CHEM. RES. CHINESE UNIVERSITIES 2012, 28(6), 1095—1100

Preparation and Antibacterial Activity of Copolymer of Methacryloxyethyldimethyl Dodecyl Ammonium Bromide/Acrylamide LU Gui-qian1,2, ZUO Hua-jiang1, DONG Wei-min1, WU Ding-cai1 and FU Ruo-wen1,2* 1. Key Laboratory for Polymeric Composite and Functiond Materials, Ministry of Education, 2. Design and Synthesis of New Polymer Materials and Application Laboratory, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, P. R. China Abstract Copolymers PMDAB-co-AA with excellent antibacterial activity were designed and prepared (MDAB/AA=methacryloxyethyldimethyl dodecyl ammonium bromide with acrylamide). The chemical structures of the copolymers were characterized by Fourier transform infrared(FTIR), 1H NMR, 13C NMR and chemical titration. The antimicrobial activities and kinetics of the copolymer against E. coli and S. aureus were examined by viable cell counting method. The compositions of the copolymers could be easily controlled by the feed molar ratio of MDAB to AA. Furthermore, the highest antibacterial activity was achieved when the molar fraction of MDAB was in a range of 5%―20%, and the killing rate reached 100% under the test conditions. In addition, the antibacterial activity of PMDAB-co-AA was maintained and stable without any loss after 15 times of repeated usage. It was proved that the PMDAB-co-AA samples targeted at cell membrane, and caused serious damage to cell integrity and inner membrane permeation. The surrounding conditions, such as pH and inorgainc salts concentrations(CaCl2 or NaCl), also affected their antibacterial activities. Keywords Dimethylaminoethyl methacrylate; Acrylamide; Quaternary ammonium salt; Antibacterial activity Article ID 1005-9040(2012)-06-1095-06

1

Introduction

Quaternary ammonium salts(QASs) are among the most commonly used antimicrobial agents[1,2]. Generally, the monoor bis-QAS with an alkyl chain having more than 8 carbons show excellent antibacterial activity. In our previous work[3], four QAS monomers have been synthesized by the quaternization of dimethylaminoethyl methacrylate(DMAEMA) with different alkyl agents. One of them is methacryloxyethyldimethyl dodecyl ammonium bromide(MDAB). However, this type of small molecular QAS is highly toxic to environment[4]. To obtain biocidal effect without releasing biocide into the environment, antimicrobial agents should be covalently grafted to functional surfaces. Some studies have reported the successful covalent attachment of polymeric antimicrobial materials onto glass[5], polymers[6], paper[5] and metals[7]. However, in some cases, the polymerization of QAS monomers resulted in the decrease of antibacterial activity. It was found that though MDAB exerted high antibacterial activity[minimum bactericidal concentration(MBC)=12―24 µg/mL] against both E. coli and S. aureus, the corresponding homopolymer PMDAB did not show obvious antibacterial activity[8]. That might be attributed to the increase of inter- or intramolecular hydrophobic interaction resulted from the long alkyl

side-chains, which leads to the coiling and aggregation of polymer chains in aqueous solution. Thus the contact of QAS groups with cells could be significantly decreased, consequently weakening their antibacterial activity. We have thought that if a certain amount of MDAB monomers is dispersed separately into a structural similar polymer chain by copolymerization, they seem to be dispersed in a polymer solution. In this case, the inter- or intramolecular hydrophobic interaction of MDAB monomers decreases, so that the MDAB component in the copolymer is expected to present excellent antibacterial activity as corresponding monomers. Based on this new idea, we designed and synthesized the copolymer of MDAB with water soluble monomer, acrylamide(AA) in this work. AA is a common water-soluble monomer. Its corresponding homopolymer polyacrylamide(PAA) has been widely applied in the fields of mining, waste water treatment, coal mining, paper making, agriculture and construction engineering, and so on[9,10]. Though AA itself does not exhibit any antimicrobial activity, it can copolymerize with antibacterial MDAB to realize the loose distribution of QAS groups along the polymer chain, so that the antibacterial activity of MDAB could be maintained. Additionally, the high toxicity attributed to the small molecular monomer could be eliminated.

——————————— *Corresponding author. E-mail: [email protected] Received January 16, 2012; accepted July 1, 2012. Supported by the National Natural Science Foundation of China(No.50673101) and the Project of the Department of Education of Guangdong Province, China(No.cgzhzd0901).

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Herein, antibacterial PMDAB-co-AA copolymers were designed and prepared by the copolymerization of MDAB with AA by free radical copolymerization. The composition of PMDAB-co-AA was mainly controlled by the feed molar ratio of MDAB to AA. The structure of representative PMDABco-AA was characterized by Fourier transform infrared(FTIR) spectrometry, 1H NMR and 13C NMR. The cationic density(CD) and molecular weight(Mw) were determined by chemical titration and multiangle laser light scattering method, respectively. The antimicrobial activities of PMDAB-co-AA against E. coli and S. aureus were examined by viable cell counting method. The antibacterial mechanism of PMDAB-co-AA was investigated by determining the nutrient constituents release. The effects of surrounding conditions, such as pH value and the concentrations of inorganic salts(CaCl2 or NaCl) on antibacterial action were also investigated.

2 2.1

Experimental Materials

DMAEMA(98%) was purchased from Aldrich and distilled under reduced pressure prior to usage. AA and 2,2-azobis(isobutyronitrile)(AIBN), purchased from Tianjin No.1 Chemical Reagent Factory(China), were purified by recrystallization twice from distilled water and methanol, respectively, and then dried in vacuum at room temperature for 24 h before use. Dodecyl bromide and solvents used in the synthesis were purchased from China Medicine Group Shanghai Chemical Reagent Corporation(China) and Guangzhou Chemical Reagent Factory(China), respectively and used as received. Nutrient agar[approximate formula(per liter): peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar 15 g] and nutrient broth[approximate formula(per liter): peptone 10 g, beef extract 3 g, sodium chloride 5 g] were purchased from Guangzhou Huankai Microbial Sci. & Tech., Co., Ltd.(China). E. coli (ATCC 25922) and S. aureus(ATCC 25923) were purchased from Guangdong Microbe Institute(China) and used as received.

2.2

Synthesis of MDAB

MDAB monomer was synthesized according to the procedure described in the literature[3]. In a 100 mL volume flask equipped with a magnetic stirrer, a cooler and a thermometer were charged 10 mL of DMAEMA, 16.27 g of dodecyl bromide and a small amount of hydroquinone. Subsequently, 30 mL of acetonitrile was added to it as solvent. After that, the mixture was stirred at 45 oC for a period of time. Finally, white needle crystals were obtained by cooling and filtering the reaction solution, followed by washing with dry ether several times and drying under vacuum at room temperature. The yield of MBAB was 85%.

2.3

Synthesis of PMDAB-co-AA

A series of PMDAB-co-AA with various compositions was prepared as follows. A certain amount of MDAB and AA monomers was dissolved in a mixture of water and ace-

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tone(volume ratio, 20:10) in a three-necked flask equipped with a condenser, a magnetic stirrer and a nitrogen inlet. After the removal of oxygen in the flask by flowing nitrogen for 0.5 h, the reactants were heated to 60 °C, to which a desired amount of AIBN was then added. The mixture was stirred under flowing nitrogen for 10 h. The obtained PMDAB-co-AA was precipitated in acetone, dissolved in water and then precipitated again in acetone in turn, followed by drying under vacuum at 50 °C for 24 h. White solid PMDAB-co-AA was obtained as a final product.

2.4

Determination of CD

The cationic density(the content of N+) in the copolymers was determined according to the procedure described in the literature[11]. First, 10 mL of 2% PMDAB-co-AA aqueous solution was mixed with 25 mL of 0.05%(mass fraction) methylene blue in a 100 mL test tube, to which 15 mL of chloroform was then added. The resultant mixture was shaken vigorously and titrated with a standard dodecyl sodium sulfate aqueous solution until a blue color appeared. The corresponding CD was calculated as follows: c1 (V2 − V1 ) CD = c1 (V2 − V1 ) + [ ρV × 10−3 − c1 (V2 − V1 ) M 1 ]/M 2 where ρ and c1 are the concentrations of copolyemer solution (g/L) and standard sodium dodecyl sulfate(SDS) aqueous solution(mol/L), respectively; V is the volume of copolyemer solution(L); V1 and V2 are the volumes of standard SDS aqueous solutions used in control titration(mL) and in copolyemer titration(mL), respectively; M1 and M2 are the molecular weights of cationic and AA structural unit in the copolymer, respectively.

2.5

Determination of Mw

Mw of PMDAB-co-AA samples was determined by static multiangle laser light scattering method as follows. PMDAB-co-AA solutions with a series of varied concentrations were prepared in distilled water, filtered with a syringe filter, and then placed in scintillation vials. Scattering intensities at seven angles were collected over a 5-min period via a model Wyatt Dawn DSP-F static MALLS at 532 nm.

2.6

Characterization

The IR spectra of the monomers and synthesized copolymers were recorded on an IR spectrophotometer(Nicolet210, USA) via KBr pellets. 1H NMR and 13C NMR spectra were recorded on an FTNMR spectrometer(INOVA-500, USA) with D2O as the solvent.

2.7

Antibacterial Assessment

2.7.1

Antibacterial Activity

The antibacterial activities of the copolymers with different compositions were monitored with gram-positive S. aureus and gram-negative E. coli as test organisms by viable cell counting method as described previously[3].

2.7.2

Time-kill Study

Time-kill determination was performed to investigate the

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kinetics of bacteria killed by the copolymers as previously described[3]. Briefly, the copolymer water solution was mixed with bacterial suspension, adjusted to final concentrations of 0.5 mg copolymer/mL and 106 bacterial cells/mL, respectively, and then incubated at 36―37 °C with gentle agitation in a shaked water bath. At different time intervals of incubation, 1 mL of the solution was taken out and diluted with 9.0 mL of saline distilled water. The surviving bacteria in these solutions were counted by the spread plate method. Each test was repeated three times.

2.7.3

E. coli Inner Membrane(IM) Permeability

The ability of copolymers to permeate the E.coli IM was investigated as a function of the release of cytoplasmic β-galactosidase. The amount of active β-galactosidase released into the medium can be easily measured by its catalytic hydrolysis activity of o-nitrophenyl-β-D-galactopyranoside(ONPG) substrate to a bright yellow product, o-nitrophenol, which has an obvious UV absorbance at 420 nm. Briefly, 1.5 mL of the culture containing 107 cells/mL and 1.5 mL of 25 mmol/L ONPG solution were mixed with 11 mL of phosphate buffer solution(PBS). After 15 min, 15 mL of 0.4 mg/mL copolymer solution was added in the mixture, and the mixture was then diluted to 30 mL. The change of optical density of the mixed solution with time at 420 nm was recorded via a UV spectrophotometer(UV756MC, Shanghai Precision & Scientific Instrument Co., Ltd., China).

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these cultures were detected by means of viable cell counting method as described previously[4].

3 3.1

Results and Discussion Synthesis of PMDAB-co-AA

A series of PMDAB-co-AA random copolymers was prepared by changing the feed molar fraction of MDAB from 0 to 30%, and the chemical structure is shown in Scheme 1.

Antibacterial Durability Test

2 mL of fresh 106 cells/mL E. coli was added in 20 mL(0.5 mg/mL or 3.0 mg/mL) of the copolymer solution. The procedure was repeated every 2 d for 30 d. Each time, after adding culture, the mixture solution was incubated at 37 oC for 3 h, and the growth colonies were detected by viable cell counting method.

2.8

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Scheme 1

Structure of PMDAB-co-AA random copolymers The correlation between their corresponding CD value and feed molar fraction of MDAB is shown in Fig.1. The CD value increased linearly with the feed molar fraction of MDAB up to 20%. Therefore, the final composition of PMDAB-co-AA could be easily controlled by the feed molar fraction of MDAB. However, when the feed molar fraction of MDAB exceeded 20%, the hydrophobic interactions within the macromolecules was so strong that the copolymers turned from water-soluble to water-insoluble, and a lot of QAS groups was increased inside. Accordingly, the CD value reduced to 1.8%―3.5% when the feed molar fraction of MDAB was in a range of 25%―30%.

Integrity of S. aureus Membrane

The obvious absorption of DNA and RNA at 260 nm could be monitored to investigate the integrity of S. aureus membrane as DNA and RNA could not be transported through an intact cell membrane. After overnight incubation, S. aureus suspension was centrifuged at 2700 r/min for 10 min to remove the supernatant. The cells were washed twice with sterile distilled water and resuspended at a final concentration of 107 cells/mL. A certain amount of the copolymer was then added in it, and the final concentration was 0.2 mg/mL. The change of optical density of the mixed solution with time at 260 nm was recorded on a UV spectrophotometer(UV756MC, Shanghai Precision & Scientific Instrument Co., Ltd., China).

Fig.1

Relationship between CD of PMDAB-co-AA and feed molar fraction of MDAB As seen from Fig.2, the yields of the copolymers reached 70%―83% when the feed molar fraction of MDAB was in a range of 3%―30%, which proves the successful copolymerization of MDAB and AA. However, the yield decreased gradually

2.10 Influences of pH Value and Inorganic Salts on the Antibacterial Activity The liquid culture was adjusted to the expected pH values by HCl or NaOH, or to different concentrations by CaCl2 or NaCl. Then, the antibacterial activities of the copolymers in

Fig.2

Copolymerization yield of PMDAB-co-AA vs. feed molar fraction of MDAB

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with the increase of the feed molar fraction of MDAB because of the low polymerization activity of MDAB with a large side group. Meanwhile, though the corresponding Mw was high(all exceeding 7.9×104), the Mw also decreased obviously from 9.3×104 to 7.9×104 when the feed molar fraction increased from 3% to 20%. This means the comparativeley low reaction rate of MDAB. With the increase of feed molar fraction of MDAB, both yield and Mw of the copolymer decreased, since less monomer was introduced into the polymer chain. Therefore, based on the CD value, polymerization yield, and Mw of the copolymers, the recommended feed molar fraction of MDAB was in a range of 5%―20%. The representative samples of PMDAB-co-AA were characterized by FTIR(Fig.3), 1H NMR[Fig.4(A)] and 13C NMR [Fig.4(B)].

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1

H NMR(D2O), δ: 3.52(6H, H-h), 3.57(2H, H-i), 1.75(20H, H-j), 4.2(2H, H-g), 4.5(2H, H-f), 4.70(2H, H-c), 2.68(H, H-b), 2.1―2.3(4H, H-a and H-d); 13C NMR(D2O), δ: 179.6(C3 and C7) , 51.5―58.6(C8 and C9), 28.4―36.5(C11, C12). Meanwhile, the absorption peaks concerning ―C=C―, e.g., 1633 cm–1(C=C stretching vibration, FTIR), 3092 cm–1 (=CH stretching vibration, FTIR), δ 5.61 and 6.02 (―CH2=CH, 1H NMR), δ 125.6 and 136.0(―CH2=CH, 13 C NMR) all disappeared. These data are in agreement with the chemical structure in Scheme 1, which proves the successful copolymerization of MDAB and AA. Therefore, the antibacterial units MDAB were diluted with AA in a single chain like that in the polymer solution. This kind of structure is beneficial to the antibacterial activity of MDAB. Simultaneously, the decrease of toxicity could be gained.

3.2

Antibacterial Ability of the Copolymers

As shown in Fig.5, the antibacterial activity of the PMDAB-co-AA against S. aureus and E. coli increased with increasing the feed molar fraction of MDAB at first and reached the highest level when the feed molar fraction of MDAB ranged from 5% to 20%. These results indicate that the MDAB segments introduced in the PMDAB-co-AA chains presented excellent antibacterial activity as the precursory monomer. Fig.3

FTIR spectra of PAA(a) and PMDAB-co-AA (b―d) with different feed molar fraction of MDAB

b. PMDAB-co-AA(3% of MDAB); c. PMDAB-co-AA(10% of MDAB); d. PMDAB-co-AA(20% of MDAB).

Fig.5

Antibacterial activity of PMDAB-co-AA with different compositions against E. coli(a) and S. aureus(b)

Suspension: 106 cells/mL E. coli and S. aureus; contact time: 1 h; concentration of copolymer: 0.5 mg/mL.

H NMR(A) and 13C NMR(B) spectra of copolymer PMDAB-co-AA[n(MDAB):n(AA)=7:93] The characteristic absorption peaks are shown as follows. FTIR, ߥ෤/cm–1: 3433(NH2 absorption), 2930―2845(saturated C―H stretching vibration), 1660(C=O absorption of amide), 1720(C=O absorption of ester), 1205(C―O stretching vibration of ester), 643(CH2)n(n≥2) rocking vibration. Fig.4

1

Several researchers[12―14] have reported that the increased cationic charge density can enhance significantly the ability of the copolymer to be adsorbed onto charged bacterial cells, resulting in obvious improvement of biological activity. Therefore it is assumed that the increased content of MDAB in the copolymers would lead to the increased CD value and thus result in a high antibacterial activity. The increasing of antibacterial ability with CD value is also schematically shown in Fig.6. Certain amounts of antibacterial MDAB component should be introduced into the copolymer matrix in order to provide suitable cationic charge density. However, the feed molar fraction of MDAB higher than 20% resulted in too high segment density of MDAB in the copolymer and therefore hydrophobicity and opposite effect were observed, namely, their antibacterial activity decreased. That is why the PMDAB-co-AA with suitable MDAB content

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in comparison to AA units has much higher antibacterial activity than homopolymerized PMDAB.

Fig.6

Antibacterial activity of the copolymers with different cationic densities

Suspension: 106 cells/mL E. coli; contact time: 3 h; concentration of copolymer: 0.5 mg/mL.

The effect of contact time on the antibacterial activity against E. coli was investigated by two representative PMDAB-co-AA samples with MDAB feed molar fraction of 10% and 20%, respectively. It was found that both the samples had excellent antibacterial dynamics(Fig.7). The tested samples with MDAB feed molar fraction of 20% and 10% killed bacterial cells completely within 10 and 30 min, respectively.

Fig.7

antibacterial activity of PMDAB-co-AA(MDAB feed molar fraction 10%) was maintained during an examination period of 30 d. The copolymer could kill 100% of E. coli in each test repeated 15 times within 30 d, no matter what the copolymer concentration was, that is, the concentration of the copolymer used was low(e.g., 0.5 mg/mL) or high(e.g., 3.0 mg/mL). In order to explore the antibacterial action between copolymer and cell, the permeability of PMDAB-co-AA into the inner membrane of E. coli was assessed by measuring β-galactosidase activity in the cells. It was reported that the ONPG which is produced by fractured E. coli cells could be changed to o-nitro phenol(ONP) under the catalysis of β-D-galactosidase. Therefore, it was possible to estimate whether the membrane of E. coli cells was disrupted or not by determining the amount of ONP in culture or observing the change of culture’s color because of ONP showing yellow in solution. Fig.9 shows the absorbance of ONP at 420 nm with test time. The enhancement of the absorbance at 420 nm with test time indicates that an amount of β-D-galactosidase released from fractured cells, suggesting that the inner membrane of E. coli cells was the reaction site as contacting with PMDAB-co-AA. This conclusion was also supported by the change of solution color. It was observed that the color of the culture mixture with copolymer and cells became more and more yellow with the prolongation of test time, suggesting that much more β-D-galactosidase released from the fractured cells.

Influence of contact time on the antibacterial activity of PMDAB-co-AA with MDAB feed molar fraction of 10%(a) and 20%(b)

Suspension: 106 cells/mL E.coli; concentration of copolymer: 0.5 mg/mL.

It appeared that the copolymer concentration of 0.5 mg/mL or higher ones were enough to kill 100% of tested bacteria within 1 h, when PMDAB-co-AA samples with MDAB feed molar fraction of 10% and 20% were used for tests(Fig.8). The results of antibacterial durability test show that the

Fig.8

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Influence of PMDAB-co-AA concentration on antibacterial activity

Feed molar fraction of MDAB: a. 10%; b. 20%. Suspension: 106 cells/mL E. coli; contact time: 1 h.

Fig.9

Absorbance of ONP at 420 nm vs. test time

Feed molar fraction of MDAB: 10%; suspension: 107 cells/mL E. coli; concentration of copolymer: 0.5 mg/mL.

The membrane integrality of S. aureus was examined by the release of DNA and RNA which are described as “260 nm absorbing materials” [15,16]. As shown in Fig.10, an amount of 260 nm absorbing materials released from S. aureus and

Fig.10

Absorbance at 260 nm vs. test time

Feed molar fraction of MDAB: 10%; suspension: 107 cells/mL E. coli; concentration of copolymer: 0.2 mg/mL.

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gradually increased with contact time. It can therefore be concluded that PMDAB-co-AA copolymers strongly destroyed the cytoplastic membranes of S.aureus and resulted in the death of bacterial cells.

3.3 Effect of Surrounding Conditions on Antibacterial Activity It can be seen from Fig.11 that as pH10.4, the solution without any copolymers in it naturally exhibited a high antibacterial activity and could kill over 70% of the test bacteria. That is, strong acidic or basic surrounding condition naturally provides with potent antibacterial ability[17]. The as-prepared PMDAB-co-AA showed an excellent antibacterial activity against E. coli within a pH range of 6―12. An opposite activity which was detected at pH from 3 to 6, was most likely due to the reduced adsorption force of the PMDAB-co-AA onto the bacterial cells because of the presence of H+[18,19].

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significantly decreased. This phenomena could be explained by the morphology of PMDAB-co-AA chains in concentrated salt solution. Higher salt concentration would make the polymer chains gradually huddle, so much as to separate out due to strong ions interaction. This could result in a limited contact between antibacterial functional groups and bacterial cells, and consequently lowered inhibition ability. Similar behavior was already reported[3].

4

Conclusions

PMDAB-co-AA copolymers with various amounts of antibacterial components were successfully prepared by free radical copolymerization of MDAB and AA. These copolymers demonstrated the highest antibacterial activity when the feed molar fraction of MDAB ranged from 5% to 20%. Their antibacterial activity increased with increasing cationic density, contact time and concentration. They showed a high and stable antibacterial efficiency within 30 d in 15 tests. The PMDAB-co-AA targeted at cell membrane, and caused serious damage to cell integrity and inner membrane permeation. The copolymer antibacterial activity was influenced by the pH and salt concentration.

References [1] Chen C. Z., Beck-Tan N. C., Dhurjati P., van Dyk T. K., LaRossa R. A., Cooper S. L., Biomacromolecules, 2000, 1, 473

Fig.11 Antibacterial activity with(a) or without(b) copolymer in different pH solutions 6

Feed molar fraction of MDAB: 10%; suspension: 10 cells/mL E. coli; contact time: 1 h; concentration of copolymer: 0.5 mg/mL.

The experimental results show that the mass fraction of CaCl2 in water solution less than 2.5% was too low to kill the cells, but obviously contributed to the growth of a number of cells, while for the sufficiently higher mass fraction(>5%) increased inhibition ability was detected(5.0% of CaCl2 could kill >90% of test bacteria)(Fig.12). NaCl solution showed a similar behavior as CaCl 2. When a certain amount of PMDAB-co-AA copolymer was added in the dilute salt solution(less than 0.5% of salt), it showed a high antibacterial activity. However, when the mass fraction of the salt was over 0.5%, the antibacterial efficiency of the PMDAB-co-AA

[2] Shirai A., Maeda T., Nagamune H., Matsuki H., Kaneshina S., Kourai H., Eur. J. Med. Chem., 2005, 40, 113 [3] Lu G., Wu D., Fu R., React. Funct. Polym., 2007, 67, 355 [4] Song J., Kong H., Jang J., Chem. Commun., 2009, (36), 5418 [5] Lee S. B., Koepsel R. R., Morley S. W., Matyjaszewski K., Sun Y., Russell A., J. Biomacromolecules, 2004, 5, 877 [6] Cen L., Neoh K. G., Kang E. T., Langmuir, 2003, 19, 10295 [7] Yuan S. J., Xu F. J., Pehkonen S. O., Ting Y. P., Neoh K. G., Kang E. T., Biotech. Bioeng, 2009, 103, 268 [8] Kenawy E. R., Abdel-Hay F. I., El-Shanshoury A. E. R. R., El-Newehy M. H., J. Polym. Sci. Pol. Chem., 2002, 40, 2384 [9] Lu S., Liu R., Sun X. A., J. Appl. Polym. Sci., 2002, 84, 343 [10] Solberg D., Wågberg L., Colloid Surface A, 2003, 219, 161 [11] Lu G., Studies on Preparation and Structure-activity Relationships of Polymeric Antibacterial from Dimethylaminoethyl Methacrylate, Sun Yat-Sen Univeristy, 2008 [12] Ignatova M., Voccia S., Gabriel S., Gilbert B., Cossement D., Jerome R., Jerome C., Langmuir, 2009, 25, 891 [13] Shi Z., Neoh K. G., Kang E. T., Biomaterials, 2005, 26, 501 [14] Murata H., Koepsel R. R., Matyjaszewski K., Russell A., J. Biomaterials, 2007, 28, 4870 [15] Chen C. Z., Cooper S. L., Biomaterials, 2002, 23, 3359 [16] Hu F. X., Neoh K. G., Cen L., Kang E. T., Biotechnol. Bioeng., 2005, 89, 474 [17] Kamberi M., Tsutsumi K., Kotegawa T., Kawano K., Nakamura K., Niki Y., Nakano S., Antimicrob. Agents Ch., 1999, 43, 525

Fig.12 Antibacterial activity with or without copolymer in different CaCl2(NaCl ) solution 6

Feed molar fraction of MDAB: 10%; suspension: 10 cells/mL E. coli; contact time: 1 h; concentration of copolymer: 0.5 mg/mL.

[18] Gao B., He S., Guo J., Wang R., J. Appl. Polym. Sci., 2006, 100, 1531 [19] Gao B., He S., Guo J., Wang R., Mater. Lett., 2007, 61, 877