Secondary and Tertiary Polydiallylammonium Salts - ACS Publications

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Biomacromolecules 2009, 10, 2976–2986

Secondary and Tertiary Polydiallylammonium Salts: Novel Polymers with High Antimicrobial Activity Larisa M. Timofeeva,*,† Natalia A. Kleshcheva,† Antonina F. Moroz,‡ and Lyubov V. Didenko‡ A. V. Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences, Leninsky prosp. 29, Moscow 119991, Russia, and N. F. Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Gamaleya street 18, Moscow 123098, Russia Received April 16, 2009; Revised Manuscript Received August 12, 2009

Antimicrobial activity of secondary and tertiary poly(diallylammonium) salts (PDAAs) had not been reported before. Due to difficulties with preparation of polymers from the monomers of the diallylamine (DAA) series in the nonquaternary form, up to recently it was not possible to obtain PDAAs with a sufficiently high molecular mass. Here, we describe the investigations of antimicrobial activity of novel water-soluble cationic polyelectrolytes of the PDAA series, namely secondary poly(diallylammonium trifluoroacetate) (PDAATFA) and tertiary poly(diallylmethylammonium trifluoroacetate) (PDAMATFA), in synthesis of which we have recently succeeded, against gram-positive and gram-negative bacteria, and fungi. We have studied the effect of molecular weight (polymeric chain length) and ionic strength of solution on the biocidal efficiency of those polymers; in addition, the concentration dependences of PDAATFA reduced viscosity in salt-free and KCl aqueous solutions have been investigated. The antimicrobial properties of polybase polydiallylamine (BPDAA), which was obtained in an aqueous solution of PDAATFA in presence of alkali, have been also studied as well as biocidal activity of commercial open-chain polybase branched PEI. Those PDAATFA, BPDAA and PEI polymers served as the systems to study the structure-activity relationships. Transmission electronic microscopy study was carried out to characterize the mode of antimicrobial action of PDAATFA using E. coli. It was shown that the synthesized PDAATFA and PDAMATFA exhibit, unlike the quaternary polymers of this series, a rather high biocidal efficiency that is comparable with the activity of known effective cationic polymer biocides or exceeds it. Novel polyelectrolytes exhibit quite strong biocidal properties at different conditions including aqueous solutions of moderate ionic strength (serum, 0.01 M/0.1 M) and aqueous-alkaline solutions (pH 10.5) until the macrochain retains some positive charge, but complete neutralization of the polyelectrolyte in a 1 M salt solution results in the loss of its biocidal activity. The obtained results evidence that the structure of links, which combine the hydrophobic pyrrolidinium rings with the hydrophilic secondary/tertiary ammonium groups, is responsible for the high biocidal activity of the PDAAs. Polymeric nature of the synthesized compounds is one of the most significant factors of their bactericidal efficiency, unlike their high fungicidal activity, which is evidently related to the secondary/tertiary pyrrolidinium cycle.

Introduction Cationic quaternary poly(diallyldialkylammonium) salts (QPDAAs) obtained by free-radical polymerization of the quaternary diallylammonium monomers are well studied: kinetics and mechanisms of their preparation and properties of the polymers were investigated in detail.1-3 Unlike a significant number of known cationic quaternary ammonium compounds (QACs) and polymeric quaternary ammonium compounds (PQACs) that exhibit high biocidal activity,4,5 antimicrobial activity of QPDAAs is not efficient.4a,b The first representative of QPDAA series, poly(diallyldimethylammonium chloride) (PDADMAC), exhibits weak antimicrobial effect.4a,b In the opinion of Merianos, the moderate activity of QPDAAs is related to that the quaternary nitrogen is pendant away from backbone chain and requires lipophilic groups and high charge density for biocidal effect.4a,b In relation with this, of special interest is the high biocidal efficiency of novel water-soluble nonquaternary polymers of poly(diallylammonium) salts series (PDAAs) discovered by the * To whom correspondence should be addressed. E-mail: timofeeva@ ips.ac.ru. † A. V. Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences. ‡ N. F. Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences.

authors,6 namely, the secondary poly(diallylammonium trifluoroacetate) (PDAATFA) and tertiary poly(diallylmethylammonium trifluoroacetate) (PDAMATFA), which have been synthesized recently.6,7 The antimicrobial activity of secondary and tertiary PDAAs had not been reported before. Due to difficulties in preparation of polymers from monomers of the diallylamine (DAA) series in nonquaternary form, which are related to degradative chain transfer to monomer and kinetic chain termination, up to recently it was not possible to obtain secondary and tertiary PDAAs with a sufficiently high molecular mass.8-11 We have proposed an approach that is essentially based on the creation in the polymerizing medium of a dominant amount of monomers of the DAA series in protonated form, owing to which the competitive ability of the chain transfer to monomer reaction diminishes and the degradative chain transfer transforms into the effective one.12 In accordance with this approach, the methods has been employed for the synthesis of novel monomer systems, namely trifluoroacetic salts of the monomers of DAA series, and the cationic polyelectrolytes on their bases. Polycations PDAATFA and PDAMATFA have been prepared with Mw up to 60-65 kDa (Scheme 1).6,7 In the processes of polymerization, where chain termination is controlled by bimolecular mechanism, relatively low values of the chain transfer to monomer constant CM have been obtained, for

10.1021/bm900435v CCC: $40.75  2009 American Chemical Society Published on Web 10/01/2009

Secondary and Tertiary Polydiallylammonium Salts

Biomacromolecules, Vol. 10, No. 11, 2009

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Scheme 1. Schematic Representation of the Radical Cyclopolymerization of DAATFA and DAMATFAa

a Conditions: DAATFA (DAMATFA) aqueous solutions, initiator I ammonium persulphate (APS); termopolymarization, T ) 30; 50 °C; photopolymerization, hν, T ) 20 °C.

example, for the polymerization of DAMATFA initiated by ammonium persulphate (APS) CM ) (3.5-12.2) × 10-3, and these values are comparable with magnitudes of CM ((1.4-4.2) × 10-3) for diallyldimethylammonium chloride (DADMAC) with the same initiator.2,6,7 Poly(diallylammonium) salts in nonquaternary form are promising, since their properties may be varied with pH of medium as well as by chemical modification, due to presence of ∼NH2+ or ∼NH+ groups. These amino groups make possible the specific hydrogen bond interactions along with the electrostatic interactions with chemical (or biochemical) systems that may enhance the surface activity of polymers. Presence of NH2+/ NH+ groups in pyrrolidinium rings of PDAAs links containing six -CH2- groups makes the PDAAs somewhat similar to open-chain polyamine polyhexamethylene guanidine (PGMG), which is known as biocidal polymer with quite high efficiency as well as polyhexamethylene biguanides (PHMB).4a-d,5c,d PHMB and PHMG are sufficiently hydrophobic polyelectrolyte salts with moderate molecular weight and chain length possessing high charge density (average charge per link), due to high dissociation degree in aqueous solutions, and ability to specific hydrogen bond interactions. At the same time, Klibanov and co-workers have demonstrated that nonquaternary polybase branched polyethylenimine (PEI) with the primary, secondary, and tertiary amino groups in the links is not effective in making material (glass) surface bactericidal,13a while quaternized and N-alkylated polycations PEIs attached to materials of different nature inactivate both pathogenic bacteria and influenza virus with high efficiency.13 The effect of an increase in the positive charge, the length of the alkyl tail, and the hydrophobicity of polymer on the efficiency of different PQACs have been reported and described previously.4a,b,5a-c,13a,b,14 Sellenet et al. has showed that copolymerization with hydrophilic comonomer can significantly improve both the efficacy and the biocompatibility of antibacterial quaternized poly(vinylpyridine).5f Sen et

al. has investigated antimicrobial and hemolytic (toxicity) properties of amphiphilic polycations as a function of the spatial positioning of the positive charge and the hydrophobic alkyl tail.5g The synthesized water-soluble PDAA salts combine several important properties to exhibit surface activity in aqueous solution, namely, the significant hydrophobicity due to pyrrolidinium links, a positive charge (according to quantum chemical calculations, the greater part of pyrrolidinium charge is located on ∼NH2+/∼NH+ groups and the rest on the protons of Cδ-Hδ+, Cδ-H2δ+ and Cδ-H3δ+ groups of the links),12 the ability to form hydrogen bonds (with oxygen atoms of phospholipids as well) and the corresponding hydrophilicity. One may expect that, owing to these factors, the nonquaternary PDAAs will have an advantage over both water-soluble QPDAAs and PQACs, and will acquire properties other than those of quaternary poly(diallylammonium) salts. In the present work, we focused on the study of antimicrobial potential of the novel polymers in different conditions. Herein, we describe the investigations of antimicrobial activity of PDAATFA and PDAMATFA polymers against some representative gram-positive and gram-negative bacteria and also fungi of Candida species. The latter was chosen because of Candida and, especially, Candida albicans is pathogenic to humans and causes serious infectious diseases.4e-g Candidiasis is a typical hospital-acquired infection and there is an evergrowing need for prevention of it.4e-g We have studied the effect of molecular weight (polymeric chain length) and ionic strength of solution on the biocidal efficiency of those polymers. The antimicrobial properties of polybase polydiallylamine (BPDAA), which was obtained in an aqueous solution of PDAATFA in presence of alkali, have been also studied as well as biocidal activity of commercial open-chain polybase branched PEI. Those PDAATFA, BPDAA, and PEI polymers served as the systems to study the structure-activity relationships. Transmission

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electronic microscopy (TEM) study was carried out to characterize the mode of antimicrobial action of PDAATFA using E. coli (Gram-negative bacterium) and also to make evident that the polymers exhibit bactericidal efficiency.

Experimental Section Materials. We used reagents grade chemicals purchased from CHIMMED (Russia), except DAA (Germany, Aldrich, analytical grade), and trifluoroacetic acid (TFA; Germany, “Riedel-de-Hae¨n”, g99%). Polyethylenimine (PEI; branched polymer, Mw 45 kDa, 45 wt % solution in water) was purchased from Polymin (Germany) and used without purification. Methods of Synthesis. Diallylmethylamine (DAMA) was prepared from methylamine and allyl chloride using the known procedure described elsewhere.15a Monomer salts DAATFA and DAMATFA were prepared from DAA and DAMA, respectively, and TFA according to the elaborated methods described elsewhere.6,7 PDAATFA and PDAMATFA polymer salts were prepared by free radical polymerization of DAATFA and DAMATFA, respectively, in aqueous solutions in presence of radical initiator APS (see the Scheme 1) according to the methods described elsewhere.6,7 The conditions were as follows: T ) 30 °C; (1) [M] ) 2 mol/L and [APS] ) 7 mM for preparing of samples with Mw ) 24 kDa (PDAATFA, [η] ) 18 mL/g, and PDAMATFA, [η] ) 16 mL/g); (2) [M] ) 3 mol/L and [APS] ) 1 mM for preparing of sample PDAATFA with Mw ) 62 kDa ([η] ) 24 mL/g); (3) [M] ) 3 mol/L and [APS] ) 5 mM for preparing of sample PDAMATFA with Mw ) 55 kDa ([η] ) 24 mL/g). Composition and structure of these polymers were confirmed by elemental analysis and 1 H and 13C NMR data. Methods of Analysis. The 13C NMR spectrum was observed on a Bruker DRX 500 spectrometer at 303 K, the chemical shifts are reported relative to acetone (δc 31.45). The 1H and HSQC spectra were observed on a Bruker AVANCE 600 spectrometer, 303 K. The chemical shifts in 1H NMR spectrum are reported relative to internal TSP (δH 0.0 ppm). The fluorine content of the synthesized products was evaluated by the spectrophotometric analysis using a SPECORD M-40.15b The intrinsic viscosity [η] was determined using an Ubbelohde viscometer at 30 °C in 1 N NaCl. The weight-average molecular weight Mw was determined by the sedimentation ultracentrifugation in a MOM 3180 ultracentrifuge in 1 N NaCl solution (T ) 25 °C; the rotor rotation speed was 50000 rpm). The nonstationary equilibrium method (Archibald’s method) was used.16 Characterization Data of Polymers. On the basis of analysis for fluorine, prepared polymers contain one molecule of TFA per monomeric link: Calcd for PDAMATFA (C9H14NO2F3)n (225.2)n (%): C, 48.00; H, 6.22; N, 6.22; F, 25.33. Found (%): C, 47.00; H, 6.07; N, 6.12; F, 25.03. Calcd for PDAATFA (C8H12NO2F3)n (211.18)n (%): C, 45.52; H, 5.69; N, 6.63; F, 26.99. Found (%): C, 45.47; H, 5.57; N, 6.61; F, 26.84. The structures of synthesized polymers have been established on the basis of elemental analysis data and by comparison of 1H and 13C NMR spectra of the polymers to the spectra of quaternary PDADMAC and low molecular weight polybase poly(diallylmethylamine) (BPDAMA, the weight-average degree of polymerization Pw ) 20).2,17 We have found that the cis/trans ratio for cis- and trans-substituted pyrrolidine rings for the isolated polybase BPDAMA (Mw ) 32 kDa, Pw ) 288), is equal to approximately 5:1.7,18a It corresponds to the data derived from 13C spectral characteristics for the low molecular weight BPDAMA.17b Note that in the 13C spectrum of PDAMATFA shown here (Figure 1a), there are characteristic signals of carbon atoms C13F3 (quartet, from 113.0 to 122.0 ppm) and C13OO- (163.0 ppm); the last signal has a downfield shift in comparison to the signal for nonionized COOH group in TFA (166.0 ppm) due to formation of carboxyl anion.18b-d 1 H spectra of the polymers (Figure 1b, the 1H spectrum of PDAMATFA, the assignment of signals of the backbone protons in the 1H NMR spectrum has been performed using the HSQC spectrum

Timofeeva et al. of the sample) contain low-intensity proton signals of CH2dCH- group (region from 5.5 to 6.0 ppm). The low-intensity signals of vinyl group are detected also at 127.0-130.0 ppm in the 13C spectrum of the polymers (e.g., 13C spectrum of PDAMATFA, Figure 1a). (Note that these signals do not disappear after reprecipitating a sample with Et2O and are still observable after several years storage of polymer salts.) The signals of vinyl and Me (detected in the 13C spectrum in the region from 16.5 to 12.5 ppm that is not represented in Figure 1a) groups have been assigned to the end groups in the polymer chain, which are formed in accordance with the mechanism shown on the Scheme 1.7,18a Similar signals were first observed in 1H and 13C NMR spectra of PDADMAC and were attributed to terminal groups in the polymer chain.2,17a The signals of terminal Me groups (14.6 and 19.3 ppm for cis- and trans-Me groups, respectively) were detected also for the low molecular weight BPDAMA by using inversion/recovery technique for identification of initiation and termination groups.18e Topchiev and co-workers have shown for the quaternary PDADMAC that one macromolecule contains in average one CH2dCH- end group.18f Taking into consideration the similar initiator upon polymerization of DADMAC, DAATFA, and DAMATFA, and the abovementioned comparable magnitudes of CM, we have assumed that that ratio is true for the secondary and tertiary PDAA salts.7,18a To estimate the number-average degree of polymerization Pn, we have compared the intensity of proton signals of the end CH2dCH- group with sum of the intensities of signals from macrochain protons using 1H spectra of several polymer salts samples (see Figure 1b).7,18a Thus, the polydispersity coefficient δ ) Pw/Pn ) 1.85 has been evaluated, and, correspondingly, the number-average molecular weight Mn was estimated.7,18a Method of Testing of Antimicrobial Activity. Testing of Polymers and Monomers. We analyzed the biocidal activity of nonfractionated polymer samples PDAMATFA (Mw ) 55 kDa and Mw ) 24 kDa) and PDAATFA (Mw ) 62 kDa and Mw ) 24 kDa), and branched PEI (Mw ) 43 kDa) against microorganisms from the International Collection of Museum Strains, Russian Tarasevich State Institute of Standardization and Control of Biomedical Preparations (TSISCMBP). Tests were carried out according to the Official Instruction for Analyses.19a A polymer sample to be tested was dissolved in twice-distilled water to an initial concentration of 1000 µg/mL (∼0.1 wt %), pH ) 4.6 for PDAATFA and pH ) 4.8 for PDAMATFA initial solutions. A series of 10 test tubes each containing 1 mL of polymer solution with different concentrations was prepared by sequential dilution by a factor of 2. A museum test culture was grown on beef-extract agar plate for 24 h at 37 °C; afterward a smooth [S-type] colony was selected and was subcultured at the same conditions. This final culture was suspended in twice-distilled water to a concentration of 5 × 108 colony-forming units (CFU)/mL according to TSISCMBP Optic Turbidity Standard No 10. Cell suspension in the amount of 0.1 mL (5 × 107 CFU) was added to each test tube. The control test tube contained 1 mL of twicedistilled water and 0.1 mL of suspension. After a 1.5 h long contact, seedings (using the streak seeding method) were done by the platinum loop to the corresponding nutrient medium in a Petri dish divided into 10 sections. The dish was incubated for 24 h at 37 °C. Afterward the bacterial colonies grown on the nutrient medium were counted on a light box, and the minimal bactericidal (killing) concentrations were determined, at which the total inhibition of micro-organism growth (MIC) was observed. All experiments were carried out at least 4 times, and the data are reported as the mean values ( experimental errors, which were calculated according to the recommended procedures.19b Note that in the case of the biological testing of quaternary ammonium salts, the use of phosphate buffer is not recommended by the Official Instruction to avoid possible precipitation which had been observed, in particular, upon testing of polymer guanidines in phosphate buffer.19a It should be emphasized that the values of pH of all tested polymer salts solutions increase with the dilution until pH 6.4-6.6 near to the neutral value. This allows control done at the neutral pH. The values of pH of the solutions at MIC are given in Table 2 below.



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Figure 1. 13C (a) and 1H (b) NMR spectra of PDAMATFA (Mw 55 kDa) in D2O. Signals of the end -CHdCH2 group are detected in the 13C and 1 H spectra (see Scheme 1 and text). The assignment of signals of the backbone protons in the 1H NMR spectrum has been performed using the HSQC spectrum of the sample.

The effect of the monomer salts DAATFA and DAMATFA, and TFA was investigated in the same way. Testing of PEI. Antimicrobial activity of PEI was tested as described above. A sample of PEI was dissolved to an initial concentration of 1000 µg/mL (∼0.1 wt %, pH 9.8). (Note that the values of pH of the tested PEI solutions decrease with the dilution until pH 7.4-7.2 near to the neutral value. The pH value of PEI solution at MIC is given in Table 5 below.) Testing in Serum. A normal horse serum from the Stavropol Scientific-Research Institute of Epidemiology and Microbiology was

used. A series of 10 test tubes each containing 1 mL of PDAATFA (P-50 sample, Mw ) 24 kDa) aqueous solution with different concentrations was prepared by serial dilution and 0.6 mL of serum was added to test tubes. Afterward, 0.1 mL of cell suspension was added to each test tube as described above. The control test tube contained 1 mL of twice-distilled water, 0.6 mL of serum, and 0.1 mL of cell suspension. Testing in KCl Solutions. KCl aqueous solutions (0.01, 0.1, and 1 M) were prepared in twice-distilled water. P-50 polymer sample was dissolved in a 0.01 M KCl aqueous solution to an initial concentration of 1000 µg/mL. A series of 10 test tubes, each containing 1 mL of

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P-50 with different concentrations, was prepared by serial dilution in a 0.01 M KCl; afterward, 0.1 mL of cell suspension was added to test tubes, as described above. The control test tube contained 1 mL of a 0.01 M KCl aqueous solution and 0.1 mL of cell suspension. Testing in a 0.1 M (1 M) KCl aqueous solution was carried out in the same way. Polymer + NaOH Composition. When obtaining polybase from the secondary or tertiary PDAAs polysalts by isolating it in a strong alkaline medium and storing that polybase, postpolymerizing intermacromolecular cross-linking reactions occurred.18a Therefore for studying biocidal activity of water-soluble polybase polydiallylamine (note that tertiary polydiallylmethylamine is not water-soluble),7,18a a fresh aqueous solution of PDAATFA (P-50 sample) with an equimolar amount of NaOH (the equimolar concentrations of alkali and polymer expressed in moles of monomer links) was used. According to our titration data on the PDAAs polysalts, polymer PDAATFA exists as water-soluble polybase BPDAA in the presence of an equimolar amount of NaOH:18g

Under the similar titration of polysalt PDAMATFA, water-nonsoluble polybase BPDAMA precipitated. Structures of isolated BPDAA and BPDAMA have been confirmed previously by elemental analysis and 13C and 1H NMR spectra.7,18a Because intermacromolecular crosslinking is unlikely in the fresh solution at the low polymer concentrations (∼0.1 wt % of PDAMATFA and less) in the presence of an equimolar amount of NaOH (i.e., soft alkaline conditions), one may expect that polybase BPDAA obtained from polysalt PDAATFA with Mw ) 24 kDa has Mw value of 11 kDa. P-50 polymer sample was dissolved in twice-distilled water and an 0.1 M NaOH aqueous solution was added to ensure the equimolar ratio of the polymer and NaOH; the obtained initial concentration of P-50 was 1000 µg/mL (∼0.1 wt %), pH 10.5. Afterward, a series of 10 test tubes each containing 1 mL of the equimolar composition with different concentrations was prepared by serial dilution in twice-distilled water and cell suspension was added as described above. (Note that the values of pH of the tested polymer + NaOH solutions decrease with the dilution until pH 7.4-7.2 near to the neutral value. The values of pH of polymer+NaOH solutions at MIC are given in Table 5 below.) The control test tube contained 1 mL of twice-distilled water and 0.1 mL of cell suspension. An equimolar TFA+NaOH composition was tested in the same way as the polymer + NaOH composition; the obtained initial concentration of TFA was 1000 µg/mL (∼0.1 wt %), pH 7.4. Electronic Microscopy. The biological effect of PDAAs on microorganisms was analyzed with a JEM-100 B transmission electron microscope at an operating voltage of 80 kV. The effect of PDAATFA (MW ) 24 kDa) on gram-negative E. coli bacterium was studied. A cell suspension with E. coli (the culture from TSISCMBP Museum Collection) prepared as it is described above (0.1 mL, i.e., 5 × 107 CFU) was maintained for 1.5 h in PDAATFA solutions with bactericidal (125 µg/mL) and bacteriostatic (sub-bactericidal, 65 µg/mL) concentrations and in twice-distilled water (control experiment) also. TEM samples were prepared in the following way. The bacterial cells from all of the mixtures were washed with twice-distilled water and concentrated by centrifuging at 6000 rpm (Hettich EBA 20 centrifuge, Germany) to a concentration of 108 CFU/mL. Then cells were fixed according to the standard method of Ito and Karnovsky: aqueous solution of 3% paraformaldehyde and picric acid (in the presence of 1 N NaOH) in an 0.2 M cacodylate buffer (pH 7.2) and 25% glutaraldehyde solution.20a Postfixation was carried out with 1% OsO4 in a

Timofeeva et al. 0.2 M cacodylate buffer (pH 7.2) followed by an uranyl acetate fixation, 1% aqueous solution in an 0.2 M maleate buffer (pH 6.0).20b Then bacterial cells were dehydrated in alcohol solutions of increasing concentrations (from 50 to 70 to 96 and to 100%) and covered with methacrylic gum, LR White, according to a method described elsewhere.20c Ultrathin sections (thickness 200-300 Å) were cut with an LKB 3 ultratome (LKB, Sweden) using a glass knife. A section was placed on a 200 mesh Formvar coated nickel grid (Polyscience, Ltd., Germany). The sections were stained according to Reynolds method and analyzed with the electronic microscope.21 A total of 10 grids were prepared for every one of the samples. The conclusion about lysed or “living” cells was obtained by means of analysis of at least 20 random TEM fields of view on every grid and by observing at least 200 cells for every sample.

Results and Discussion Study of Antimicrobial Activity. One can see in the Table 2 that the polymers PDAATFA and PDAMATFA exhibit high biocidal activity against a significant number of bacteria and Candida fungi. It is important that the corresponding monomer salts and TFA exhibit activity only in the case of the highest initial experimental concentration or do not exhibit it at all. The efficiency of the polymers depends on the microorganism and is distinct for the investigated polymers: substitution of a H atom on nitrogen with a Me group affects the activity in different ways and may result in both reduction (or increase) in the MIC against different microorganisms by a factor of 2-4 (Table 2). A significant decrease (by a factor of ∼2.5) in molecular weight (polymeric chain length) results in growth of the MIC against studied E. coli and S. aureus by a factor of ∼10-20, while the fungicidal activity virtually does not change (see Table 2). It should be stressed that, in each case, the antimicrobial effect of a polymer sample with a given Mw value is actually averaged over the effect of polymers with molecular weights Mw-Mn within the polydispersity range (δ ) 1.85). In comparing the effects of samples with different Mws, it is important to take into account that if the value of Mw is decreased by a factor of ∼2.5, the corresponding ranges of Mw-Mn are not overlapping. The MIC of PDAATFA and PDAMATFA quoted for the samples with higher MW (see Table 2) are lower, in particular, against S. aureus than the minimal bactericidal concentration of 6.6 to 10 µg/mL (6.6-10 ppm) obtained for PQAC with positively charged nitrogen atoms located in the backbone of macrochain and high charge density, namely, polyionene, with an optimal Mw value (for bactericidal efficiency) under comparable conditions of experiment.4a,b The activity of PDAATFA and PDAMATFA against S. aureus is close to that of poly(dodecyldimethyl[vinylbenzyl]ammonium chloride), whose alkyl radical C12 significantly enhances the antimicrobial effect; the corresponding MIC value is 0.5 µg/mL (0.5 ppm).4a,b As a whole, the optimal values of MIC of secondary and tertiary PDAAs with higher MW, which range from 1.5 to 30 µg/mL, are comparable to the range of MICs of known efficient QACs, PQACs, and poly(biguanides), which are from 10 to 50 µg/mL.4a,b Polycations PDAATFA and PDAMATFA exhibit higher bactericidal activity against gram-positive bacteria than gram-negative bacteria (see Tables 1 and 2). Such behavior is similar to that observed for cationic PQAC and polymeric biguanides.4a-d,5d McDonnell and Russell note that, as a whole, gram-negative microorganisms exhibit stronger resistance to antiseptics and disinfectants than gram-positive ones (with the exception of gram-positive mycobacteria) and fungi of the Candida genus occupy an intermediate position.23



Table 1. Solubility of the Synthesized Monomers and Polymer Salts in Various Solventsa solvent sample DAATFA PDAATFA DAMATFA PDAMATFA a

water ether acetone chloroform methanol hexane benzene s s s s

s i s i

s i s s

s i s i

s s s s

i i i i

s i i i

s ) soluble; i ) insoluble.7

It should be emphasized that the pH values of the all polymer solutions at MIC are near to the neutral value unlike low pH of the monomer salts and TFA at the highest initial concentration, at which they do not exhibit biocidal efficacy (Table 2, see also ref about the biological action of TFA). Thus, there is evidently no biocidal impact of pH in the case of the polymers biocidal action. Electronic Microscopy Study. The TEM study of three samples prepared from the bacterial cells shows that morphologic changes in E. coli caused by the effect of bacteriostatic and bactericidal PDAATFA concentrations are basically the same. The analysis of TEM images of the test (Figure 2a-c) and control (Figure 2d) bacterial cells shows that the changes that occur in submicroscopic organization of the gram-negative bacteria involve all structural components of the cell: cell wall (CW), cytoplasmic membrane (CPM), cytoplasm (CP), ribosomes, and nucleoid. If the bactericidal concentration is used, the number of fully damaged cells was significantly higher than in the case of bacteriostatic concentration. In the TEM image in Figure 2a, one can clearly see a segment of outer membrane of the bacterium CW covered with granular structures having an average diameter of ∼21.8 nm that may be identified with polymer globules. The size of the granules and the shape of their contact with the CW agree well with those of similar structures on a surface of model negatively charged lipid membrane (cardiolipin) that were observed at negative-stain electron micrographs of the membrane interacting with PDAATFA (MW ) 24 kDa).22 An increase in the electronic image density of the outer membrane is observed at the contact sites (Figure 2a). In some places of contact with the polymer, partial or total rupture of the CW and CPM of bacteria is noted (Figure 2b). A significant increase in periplasmic space is most probably evidence of a disturbance of the water-salt metabolism in the bacterial cells (Figures 2a,b). A boost of the total image density of the CP and the formation of lysis zones in it are observed that may be linked to a coagulation of intracellular components (Figure 2b). It should be noted that structures of the types (a) and (b) are observed in micrographs of the cells maintained at the bacteriostatic concentration of the polymer. The final result of the PDAATFA action is complete disruption of the bacterial cells and formation of detrital masses (Figure 2c). Images of this type are characteristic of the cells maintained at the bactericidal concentration of the polymer. In the sample prepared from the bacterial cells maintained at the bactericidal concentration, detrital masses have been mainly observed and only single destructive cells with heavy damage have been discovered in several TEM fields. In the control experiment, the E. coli cells have the structure that is typical of gramnegative bacteria (Figure 2d). The obtained results confirm the concept of Franklin and Snow of the effect of polymeric biocides, which involves disorganization of CPM, followed by rupture of the membrane leading to death of bacterium.24 The main features of the changes in the E. coli cells under the effect of PDAATFA

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(Figure 2a-c) are close to those described by McDonnell and Russell for different microorganisms that underwent the effect of cationic PQAC and polymeric biguanides.23 In the case of the interaction of PHMB with E. coli, Maillard distinguishes a number of sequential events, including specific adsorption on negatively charged groups of the outer membrane compounds, damage of the outer membrane, binding with CPM phospholipids, an increase in the permeability of the membrane with release of K+ ions, and full loss of the membrane functions and coagulation of intracellular components that result in a bactericidal effect.25 Numerous investigations of interactions of different cationic polyelectrolytes with model lipid membranes showed that the complex is formed between the membrane and the polycation, which is stabilized by electrostatic interaction of free links of the polycation and negatively charged groups of lipid molecules.26 Complexation is accompanied by migration of the negatively charged molecules to the outer leaflet of the membrane and lateral segregation of the negatively charged lipids.26 Ikeda et al. showed that PHMB by interacting with negatively charged bilayer lipid membranes causes aggregation of acid lipids in the adsorption area.27 An increase in the electronic-optical image density of the outer membrane of the E. coli cell in the area of contact with the polymer (Figure 2a) is most probably a consequence of the aggregation in the lipid bilayer of the outer membrane. Effect of Molecular Weight (Polymer Chain Length). To assess the effect of molecular weight Mw on the antimicrobial activity of polymers, it is useful to represent MIC of polymers in terms of the number of polymer molecules NMIC in 1 mL of the solution. One can see in Table 3 that when Mw values increase by a factor of ∼2.5, corresponding NMIC values diminish by a factor of 20-50 upon interaction of PDAAs with grampositive and gram-negative bacteria. It may be related to several factors, which can enhance biocidal efficiency with the growth of Mw. Our study of concentration dependencies of equivalent electric conductivity of PDAATFA polysalts aqueous solutions (specific conductivity per mole of monomer links) shows that the ionization degree of the polyelectrolyte with Mw 62 kDa (Pw ) 294) is very close to that of PDAATFA with Mw 24 kDa (Pw ) 114) at equal concentrations in the studied concentration range, including highly diluted solutions.28 Therefore, the observed enhancement of the bactericidal efficiency is initially related to such evident factors as (i) the growth of hydrophobic mass of polymer, (ii) an enlargement of a polymer coil, and (iii) an increase in a net charge and absolute number of active cationic centers on a single polyelectrolyte molecule. One may expect that the above-mentioned changes of polymer characteristics should result in (i) an increase in a probability of adsorption of polymer molecules from solution on a cell and, simultaneously, (ii) a decrease in average number of polymer molecules that is necessary and sufficient to achieve the biocidal effect. Consequently, polycation with the higher Mw is able to produce inhibitory action at a concentration less than MIC of the polymer with the lower Mw value. Corresponding diminution of the inhibitory concentration leads to an increase in the dissociation and ionization degree of the polycation (dependence of dissociation degree of polyelectrolyte on its concentration in aqueous solution is considered in the next section) that causes additional boost in its biocidal efficiency and makes possible a further reducing of the inhibitory concentration. One may assume that this positive feedback results in such significant decreases in MIC and NMIC values of the PDAAs (limited by

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Table 2. Minimal Bactericidal Concentrations (MIC) of Aqueous Solutions of Polymersa MIC, µg/mLb microorganisms

PDAATFA, Mw 62 kDac

PDAMATFA, Mw 55 kDad

PDAATFA,Mw 24 kDae

PDAMATFA, Mw 24 kDaf

momomer salts, TFA

Escherichia coli, ATCC 25922 Staphylococcus aureus, ATCC 6538 P Candida albicans, ATCC 865-653 Pseudomonas aeruginosa, ATCC 9027 Proteus mirabilis, 47 Klebsiella pneumoniae, ATCC 13883

15 ( 1.8 1.5 ( 0.3 1.5 ( 0.3 125 ( 7.5 31 ( 3.1 15 ( 1.8

7 ( 1.0 7 ( 1.0 3.5 ( 0.6 31 ( 3.1 31 ( 3.1 62 ( 5.0

125 ( 7.5 31 ( 3.1 3.5 ( 0.6

62 ( 5.0 62 ( 5.0 3.5 ( 0.3

inactive at 500g 500g inactive at 500g

a All experiments were carried out at least four times, and the mean values and experimental errors were calculated according to the recommended procedures.19b b The values of pH of the polymer solutions increase from 6.2 to 6.6, with a variation of a concentration from 125 to 1.5 µg/mL. c Pw ) 294. d Pw ) 244. e Pw ) 114. f Pw ) 107. g 500 µg/mL is the highest studied concentration of items; at this concentration of items, the values of pH are following: pH 4.8-5.0 for the monomer salts solutions, and pH 2.9 for TFA solution.

Figure 2. Transmission electron micrographs. TEM images of the E. coli bacterium, the culture of which was prepared and maintained during 1.5 h in solutions of PDAATFA (Mw ) 24 kDa) at the bactericidal (125 µg/mL) and bacteriostatic (62 µg/mL) concentrations (a-c), as it is described in the text, and then analyzed by TEM; control culture maintained in twice-distilled water (d): (a) sticking of polymer to the outer membrane of the CW (noted by arrows), v 152000× magnification; (b) increased periplasmic space (noted by point arrows), increase in the image density of the CP (noted by dashed arrow), and the lysis zones in the CP (noted by arrow), v a 34000× magnification; (c) cellular detritus, v a 36000× magnification; (d) control culture of E. coli, v a 26000× magnification. See text for details. Table 3. Minimal Bactericidal Concentrations of Aqueous Solutions of Polymers Expressed in Terms of the Number of Polymer Molecules NMIC × 10-12/1 mLa PDAATFA

PDAMATFA

microorganisms

Mw 62 kDa

Mw 24 kDa

Mw 55 kDa

Mw 24 kDa

Escherichia coli, ATCC 25922 Staphylococcusaureus, ATCC 6538 P Candida albicans, ATCC 865-653

146 15 15

3128 776 88

77 77 38

1550 1550 87

a The number NMIC of polymer molecules in 1 mL at MIC of polymer is obtained from the formula: NMIC ) C × NA /Mw/mw where C (in moles of monomer links/mL) is equal to MIC (the mean values from Table 2); mw is the molecular weight of one link of a polymer, and NA ) 6.02 × 1023 is the Avogadro constant.

the properties of given system “polymer biocide-microbe”) with the growth of Mw by a factor of ∼2.5. The results on the influence of molecular weight on the antibacterial activity correlate with the data for PQAC and polymeric biguanides and guanidines.4,5a,c,d,24,26 Klibanov and co-workers demonstrated that quaternized N-alkylated PEI of low molecular weight immobilized onto material surfaces have only a weak bactericidal efficiency.13b However, it is known from the studies of Ikeda and Tazuke, Merianos, and some investigations described by Kenawy et al. that the dependence of antibacterial activity of different PQAC and PHMB on their Mw has a bell-like shape, that is, there is a region of optimal Mw values where the polymers exhibit the highest bactericidal efficiency.4a,b,5a,14 Comparison with the effect of these polymers directly on protoplasts and spheroplasts, which enhance with growth of Mw, indicates that the adsorption ability and capability to penetrate through CW are the primary factors that control antimicrobial activity of polycations.14,29 Our data do not allow us to make a certain conclusion whether the nonfractionated polymer samples with Mw ) 62 kDa with

Mw ) 55 kDa (with the consideration for the range of polydispersity Mw-Mn) exhibit the most strong effect against studied microorganisms, thus, these values of Mw are optimal. Franklin and Snow note that the structures of CW of bacteria and fungi are mainly open networks of macromolecules and usually do not offer an efficient barrier against penetration of compounds of molecular mass less than 50 kDa (with the exception of mycobacteria).24 In our case, it is not excluded that the effect of the nonfractionated samples with the relatively high Mw values is optimal owing to the fact that a polymer fraction with larger weight (and strong surface active properties) facilitates an efficient damage of CW permeability, thus creating conditions for the transport of polymer fractions with lower weights. The weak dependence of fungicidal activity of PDAATFA and PDAMATFA on Mw and virtually complete absence of such activity for the monomer salts and TFA (for the studied initial concentrations) seem to indicate that the high efficiency against fungi are related to the presence of pyrrolidinium cycles in the structure of these polymers. This conclusion is indirectly



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Table 4. Minimal Bactericidal (MIC) and Bacteriostatic Concentrations (µg/mL) of PDAATFA (Mw ) 24 kDa, P-50 Sample) in Solutions of Different Ionic Strengtha,b microorganism item b

P-50 P-50 in serum aqueous solutionc P-50 in 0.01 M KCl P-50 in 0.1 M KCl P-50 in 1 M KCl

E. coli 125 ( 7.5 250 ( 10.0

S. aureus 31 ( 3.1 62 ( 5.0

C. albicans 3.5 ( 0.6 7 ( 1.0

250 ( 10.0 62 ( 5.0 (31, V) 7 ( 1.0 250 ( 10.0 62 ( 5.0 (31, V) 15 ( 1.8 (7, V) inactive at 500d inactive at 500d 500 ( 10.0

a See footnote a in Table 2. b Bacteriostatic concentrations (if they were registered) are listed in brackets, symbol “V” designates that growth is less than in control. c Note that in the case of serum aqueous solutions the volume of the testing solution is equal to 1.6 mL, thus, the real concentration of the polymer is less by a factor of 1.6, as well as the concentration of a culture, by comparison with the experiment in aqueous solutions. d 500 µg/mL is the highest studied concentration of items.

confirmed by the data on the activity against Candida albicans of a polymer of the QPDAA series at a concentration of 0.1 wt % (1 mg/mL) reported previously.4a,b In summary, the results on the effect of molecular weight (polymeric length) on the antimicrobial activity emphasize that polymeric nature of the synthesized compounds is one of the most significant factors of their bactericidal efficiency, while their fungicidal activity is virtually independent of the Mw. Effect of Ionic Strength of Solution. Because according to the proposed mechanism of polycations biocidal action, efficiency of PDAA polysalts is due to a significant extent to their cationic charge, we studied influence of ionic strength of solution on the polymer antimicrobial activity. The PDAA activity was assessed in presence of blood (horse) serum (because of importance for potential clinic applications) and in solutions of a simple salt (KCl) with different concentrations. The results obtained in those studies using PDAATFA (P-50 sample) against gram-positive and gram-negative bacteria and fungi are presented in Table 4. In the presence of serum (concentration of NaCl in serum is 0.9 wt % that corresponds to a 0.15 M, hence the ionic strength of 0.6 mL of serum in 1.6 mL of total mixture is 0.056 M) and in a 0.01 M KCl solution, the MIC values do not change or increase by a factor of 2, depending on the type of microorganism in comparison with their magnitudes in saltfree aqueous solutions, and in a 0.1 M KCl aqueous solution, the MICs increase by a factor of 2-4, depending on the type of microorganism. However, in a 1 M KCl solution, the polymer loses its activity in the studied range of concentrations (the only exception is the fungicidal activity at the maximum concentration). The observed decrease in the biocidal activity is evidently related to that in presence of added salt, electrostatic interactions are increasingly screened with growth in the added salt concentration and ionic strength of solution that results in a diminution in the degree of dissociation and ionization of polyelectrolyte. The obtained results correlate with the data on the behavior of P-50 reduced viscosity concentration dependences in salt-free and KCl aqueous solutions (Figure 3, curves 1-4). One can see in Figure 3 that “polyelectrolyte effect”, which is related to electrostatic repulsion of the similarly charged ionized links of polycation and expansion of polyelectrolyte coils, due to an increase in the dissociation degree of polyelectrolyte with dilution in salt-free solution (curve 1), is gradually diminished at low concentrations of added salt where reduced viscosity dependencies is nonmonotonic (curves 2 and 3); in a 1 M KCl solution, PDAATFA behaves as an uncharged polymer (curve 4). This effect is fairly typical of polyelectrolyte.30 For

Figure 3. Concentration dependences of PDAATFA (P-50 sample) reduced viscosity in salt-free (1) and KCl aqueous solutions: (2) 0.01 M, (3) 0.1 M, and (4) 1 M KCl. All measurements were carried out at least five times for every concentration point, and the mean values and experimental errors were calculated according to the recommended procedures.19b

the quaternary PDADMAC, Negi et al. showed that the viscosity curves in 1 and 2 N solutions of NaCl virtually coincide indicating that the polyelectrolyte neutralization occurs in aqueous solution at a 1 N salt concentration.30e If the ionic strength Ipol of a salt-free polyelectrolyte aqueous solution is the molar concentration of the mobile counteranions in solution than the maximum Ipol value can not exceed 1/2M, where M is the molar concentration of monomer links, upon hypothetical full dissociation of the polyelectrolyte (in salt-free polyelectrolyte solution the degree of dissociation of given polyelectrolyte depends on its molecular weight and concentration). Consequently, at the minimal bactericidal concentrations of the PDAA salts belonging to a range 125-1.5 µg/mL (that corresponds to ∼6 × 10-4 M - ∼10-5 M of monomer links), the corresponding Ipol has very low values of less than 3 × 10-4 M. The ionic strength of the initial PDAAs solutions (at the concentration of monomer links ∼0.002 M) is less than 10-3 M. Thus, the boost of ionic strength of the initial polymer solution until the values of 10-2 M or 10-1 M results in only a moderate decrease in the biocidal efficiency of the polycation. Summarizing the results obtained in this section and comparing the data in Table 4 with Figure 3, one may conclude that the polyelectrolyte of the PDAA series exhibits quite high biocidal activity until the macrochain retains some positive charge and there are some active nitrogen centers unscreened by counteranions. The complete neutralization of the polyelectrolyte in a 1 M salt solution results in the loss of its biocidal activity. The obtained results reveal the crucial role of electrostatic interactions in the mechanism of biocidal action of the PDAA polyelectrolytes. Structure-ActiVity Relationships. Secondary and tertiary forms of the PDAA salts allow to obtain these polyelectrolytes as polybases. This makes it possible to assess antimicrobial activity of water-soluble polyamine composed of pyrrolidine links with secondary amino groups (as pointed above, the tertiary polybase of this series is not water-soluble) and to compare its effect with that of a known polyamine of open-chain structure, in particular, with the commercial polybase PEI containing primary, secondary, and tertiary amino groups in its links.

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Table 5. Minimal Bactericidal (MIC) and Bacteriostatic Concentrations (µg/mL) of (i) PDAATFA (Mw ) 24 kDa, P-50 Sample) in Aqueous Solution and in P-50 + NaOH Equimolar Composition in Aqueous Solution (See Text); (ii) TFA in TFA + NaOH Equimolar Composition in Aqueous Solution; (iii) PEI (Branched Polymer, Mw ) 45 kDa) in Aqueous Solutiona microorganism item

E. coli

S. aureus

C. albicans

P-50 P-50 in P-50 + NaOH equimolar compositionc TFA in TFA + NaOH equimolar compositiond PEI

125 ( 7.5 250 ( 10.0 inactive at 500e inactive at 500e

31 ( 3.1 125 ( 7.5 (62, V) inactive at 500e (500, V)e

3.5 ( 0.6 7 ( 1.0 inactive at 500e 62 ( 5.0f (31, V, 2 colonies; 15, V, 6 colonies)

b

a See footnote a in Table 2 and footnote b in Table 4. b The values of pH of P-50 solutions at MIC are 6.4-6.6 (see footnote b in Table 2). c Equimolar ratio of P-50 sample (evaluated in moles of monomer links/L) and NaOH in aqueous solution (see text); the values of pH of the solutions at MIC are 7.4-7.2. d Equimolar ratio of TFA and NaOH in aqueous solution (see text). e 500 µg/mL is the highest studied concentration of items; at this concentration of items, the values of pH are following: pH 9.5 for PEI solution and pH 7.4 for TFA + NaOH equimolar composition. f pH 7.4 for PEI solution at the MIC (see text).

For this purpose, a water-soluble polybase BPDAA was obtained in a fresh aqueous solution of the equimolar composition of PDAATFA (P-50 sample) with NaOH (as described above, polysalt PDAATFA exists as a water-soluble polybase BPDAA at these conditions). Both the value of pH 10.5 of the initial PDAATFA + NaOH composition (especially in comparison with the neutral pH 7.4 of the TFA + NaOH mixture) and pH 9.8 of the initial PEI aqueous solution indicate the partial protonation of the amino groups of these polybases, that is, BPDAA and PEI have some positive charge in the solutions. (Note that the largest degree of protonation of branched PEI is 66% in the presence of an acid at pH 4.4.31) The possible biocidal activity of BPDAA obtained in the P-50 + NaOH composition and the activities of TFA + NaOH equimolar composition and PEI were studied against grampositive and gram-negative bacteria and fungi (Table 5). One can see in Table 5 that the tested mixture TFA + NaOH does not exhibit activity even at the level of bacteriostatic effect, while PDAATFA + NaOH composition is rather active. The MIC values of the latter are only two to four times higher than that of PDAATFA in aqueous solution. Hence, polybase BPDAA exhibits the noticeable biocidal effect, albeit lower than the initial polysalt PDAATFA. Comparison of these results with those presented in Table 4 (see the second and third lines) shows that the efficacy of BPDAA (obtained from P-50 polysalt) is close to that of P-50 in the presence of serum or in a 0.01 M KCl aqueous solution. Unlike the BPDAA polybase, PEI is inactive against the gram-positive and gram-negative bacteria (in the studied range of concentrations). This behavior of PEI in aqueous solutions was partly expected, because PEI is an extremely hydrophilic and only slightly hydrophobic polymer (one nonquaternary amino or ammonium group per one -CH2-CH2- group).31 Klibanov and collaborators have revealed these disadvantages of PEI as a biocidal agent in developing novel bactericidal materials.13a Though, Oku et al. had showed that the branched polyethylenimine exhibited some membranotropic activity (the addition of PEI caused increase in permeability of liposomal membranes containing phosphatidylserine).26a Based on the results presented in Table 5, it would not be correct to conclude that the secondary/tertiary ammonium groups need to be part of a ring for the achievement by polymer of the antimicrobial activity (for instance, biocidal open-chain polymers PHMG and PHMB), because the different ratios of the hydrophilic (amino groups) and hydrophobic (CH2/CH) groups in the links of BPDAA and PEI predetermine the significantly stronger hydrophobicity of BPDAA. Obviously, relatively rigid structure of the pyrrolidine ring boosts the hydrophobic behavior of BPDAA, as well as of PDAA polysalts, still more. The comparative results on the antibacterial properties of BPDAA

and PEI polyamines, which have some positive charge due to protonation, are an evidence of that the presence of primarytertiary ammonium (and amine) centers in polymer structure is not enough, and the polymer must be sufficiently hydrophobic, like, for instance, BPDAA to exhibit strong bactericidal effect. The results of studies of the fungicidal properties are of special interest. Unlike the bactericidal activity, PEI exhibits the quite noticeable fungicidal effect (see Table 5), which is comparable (with account of the bacteriostatic effect) to that of BPDAA. Based on these results as well as on the abovementioned virtual independence of the fungicidal activity of PDAATFA and PDAMATFA on their Mw (i.e., polymeric length; see Tables 2 and 3), one can conclude that the hydrophobic properties, variation of the ionization degree, and a net charge of polymer are not so significant for the achievement of the fungicidal effect as for the bactericidal one. These results indicate that the secondary or tertiary pyrrolidinium ring is responsible for the high fungicidal activity of PDAA polymers, whose structures is much more effective as compared with the open-chain PEI. Comparison of the strong biocidal properties of PDAA polysalts as well as BPDAA polybase with the low activity of quaternary PDADMAC reveal the essential role of the hydrophilic NH2+/NH+ groups in the hydrophobic structure of pyrrolidinium (pyrrolidine) rings in the mechanism of biocidal action of PDAA polymers. One may suppose there are several reasons for this: (i) the electrostatic polycation-lipid interactions should be strong in the cases of NH2+ · · · Oδ- or (Cδ-H3δ+)NH+ · · · Oδ- intermolecular complexes formed with the secondary or tertiary pyrrolidinium rings of the PDAAs, unlike the weak electrostatic interaction in N+(Cδ-H3δ+)2 · · · Oδintermolecular complex formed with the quaternary pyrrolidinium rings of DADMAC due to bulky polar -Cδ-H3δ+ groups, which screen electrostatic interaction between atoms Oδ- and N+; (ii) the electrostatic interaction should be strengthened by N-Hδ+ · · · Oδ- hydrogen bonding that should result in more effective damage of the structural organization and integrity of cell membranes; (iii) the high hydrophilicity of the PDAAs due to NH2+/NH+ groups can facilitate diffusion of the hydrophobic polycation, in particular, across the water-filled pores of gramnegative outer membrane. The secondary/tertiary amino groups can form hydrogen bonds with phosphate, carbonyl, or hydroxyl oxygen atoms of the molecules of phospholipids, polysaccharides, or teichoic acids imbedded in the structure of outer membranes of gram-negative bacteria or gram-positive cell wall, thus increasing lateral segregation of negatively charged lipids and permeability of outer membrane/CW.32 The significant membrane activity of the PDAAs is evidenced by experiments on a negatively charged plane bilayer lipid membrane: unlike


the effect of PDADMAC, adsorption of the PDAAs causes the instantaneous rupture of the bilayers.22 Although the biocidal activity of PDAATFA is 2-4 times higher than the activity of the PDAA polybase, one should not relate directly this effect to the influence of trifluoroacetate anion because of different ionization degree, hydrophobic properties, conformations, and number of unscreened active nitrogen centers of the polysalt and polybase. Nevertheless, ions CF3COO- and H+ probably make some contribution to the antimicrobial effect, like, for instance, acetic acid used as preservative for food products.25 Nevertheless, neither monomer salts DAATFA and DAMATFA nor TFA and TFA + NaOH composition exhibit antimicrobial properties in the considered range of concentrations (in spite of the low pH of the monomers and TFA pointed above).33 Conducted investigations allow to ascertain the relationship between the structure of the studied polymers and their antimicrobial activity. The obtained results evidence that the structure of PDAA links, which combine the hydrophobic pyrrolidinium rings with the hydrophilic secondary/tertiary ammonium groups, is responsible for the high biocidal activity of the polymers of PDAA series.

Conclusions To summarize, the synthesized novel water-soluble cationic secondary and tertiary poly(diallylammonium) salts exhibit, unlike the quaternary polymers of this series, a rather high biocidal activity of a broad spectrum that is comparable to those of known effective cationic polymer biocides or exceeds it. Morphologic changes, in particular, in the E. coli cells caused by the effect of PDAATFA analyzed by TEM, involve all structural components of the cell and are close to those described for different microorganisms that underwent the action of cationic PQAC and PHMB. Our investigations allow us to draw several conclusions. The results on the effect of molecular weight (polymeric length) on the antimicrobial activity growing with Mw show that the polymeric nature of the synthesized compounds is one of the most significant factors of their bactericidal efficiency, while their fungicidal activity is virtually independent of the Mw. Behavior of the PDAA polymers in aqueous solutions with added salt is fairly typical of polyelectrolytes. The obtained results on the effect of ionic strength of solution revealed the crucial role of electrostatic interactions in the mechanism of biocidal action of the PDAA polyelectrolytes. They exhibit quite high biocidal activity until the macrochain retains some positive charge and there are some active nitrogen centers unscreened by counteranions (in serum, 0.01M/0.1 M salt solution), while complete neutralization of the polyelectrolyte (in a 1 M salt solution) results in the loss of its biocidal activity. The secondary PDAATFA retains quite high biocidal efficiency in an aqueous-alkaline solution, in particular, in the presence of an equimolar amount of NaOH where PDAATFA exists as a water-soluble polybase BPDAA. Comparison of the antimicrobial activity of BPDAA with secondary amino groups in pyrrolidine rings with that of open-chain PEI polybase with primary to tertiary amino groups in the links, both of which have some positive charges due to protonation of amino groups, shows that the presence of those ammonium groups in the polymer structure is not enough for the achievement of bactericidal activity, and the polymer must be sufficiently hydrophobic (like BPDAA) to exhibit good bactericidal proper-


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ties, while the hydrophobic behavior of the polymer is not so significant for fungicidal activity. The high activity of the PDAAs polymers against fungi is evidently related to the structure of secondary/tertiary pyrrolidinium ring. Fungicidal efficiency of these polymers is virtually independent of their Mw and remains almost equally high in all studied cases including serum/KCl and alkaline aqueous solutions with the exception of high ionic strength solutions (a 1 M KCl). Comparison of strong biocidal properties of the PDAA polysalts and BPDAA polybase with the low activity of quaternary PDADMAC reveal the essential role of hydrophilic NH2+/NH+ (or NHδ+) groups in combination with the hydrophobic structure of pyrrolidinium (pyrrolidine) rings in the mechanism of biocidal action of PDAA polymers. One may suppose there are several reasons for this: (a) strong intermolecular polycation-lipid electrostatic interactions in the cases of secondary/tertiary pyrrolidinium links with NH2+/NH+ groups, unlike weak intermolecular PDADMAC-lipid interactions due to bulky polar -Cδ-H3δ+ groups, which screen electrostatic interaction between atoms Oδ- and N+; (b) specific hydrogen bond interactions along with the electrostatic interactions at different stages of biocidal action that should result in more effective damage of structural organization and integrity of cell membranes. Thus, substitution of the quaternary ammonium group in pyrrolidinium ring by secondary NH2+ or tertiary CH3NH+ groups strongly affected antimicrobial activity of the obtained secondary and tertiary polyamines of PDAA series and resulted in their high biocidal efficiency. Novel polyelectrolytes exhibit quite strong biocidal properties at different conditions, including aqueous solutions of moderate ionic strength (serum, 0.01 M/0.1 M) and aqueous-alkaline solutions (pH 10.5). This provides the novel PDAA polymers a number of advantages over watersoluble PQACs, whose antimicrobial activity is suppressed in the presence of organic materials (in particular, blood).4a-d One may assume that the nonquaternary PDAA polyamines generate the family of novel biocidal polymers. The latter assumption, of course, implies the necessity of investigations on the effect the novel synthesized PDAA polymers have on human and animal cells (toxicity). Acknowledgment. The authors gratefully acknowledge Prof. Alexander S. Shashkov (Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences) for his valuable help in the HSQC NMR study. Authors are also grateful to Dr. Valentina B. Rogacheva (Laboratory of Polyelectrolytes, Department of Chemistry, Lomonosov Moscow State University), Dr. of Sci. Yurii A. Ermakov (Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences), and Dr. of Sci. Alexander S. Lileev (Kurnakov Institute of General and Inorganic Chemistry, Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences) for their permission to involve unpublished data in the discussion. This work was supported by the Program for Basic Research of the Department of Chemistry and Material Science of RAS (Program 10 “Biomolecular and Medical Chemistry”) and in part by the Russian Foundation for Basic Research (Project 07-03-00630).

References and Notes (1) Butler, G. B. Cyclopolymerization and cyclocopolymerization; Marcel Dekker: New York, 1992. (2) Kabanov, V. A.; Topchiev, D. A. Vysokomol. Soedin., Ser. A 1988, 30, 675–875; Polym. Sci. USSR, Ser. A 1988, 30, 667 (Engl. Transl.)

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