionic

Antibacterial Properties of Polyvinylamine

strength. In this regard, PVAm needs to be further evaluated in the immobilized state to increase the knowledge of possible paths to render a surface antimicrobial or to inhibit the growth of unwanted bacteria, based on the method of physical adsorption. Acknowledgment. SCA Hygiene AB, BASF AG, and Vinnova are acknowledged for financial support. BASF AG is gratefully acknowledged for supply of polyelectrolytes and support, both SCA Hygiene and BASF AG are acknowledged.

References and Notes (1) Denyer, S. P.; Stewart, G. S. A. B. Int. Biodeterior. Biodegrad. 1998, 41, 261–268. (2) Ikeda, T.; Hirayama, H.; Yamaguchi, H.; Tazuke, S.; Watanabe, M. Antimicrob. Agents Chemother. 1986, 30, 132–136. (3) Ikeda, T.; Yamaguchi, H.; Tazuke, S. Antimicrob. Agents Chemother. 1984, 26, 139–144. (4) Kawabata, N. Prog. Polym. Sci. 1992, 17, 1–34. (5) Kenawy, E.-R.; Abdel-Hay, F. I.; El-Shanshoury, A. E.-R. R.; ElNewehy, M. H. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 2384– 2393. (6) Tashiro, T. Macromol. Mater. Eng. 2001, 286, 63–87. (7) Tiller, J. C.; Liao, C.-J.; Lewis, K.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5981–5985. (8) Lin, J.; Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Biotechnol. Lett. 2002, 24, 801–805. (9) Cen, L.; Neoh, K. G.; Ying, L.; Kang, E. T. Surf. Interface Anal. 2004, 36, 716–719.

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(10) Abel, T.; Cohen, J. I.; Engel, R.; Filshtinskaya, M.; Melkonian, A.; Melkonian, K. Carbohydr. Res. 2002, 337, 2495–2499. (11) Cen, L.; Neoh, K. G.; Kang, E. T. J. Biomed. Mater. Res., Part A 2004, 71A, 70–80. (12) Lewis, K.; Klibanov, A. M. Trends Biotechnol. 2005, 23, 343–348. (13) Park, D.; Wang, J.; Klibanov, A. M. Biotechnol. Prog. 2006, 22, 584– 589. (14) Pasquier, N.; Keul, H.; Heine, E.; Moeller, M. Biomacromolecules 2007, 8, 2874–2882. (15) Franklin, T. J.; Snow, G. A. Biochemistry of Antimicrobial Action, 3rd ed.; Chapman and Hall: London, 1981. (16) Kuegler, R.; Bouloussa, O.; Rondelez, F. Microbiology (Reading, U. K.) 2005, 151, 1341–1348. (17) Maillard, J.-Y. Pharm. J. 2005, 275, 639–642. (18) Qiu, Y. X.; Zhang, T. H.; Ruegsegger, M.; Marchant, R. E. Macromolecules 1998, 31, 165–171. (19) Chen, X. N.; Wang, Y.; Pelton, R. Langmuir 2005, 21, 11673–11677. (20) Wang, Y.; Chen, X. N.; Pelton, R. Langmuir 2006, 22, 4952–4958. (21) Gebhardt, N.; Zeller, D.; Nilz, C.; Steuerle, U.; Johansen, C. U.S. Patent 6261581, July 17, 2001. (22) Westman, E. H.; Ek, M.; Wagberg, L. Holzforschung 2009, 63, 33– 39. (23) Champ, S.; Koch, O.; Ek, M.; Westman, E.; Wagberg, L. WO Patent 2008/055857, May 15, 2008. (24) Terayama, H. J. Polym. Sci. 1952, 8, 243–253. (25) Horn, D. Prog. Colloid Polym. Sci. 1978, 65, 251–264. (26) Pefferkorn, E.; Elaissari, A. J. Colloid Interface Sci. 1990, 138. (27) Vivares, E.; Ramos, L. Langmuir 2005, 21, 2185–2191.

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