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Controllable synthesis and antimicrobial activities of acrylate polymers containing quaternary ammonium salts
Reactive and Functional Polymers xxx (xxxx) xxx–xxx
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Reactive and Functional Polymers journal homepage: www.elsevier.com/locate/react
Controllable synthesis and antimicrobial activities of acrylate polymers containing quaternary ammonium salts Weiqiang Zhonga,1, Chenyun Dongb,1, Runqi Liuyangb, Qizhi Guob, Hong Zenga, Yaling Linb,⁎, Anqiang Zhanga,⁎ a b
College of Materials Science and Engineering, South China University of Technology, 381 Wushan Rd., Guangzhou 510641, Guangdong, China College of Materials and Energy, South China Agricultural University, 432 Wushan Rd., Guangzhou 510642, Guangdong, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Acrylate polymers containing quaternary ammonium salts Atom transfer radical polymerization Antimicrobial activity Bacterial Phytopathogenic fungi
A series of acrylate polymers containing quaternary ammonium salts (PDMAEMA-BC) having tunable molecular weights were synthesized via atom transfer radical polymerization (ATRP), and the effect of the degree of polymerization (DP) on the antimicrobial activity against bacteria (Escherichia coli, Staphylococcus albus), pathogenic fungi (Candida albicans) and phytopathogenic fungi (Rhizoctonia solani and Fusarium oxysporum f. sp. cubense race 4) was systematically assessed. The antimicrobial properties against E. coli, S. albus and C. albicans were characterized using the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) or minimum bactericidal concentration (MBC) values, whereas the antimicrobial activities against R. solani and Foc4 were evaluated using the effective concentration (EC50 and EC90), MIC and MFC values. The results indicated that the PDMAEMA-BC homopolymers showed better antimicrobial activities than the corresponding monomer, i.e., the acrylate quaternary ammonium salt monomer (DMAEMA-BC), and the optimal antimicrobial activities were obtained for moderate PDMAEMA-BC chain lengths. These results help to understand the antimicrobial mechanism of polymeric quaternary ammonium salts and highlight their potential application as fungicidal agents for controlling both human and plant diseases.
1. Introduction When various crops are planted in the field, they are confronted with serious infection by phytopathogenic fungi. Rhizoctonia solani form sclerotia when the external conditions are not suitable for their development. In addition, Fusarium oxysporum can survive for up to 30 years in the absence of bananas, and non-host weed species that are infected by the pathogen act as inoculum reservoirs [1]. The fungi are usually killed by spraying high levels of low molecular weight antimicrobial agents because traditional antimicrobial agents are easily washed away and thus need to be repeatedly sprayed. Furthermore, low molecular weight antimicrobial agents are toxic to the environment and have poor chemical stability [2]. To properly solve this problem, antimicrobial agents are designed based on polymers that contain antimicrobial functional groups. Their advantages, including better efficacy, low toxicity, no volatility, high chemical stability and prolonged lifetimes, have attracted a great deal of attention [2–4]. The mode of action of low molecular weight cationic biocides has been summarized as follows: (i) adsorption onto the bacterial cell
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1
surface; (ii) diffusion through the cell wall; (iii) binding to the cytoplasmic membrane; (iv) disruption of the cytoplasmic membrane; (v) release of cytoplasmic constituents, such as K+ ions, DNA, and RNA; and (vi) death of the cell [5]. According to our previous studies, the bactericidal mechanism is very complicated and involves several targets in fungal cells, including the disruption of cellular structures, such as the cell wall and plasma membrane; the induction of lipid peroxidation; mitochondrial dysfunction and interference with genomic DNA [6]. Cationic antimicrobials are well-known in the development of selfsterilizing surfaces and are used in many applications, such as hospital surfaces, surgical equipment, protective hospital clothes, medical implants, wound dressings, food packaging materials, and everyday consumer products [7]. Among these cationic antimicrobials, quaternary ammonium compounds are probably the most explored and widely deployed [8–10]. The antimicrobial activity of polymers containing quaternary ammonium salts was associated with complex factors, such as molecular weight, the types of counter anions, charge density, alkyl chain length and steric hindrance, the hydrophilic–hydrophobic balance, and the type of bacteria species [2,11]. The molecular weight
Corresponding authors. E-mail addresses: [email protected] (Y. Lin), [email protected] (A. Zhang). These authors contributed equally.
http://dx.doi.org/10.1016/j.reactfunctpolym.2017.10.010 Received 25 July 2017; Received in revised form 4 October 2017; Accepted 20 October 2017 1381-5148/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Zhong, W., Reactive and Functional Polymers (2017), http://dx.doi.org/10.1016/j.reactfunctpolym.2017.10.010
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quaternization of PDMAEMA to afford PDMAEMA-BC, as shown in Scheme 1. PDMAEMA was polymerized via ATRP technology, and a brief description of the polymerization procedure is as follows: 10.0 g (0.06 mol) of DMAEMA and pre-determined amounts of EtBriB and PMDETA were dissolved in 50 mL of isopropanol in a 200 mL Schlenk flask. The reaction system was subjected to three freeze-pump-thaw cycles. Next, a calculated amount of CuBr was quickly added into the flask under a nitrogen atmosphere. During ATRP polymerization, certain polymer molecular weights were targeted by selecting appropriate initial monomer and EtBriB concentrations. The ratio of [DMAEMA]0/ [EtBriB]0 (mol/mol) were set at 4/1, 8/1, 15/1, 30/1, 60/1, 70/1 and 80/1 to yield a theoretical degree of polymerization (DP) of 4, 8, 15, 30, 60, 70 and 80, respectively. The mixture was stirred at 60 °C for 8 h under a nitrogen atmosphere. The reaction was stopped by exposing it to air and dried under vacuum at 60 °C (approximately 70% yield). The catalyst was removed by neutral alumina column chromatography with acetone as the eluent. For convenience, the products were named PDMAEMA-n, and n represents the DP of PDMAEMA. 1 H NMR (400 MHz, D2O, δ, ppm): 0.97 (s, CeCH3), 1.98 (s, CeCH2eC), 2.37 (s, N(CH3)2), 2.77 (t, NeCH2eCH2), 4.20 (t, OeCH2eCH2). FT-IR (KBr, cm− 1): 2863–2947 (νCH), 1732 (νC]O), 1020,1047 (νCeN). PDMAEMA-BC was synthesized in a quaternization reaction between PDMAEMA and benzyl chloride (BC), as briefly stated in the following: 6.0 g of PDMAEMA and 4.8 g of BC were dissolved in 20 g of an ethyl alcohol/methylbenzene (1/1, m/m) mixture in a 100 mL Schlenk flask. The mixture was subjected to three freeze-pump-thaw cycles and then stirred at 70 °C under a nitrogen atmosphere for 24 h; subsequently, the mixture was precipitated with anhydrous diethyl ether, and the precipitate was washed with anhydrous diethyl ether three times and dried under vacuum at 40 °C overnight (approximately 90% yield). For convenience, the products were named PDMAEMA-BCn, and n represents the DP of PDMAEMA-BC. 1 H NMR (400 MHz, D2O, δ, ppm): 1.06 (s, CeCH3), 3.14 (s, N+(CH3)2), 3.87(t, CH2eCH2eN+), 4.35–4.76 (m, N+eCH2eΦ, OeCH2eCH2), 7.59 (s, ΦeH). FT-IR (KBr, cm− 1): 2870–2979 (νCH), 1726 (νC]O), 1041 (νCeN), 768 (γΦeH).
plays an important role in determining the antimicrobial properties. Homopolymers of quaternary ammonium salts exhibited far better antimicrobial activities than the corresponding monomers [12,13]. Additionally, Ikeda and his co-workers investigated the antimicrobial activity of polymethacrylate containing pendant biguanide units and found that polymer samples with low and high molecular weights exhibited lower bactericidal activity against S. aureus than those in the intermediate range [14]. Chen and co-workers determined that the antimicrobial properties of quaternary ammonium functionalized poly (propyleneimine) dendrimers have a parabolic dependence on molecular weight [3]. Huang and co-workers prepared polypropylene (PP) coated with a non-leachable biocide by chemically attaching poly (quaternary ammonium) (PQA) to the surface of PP and found that polymers with relatively high molecular weight (Mn > 10 kDa) showed almost 100% killing efficiency (against E. coli), while shorter PQA chains (Mn = 1.5 kDa) demonstrated less activity with the same grafting density [15]. The difference in cell structure between fungi and bacteria leads to differences in the antimicrobial activities of quaternary ammonium salts against bacteria and fungi. Therefore, it is very essential to conduct research on the relationship between the antifungal activity and molecular weight of poly quaternary ammonium salts. To this end, in this paper, we synthesized a series of acrylate polymers containing quaternary ammonium salts (PDMAEMA-BC) with tunable molecular weights via ATRP and systematically assessed the antimicrobial activities against two typical phytopathogenic fungi (Rhizoctonia solani (R. solani), whose main morphology is a mycelium and Fusarium oxysporum f. sp. cubense race 4 (Foc4), whose main morphology is a spore), Gramnegative bacteria (Escherichia coli (E. coli)), Gram-positive bacteria (Staphylococcus albus (S. albus)) and pathogenic fungi (Candida albicans (C. albicans)) using DMAEMA-BC and PDMAEMA-BC with various molecular weights. 2. Experimental 2.1. Materials 2-(dimethylamino)ethyl methacrylate (DMAEMA, 99%), ethyl 2bromoisobutyrate (EtBriB, 98%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 98%) and copper(I) bromide (CuBr, 99%) were supplied by Shanghai Macklin Biochemical Technology Co. Ltd. (Shanghai, China). Benzyl chloride (BC, 99%) was supplied by Aladdin Reagent Co. Ltd. (Shanghai, China). Beef extract was supplied by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Peptone was supplied by Guangdong Ring Kay Microbial Technology Co., Ltd. (Guangzhou, China). Agar was supplied by MYM Biological Technology Company (Shanghai, China). RPMI-1640 liquid medium was supplied by HyClone Company of America (Utah, USA). All microorganisms were kindly supplied by the Fungus Laboratory, Department of Plant Pathology, South China Agricultural University.
2.3. Synthesis of an acrylate quaternary ammonium salt monomer (DMAEMA-BC) An acrylate quaternary ammonium salt monomer (DMAEMA-BC) was synthesized via a quaternization reaction between DMAEMA and BC. 6.0 g of DMAEMA and 4.8 g of BC were dissolved in 20 g of an ethyl alcohol/methylbenzene (1/1, m/m) mixture in a 100 mL Schlenk flask. The mixture was subjected to three freeze-pump-thaw cycles and then stirred at 40 °C under a nitrogen atmosphere for 24 h; subsequently, the mixture was precipitated with anhydrous diethyl ether, and the precipitate was washed with anhydrous diethyl ether three times and dried under vacuum at 40 °C overnight (approximately 90% yield). 1 H NMR (400 MHz, D2O, δ, ppm): 1.98 (s, CeCH3), 3.18 (s, N+(CH3)2), 3.83–3.86 (t, CH2eCH2eN+), 4.65(s, N+eCH2eΦ), 4.73–4.75 (t, OeCH2eCH2), 5.82, 6.21 (d, CH2]C(CH3)), 7.62 (s, ΦeH). FT-IR (KBr, ν, cm− 1): 2853–2970 (νCH), 1722 (νC]O),1635 (νC]C), 1041 (νCeN), 768 (γΦeH).
2.2. Synthesis of poly(2-(dimethylamino)ethyl methacrylate) containing quaternary ammonium salts (PDMAEMA-BC) Poly(2-(dimethylamino)ethyl methacrylate) containing quaternary ammonium salts (PDMAEMA-BC) was prepared in a two-step process, i.e., first, the controlled synthesis of PDMAEMA, followed by
Scheme 1. Synthetic route to PDMAEMA and PDMAEMA-BC.
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100 μL of the polymer solutions with different concentrations were transferred to a 96-well plate. Medium was added instead of polymer solution as a control plate. Then, the mixtures were incubated at 28 °C (R. solani, Foc4 and C. albicans) for 48 h or 37 °C (E. coli and S. albus) for 24 h, after which 50 μL of a 0.5 wt% 2,3,5-triphenyl tetrazolium chloride (TTC) solution was added into the 96-well plate, and the contents of each well were mixed by vortex shaking prior to incubation in the dark at 28 °C (R. solani, Foc4 and C. albicans) or 37 °C (E. coli and S. albus) for 24 h. The MIC value was the lowest concentration at which there was no visible growth, i.e., no red color due to the reduction of TTC [17]. The MFC or MBC test is the most common estimation of fungicidal or bactericidal activity and is defined as the lowest concentration of antimicrobial agent needed to kill 99.9% of the initial inoculums after incubation [18]. To determine the MFC or MBC values, 100 μL of the mixtures in the wells that were at or above the MIC value were plated on medium and incubated at 28 °C (R. solani, Foc4 and C. albicans) for 48 h or 37 °C (E. coli and S. albus) for 24 h. The MFC or MBC value was the lowest concentration at which fewer than 5 colony-forming units were observed. Additionally, the antimicrobial agent was classified as a bactericide or fungicide based on its MFC/MIC or MBC/MIC ratio. If this ratio was above 4, the antimicrobial agent was classified as bacteriostatic or fungistatic, otherwise they would be classified as bactericide or fungicide, respectively [19,20].
2.4. Characterization FT-IR spectra were collected on a VERTEX-70 spectrometer (Bruker Instrument Corp., Germany) using KBr pellets. 1H NMR spectra were obtained using a Bruker Avance III-400 (Bruker Instrument Corp., Germany) spectrometer with CDCl3 or D2O as the solvent. Size exclusion chromatography (SEC) was performed on a Waters system (Waters Corp., USA) equipped with a Waters 515 pump, a Waters 2414 differential refractive index detector, and three μ-Styragel columns. The samples were analyzed at 30 °C using THF (1 mL/min) as the eluent. The instrument was calibrated using polystyrene standards. Critical micelle concentrations (CMC) were determined using a DDS-11A conductivity meter (Shanghai Hongyi Instrument Co. Ltd., China). Zeta potentials and particle size distributions were analyzed using a Brookhaven BI-90 Plus particle size analyzer (Brookhaven Instrument Corp., USA) using ultrapure water as the solvent. The morphologies of the micelles were analyzed with a JEM-2100 transmission electron microscope (JEOL Ltd., Japan). 2.5. Antimicrobial tests 2.5.1. Test microorganisms and media The microorganisms included two typical phytopathogenic fungi (Rhizoctonia solani (R. solani) and Fusarium oxysporum f. sp. cubense race 4 (Foc4)), Gram-negative bacteria (Escherichia coli (E. coli)), Gram-positive bacteria (Staphylococcus albus (S. albus)) and pathogenic fungi (Candida albicans (C. albicans)). The media were prepared according to the literature [13,16].
3. Results and discussion 3.1. Design and synthesis of DMAEMA-BC and PDMAEMA-BC
2.5.2. Determination of mycelial growth inhibition (MGI) The antifungal activities of the samples were tested using the inhibition of mycelial growth. To prepare polymer solutions with different concentration, the samples were dissolved in sterilized water. Next, 45 mL of PDA and 5 mL of polymer solution were poured into sterile Petri dishes (diameter of 90 mm). Sterilized water was added instead of a polymer solution as control Petri dish. Then, a 6-mm disc containing mycelia was transferred to the center of the Petri dishes and incubated at 28 °C. Each experiment was conducted in triplicate. The diameter of the fungal colony was measured using a cross method three times and averaged. MGI was calculated using Eq. (1).
MGI = (R C − RT)/R C × 100%
By designing and synthesizing a series of PDMAEMA-BC polymers with tunable molecular weights, we could then examine the relationship between DP and antimicrobial activity. The chemical structures of the products were confirmed by FT-IR and 1H NMR spectroscopies. As a multifunctional monomer containing double bonds and tertiary amino groups, 2-(dimethylamino)ethyl methacrylate (DMAEMA) could be polymerized by ATRP and then quaternized by haloalkanes for the preparation of antibacterial materials [9]. Quaternized PDMAEMA is considered a promising antimicrobial agent [21]. By adjusting the mole feeding ratio of [DMAEMA]/[EtBriB] from 4/1 to 80/1, a series of PDMAEMA polymers with various molecular weights were synthesized by ATRP. The molecular weights of PDMAEMA, determined by SEC, are shown in Table 1. As shown in Figs. 1 and 2, the absorption peaks at 2863–2947, 1732, 1047 and 1020 cm− 1 represented the infrared absorption bands of saturated CeH, C]O and CeN, respectively, and the signals for the CeCH3, CeCH2eC, N(CH3)2, NeCH2eCH2 and OeCH2eCH2 protons appeared at 0.97, 1.98, 2.37, 2.77 and 4.20 ppm, respectively. A series of PDMAEMA-BC polymers were obtained from a quaternization reaction between PDMAEMA and benzyl chloride (BC). As
(1)
whereby RC and RT were the radial growth of the fungi colonies in the control plate and the treatment plate, respectively. The radial growth of the fungi colony was calculated by Eq. (2):
Radial growth (mm) = Average diameter of fungi colony (mm) − 6 (mm) (2) 2.5.3. Determination of effective concentration (EC) The antimicrobial activities of DMAEMA-BC and PDMAEMA-BC against R. solani and Foc4 were evaluated using the EC50 and EC90 values. The regression equation was obtained by using the natural logarithm of the polymer solution concentration as the abscissa and the biological statistical probability, converted to MGI, as the ordinate. The EC50 and EC90 values were calculated using the regression equation when the MGI values were 50% and 90%, respectively.
Table 1 Molecular weights of the PDMAEMA polymers prepared with different [DMAEMA]/ [EtBriB] feeding ratios.
2.5.4. Determination of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and minimum bactericidal concentration (MBC) The antimicrobial properties of DMAEMA-BC and PDMAEMA-BC against R. solani, Foc4, E. coli, S. albus and C. albicans were evaluated by the TTC coloration method [17]. Bacterial suspensions (108 CFU/mL) were incubated in medium and diluted 1000 times to obtain a concentration of 105 CFU/mL. Next, 100 μL of the bacterial suspension and
Samples
[DMAEMA]/[EtBriB] feeding ratio, mol/mol
Mna (g/mol)
Mwa (g/mol)
PDI (Mw/ Mn)a
PDMAEMA-4 PDMAEMA-8 PDMAEMA-16 PDMAEMA-30 PDMAEMA-60 PDMAEMA-70 PDMAEMA-80
4/1 8/1 16/1 30/1 60/1 70/1 80/1
0.71 × 103 1.41 × 103 2.99 × 103 5.16 × 103 9.25 × 103 11.6 × 103 14.7 × 103
0.84 × 103 1.58 × 103 4.52 × 103 6.97 × 103 1.09 × 104 13.7 × 103 23.3 × 103
1.18 1.12 1.51 1.35 1.18 1.18 1.59
a
3
Based on SEC results.
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Fig. 1. FT-IR spectra of PDMAEMA, DMAEMA-BC and PDMAEMA-BC. Fig. 3. CMC values of DMAEMA-BC and the PDMAEMA-BC polymers with different molecular weights.
shown in Figs. 1 and 2, the absorption peaks at 2870–2979, 1726, 1041 and 768 cm− 1 represented the infrared absorption bands of saturated CeH, C]O, CeN and Φ-H, respectively, and the signals for the CeCH3, N+(CH3)2, CH2eCH2eN+, N+eCH2eΦ, OeCH2eCH2 and ΦeH protons appeared at 1.06, 3.14, 3.87, 4.35–4.76 and 7.59 ppm, respectively. After the quaternization reaction, the N(CH3)2 proton signals shifted from approximately 2.37 to 3.14 ppm and the characteristic protons signals of BC (4.35–4.76 and 7.59 ppm) appeared, which indicated that the expected structure of PDMAEMA-BC were formed. DMAEMA-BC was obtained from a quaternization reaction between DMAEMA and benzyl chloride (BC). As shown in Figs. 1 and 2, the absorption peaks at 2853–2970, 1722, 1635, 1041 and 768 cm− 1 represented the infrared absorption bands of saturated CeH, C]O, C]C, CeN and ΦeH, respectively, and the signals form the CeCH3, N+(CH3)2, CH2eCH2eN+, N+eCH2eΦ, OeCH2eCH2, CH2]C(CH3) and ΦeH protons appeared at 1.98, 3.18, 3.83–3.86, 4.65, 4.73–4.75,
5.82, 6.21 and 7.62 ppm, respectively. 3.2. Critical micelle concentrations (CMC) of DMAEMA-BC and PDMAEMA-BC in water The concentration above which micelles form is called the critical micelle concentration (CMC) [22]. We measured the CMC values of DMAEMA-BC and PDMAEMA-BC by the electrical conductivity method. Fig. S1 (Supporting information) shows the conductivity plots for DMAEMA-BC and the PDMAEMA-BC polymers with different molecular weights, and Fig. 3 shows the CMC values calculated from the curves in Fig. S1. The test results showed that the CMC value of PDMAEMA-BC with low DP is higher than those of the other DMAEMA-BC and PDMAEMA-BC samples with higher DP. It was reasonable to assume
Fig. 2. 1H NMR spectra of PDMAEMA, PDMAEMA-BC and DMAEMA-BC.
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which were consistent with the results of zeta potentials and particle sizes. 3.4. Antimicrobial activity against bacteria and pathogenic fungi Screening of the antimicrobial activities of DMAEMA-BC and PDMAEMA-BC against bacteria and pathogenic fungi was performed using E. coli, S. albus and C. albicans as test organisms because they represent Gram-negative, Gram-positive bacteria and pathogenic fungi, respectively. The MIC and MBC or MFC values of DMAEMA-BC and PDMAEMA-BC were determined using the TTC coloration method. Fig. 7 is a typical photograph from the determination of the MIC values for DMAEMA-BC and PDMAEMA-BC-4 against E. coli, S. albus, C. albicans, R. solani and Foc4. The MIC and MBC or MFC values for DMAEMA-BC and PDMAEMA-BC against E. coli, S. albus and C. albicans are shown in Fig. 8. As shown in Fig. 8, the results indicated that the MIC and MBC or MFC values of the homopolymers were far lower than those of the monomers against both E. coli, S. albus and C. albicans, which meant that the activity of the polymers was much higher than that of the corresponding monomers. The CMC values of DMAEMA-BC and PDMAEMA-BC were lower than the MIC and MBC or MFC; thus, DMAEMA-BC and PDMAEMA-BC are able to form micelles. However, in the particle size distribution test, we found that DMAEMA-BC is not capable of forming large micelles. Furthermore, although the formation of micelles in a real environment is difficult, the linkages formed by covalent bonds guarantee the aggregation of charge. It was reasonable to assume that the change from a monomer to a polymer could increase the local charge density; thus, adsorption on bacterial cell surfaces was enhanced for the polymer compared to the monomer, and binding to the cytoplasmic membrane was also facilitated by the polymer because of the presence of negatively charged species in the membrane [13]. Besides, for DMAEMA-BC and the PDMAEMA-BC polymers with different molecular weights, the best antimicrobial activities against animal pathogenic bacteria and fungi were not observed for the high and low molecular weights. The optimal antimicrobial activity against E. coli, S. albus and C. albicans were achieved when the DP of PDMAEMA-BC was about 8. It is noticed that the MIC and MBC values for PDMEMA-BC-8 against E. coli were lower than the CMC, which meant that PDMEMA-BC-8 exhibited the best antimicrobial activity against E. coli without forming micelles. Without considering the formation of micelles, the processes of adsorbing onto the bacterial cell surface and disrupting the cytoplasmic membrane are likely enhanced by increasing the molecular weight of the antimicrobial agents, whereas the process of diffusing through the cell wall is inhibited by increasing the molecular weight [14]. Peptidoglycan is a major part of bacteria cell walls with open network structures. Bacteria cell walls generally do not act as significant permeability barriers against compounds with molecular masses below 50 kDa [23]. Additionally, it was observed that the DMAEMA-BC and PDMAEMA-BC with high chain lengths gave higher zeta potentials, which led to a decrease in the particle size. Thus, the largest particle size of PDMAEMA-BC was obtained from a moderate chain length which means that the degree of aggregation was the highest when the PDMAEMA-BC chain length was moderate. In conclusion, the observed optimal molecular weight for antibacterial activity can be explained based on the combined effect of those two kinds of controlling factors. Additionally, compared with the antimicrobial activities of the PDMAEMA-BC against E. coli, the test results indicated that the antimicrobial activities of the PDMAEMA-BC against S. albus were minimally dependent on the molecular weight when the molecular weight of PDMAEMA-BC was too high. This finding can be explained using the fact that Gram-positive bacteria tend to have a loose cell wall, whereas Gram-negative bacteria have an outer membrane structure in the cell wall that forms an additional barrier to foreign molecules [3,8]. This allowed the PDMAEMA-BC polymers with high molecular weight to
Fig. 4. Zeta potentials of DMAEMA-BC and the PDMAEMA-BC polymers with different molecular weights.
that the change from a monomer to a polymer with low DP increase the degree of branch, and the hydrophobicity of the backbone was increased with increase of the chain length. However, the environment in which bacteria live is more complex than pure water. The formation of micelles by DMAEMA-BC and PDMAEMA-BC is more difficult in real environment that contains salt, leaked cytoplasmic contents, and so on. 3.3. Zeta potentials and particle size distributions of DMAEMA-BC and PDMAEMA-BC in water The zeta potentials and particle sizes of DMAEMA-BC and the PDMAEMA-BC polymers displayed in Figs. 4 and 5 showed that: (1) PDMAEMA-BC with low DP exhibited the low zeta potential, which meant it easier to aggregates; (2) PDMAEMA-BC with low DP exhibited the larger particle sizes than the samples with higher DP; (3) the particle size of PDMAEMA-BC was decreasing due to the increase of absolute value of zeta potential of PDMAEMA-BC; (4) the particle size of DMAEMA-BC was below the minimum diameter that can be measured, which meant that DMAEMA-BC was unable to form large micelles. As shown in Fig. 6, the TEM study of the micellar conformation of DMAEMA-BC and PDMAEMA-BC shows that the particle sizes of PDMAEMA-BC with low DP were larger than those of the other samples,
Fig. 5. Particle size distributions of PDMAEMA-BCs with different molecular weights. The particle size distribution of DMAEMA-BC could not be determined because the diameters were below the minimum diameter that can be measured.
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Fig. 6. Transmission electron microscope of (A) DMAEMA-BC, (B) PDMAEMA-BC-4, (C) PDMAEMA-BC-8, (D) PDMAEMA-BC-16, (E) PDMAEMA-BC-30 and (F) PDMAEMA-BC-80.
Fig. 7. Typical photograph of the MIC value determination for DMAEMA-BC and the PDMAEMA-BC-16 polymers against E. coli, S. albus, C. albicans, R. solani and Foc4.
and the PDMAEMA-BC polymers with different molecular weights, the optimal antimicrobial activity against R. solani and Foc4 were achieved when the DP of PDMAEMA-BC were 4 and 16, respectively. Similar trends were observed in the antimicrobial activities of DMAEMA-BC and PDMAEMA-BC against bacteria and pathogenic fungi. It is noticed that most of the MIC and MFC values for PDMEMA-BC against R. solani and Foc4 were lower than the CMC, which meant that the factor of
diffuse through the cell walls of the Gram-positive bacteria more easily. 3.5. Antifungal activity against phytopathogenic fungi (R. solani and Foc4) Screening of the antimicrobial activities of DMAEMA-BC and PDMAEMA-BC against phytopathogenic fungi was performed using R. solani and Foc4 as test organisms. As shown in Fig. 8, for DMAEMA-BC 6
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Fig. 8. MIC and MBC (for bacterial) or MFC (for fungi) values of DMAEMA-BC and PDMAEMA-BC against (A) E. coli, (B) S. albus, (C) C. albicans, (D) R. solani and (E) Foc4.
DMAEMA-BC and PDMAEMA-BC-16 against R. solani and Foc4. The mycelial growth inhibition by DMAEMA-BC and PDMAEMA-BC-16 against R. solani and Foc4 in PDA medium is shown in Fig. 10, and the EC50 and EC90 values were calculated using the regression equation when the MGI values were 50% and 90%, respectively. As shown in Fig. 11, PDMAEMA-BC-4 and PDMAEMA-BC-16 exhibited the best antimicrobial activity against R. solani and Foc4 respectively, and the
chain length played a dominant role compared to the factor of the formation of micelles. With an increase in the chain length of PDMAEMA-BC, the aggregated charge was more stable in the real environment due to the covalent bond linkages. However, diffusion through the cell wall became more difficult with increasing PDMAEMABC chain length, which led to a decrease in the antimicrobial activity. Fig. 9 is a typical photograph displaying the antifungal effects of
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Fig. 9. Typical mycelial growth of R. solani and Foc4 in PDA medium after application of DMAEMA-BC and PDMAEMA-BC-16. The data were based on three replicates, and the test was repeated twice with similar results. Representative results are shown.
Fig. 10. Mycelial growth inhibition by DMAEMA-BC and PDMAEMA-BC-16 against R. solani and Foc4 in PDA medium. The growth of R. solani and Foc4 was measured after 48 h of incubation at 28 °C.
Fig. 11. EC50 and EC90 values of DMAEMA-BC and PDMAEMA-BC against (A) R. solani and (B) Foc4. (*: The calculated EC50 and EC90 values of DMAEMA-BC against Foc4 are much larger than 20.)
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References
antimicrobial activities were lower for the higher and lower molecular weights. For PDMAEMA-BC, the optimal antimicrobial activity against animal pathogenic bacteria and fungi was achieved with an intermediate chain length, which was consistent with the results of MIC and MFC values. Compared to bacteria, the structures of fungi cells are much more complicated. From the studies of numerous fungi, the cell wall has been shown to be primarily composed of chitin, glucans, mannans and glycoproteins, and the wall composition frequently varies markedly between species of fungi [24,25]. In addition, fungi cells contain organelles, such as mitochondria, endoplasmic reticuli and Golgi bodies. Therefore, the ability of antimicrobial agents to diffuse through the cell wall and disrupt the cellular structure is likely different between fungi and bacteria and even between various fungi. Most of the MIC and MFC values for PDMAEMA-BC against R. solani and Foc4 were lower than those against E. coli, S. albus and C. albicans, which meant the PDMAEMA-BC polymers were more suitable for inhibiting phytopathogenic fungi. Additionally, the antifungal activities of the PDMAEMA-BC polymers against R. solani were shown to have a greater molecular weight dependence than those against Foc4.
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4. Conclusions In this paper, DMAEMA-BC and a series of PDMAEMA-BC homopolymers with tunable molecular weights were synthesized to quantify the relationship between their antimicrobial properties and chain length. From the present results, optimal antimicrobial (E. coli, S. albus, C. albicans, R. solani, and Foc4) activities were achieved for an intermediate DP of PDMAEMA-BC. To properly explain the relationship between antimicrobial activity and molecular weight, the bacteria structure and the ability of the antimicrobial agent to diffuse through the cell wall and disrupt the cellular structure were considered. PDMAEMA-BC with low DP exhibited the low zeta potential, which meant it easier aggregates and form micelles with large particle size. On the other hand, the factor of chain length played a dominant role and the optimal antimicrobial activity was achieved with an intermediate chain length by taking account of the influence of both aggregated charge due to the covalent bond linkages and diffusion through the cell wall. The systematic approach to examining the antimicrobial activities of PDMAEMA-BC homopolymers with varying DP is expected to guide the future design of antimicrobial polymers as potential fungicide agents for controlling plant disease. Acknowledgments This work was supported by the Science and Technology Program of Guangzhou, China, under grant 201704020084; the Science and Technology Planning Project of Guangdong Province, China under grant 2016A020210105; and the National Natural Science Foundation of China under grant 31201552. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.reactfunctpolym.2017.10.010.
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Contents lists available at ScienceDirect
Reactive and Functional Polymers journal homepage: www.elsevier.com/locate/react
Controllable synthesis and antimicrobial activities of acrylate polymers containing quaternary ammonium salts Weiqiang Zhonga,1, Chenyun Dongb,1, Runqi Liuyangb, Qizhi Guob, Hong Zenga, Yaling Linb,⁎, Anqiang Zhanga,⁎ a b
College of Materials Science and Engineering, South China University of Technology, 381 Wushan Rd., Guangzhou 510641, Guangdong, China College of Materials and Energy, South China Agricultural University, 432 Wushan Rd., Guangzhou 510642, Guangdong, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Acrylate polymers containing quaternary ammonium salts Atom transfer radical polymerization Antimicrobial activity Bacterial Phytopathogenic fungi
A series of acrylate polymers containing quaternary ammonium salts (PDMAEMA-BC) having tunable molecular weights were synthesized via atom transfer radical polymerization (ATRP), and the effect of the degree of polymerization (DP) on the antimicrobial activity against bacteria (Escherichia coli, Staphylococcus albus), pathogenic fungi (Candida albicans) and phytopathogenic fungi (Rhizoctonia solani and Fusarium oxysporum f. sp. cubense race 4) was systematically assessed. The antimicrobial properties against E. coli, S. albus and C. albicans were characterized using the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) or minimum bactericidal concentration (MBC) values, whereas the antimicrobial activities against R. solani and Foc4 were evaluated using the effective concentration (EC50 and EC90), MIC and MFC values. The results indicated that the PDMAEMA-BC homopolymers showed better antimicrobial activities than the corresponding monomer, i.e., the acrylate quaternary ammonium salt monomer (DMAEMA-BC), and the optimal antimicrobial activities were obtained for moderate PDMAEMA-BC chain lengths. These results help to understand the antimicrobial mechanism of polymeric quaternary ammonium salts and highlight their potential application as fungicidal agents for controlling both human and plant diseases.
1. Introduction When various crops are planted in the field, they are confronted with serious infection by phytopathogenic fungi. Rhizoctonia solani form sclerotia when the external conditions are not suitable for their development. In addition, Fusarium oxysporum can survive for up to 30 years in the absence of bananas, and non-host weed species that are infected by the pathogen act as inoculum reservoirs [1]. The fungi are usually killed by spraying high levels of low molecular weight antimicrobial agents because traditional antimicrobial agents are easily washed away and thus need to be repeatedly sprayed. Furthermore, low molecular weight antimicrobial agents are toxic to the environment and have poor chemical stability [2]. To properly solve this problem, antimicrobial agents are designed based on polymers that contain antimicrobial functional groups. Their advantages, including better efficacy, low toxicity, no volatility, high chemical stability and prolonged lifetimes, have attracted a great deal of attention [2–4]. The mode of action of low molecular weight cationic biocides has been summarized as follows: (i) adsorption onto the bacterial cell
⁎
1
surface; (ii) diffusion through the cell wall; (iii) binding to the cytoplasmic membrane; (iv) disruption of the cytoplasmic membrane; (v) release of cytoplasmic constituents, such as K+ ions, DNA, and RNA; and (vi) death of the cell [5]. According to our previous studies, the bactericidal mechanism is very complicated and involves several targets in fungal cells, including the disruption of cellular structures, such as the cell wall and plasma membrane; the induction of lipid peroxidation; mitochondrial dysfunction and interference with genomic DNA [6]. Cationic antimicrobials are well-known in the development of selfsterilizing surfaces and are used in many applications, such as hospital surfaces, surgical equipment, protective hospital clothes, medical implants, wound dressings, food packaging materials, and everyday consumer products [7]. Among these cationic antimicrobials, quaternary ammonium compounds are probably the most explored and widely deployed [8–10]. The antimicrobial activity of polymers containing quaternary ammonium salts was associated with complex factors, such as molecular weight, the types of counter anions, charge density, alkyl chain length and steric hindrance, the hydrophilic–hydrophobic balance, and the type of bacteria species [2,11]. The molecular weight
Corresponding authors. E-mail addresses: [email protected] (Y. Lin), [email protected] (A. Zhang). These authors contributed equally.
http://dx.doi.org/10.1016/j.reactfunctpolym.2017.10.010 Received 25 July 2017; Received in revised form 4 October 2017; Accepted 20 October 2017 1381-5148/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Zhong, W., Reactive and Functional Polymers (2017), http://dx.doi.org/10.1016/j.reactfunctpolym.2017.10.010
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quaternization of PDMAEMA to afford PDMAEMA-BC, as shown in Scheme 1. PDMAEMA was polymerized via ATRP technology, and a brief description of the polymerization procedure is as follows: 10.0 g (0.06 mol) of DMAEMA and pre-determined amounts of EtBriB and PMDETA were dissolved in 50 mL of isopropanol in a 200 mL Schlenk flask. The reaction system was subjected to three freeze-pump-thaw cycles. Next, a calculated amount of CuBr was quickly added into the flask under a nitrogen atmosphere. During ATRP polymerization, certain polymer molecular weights were targeted by selecting appropriate initial monomer and EtBriB concentrations. The ratio of [DMAEMA]0/ [EtBriB]0 (mol/mol) were set at 4/1, 8/1, 15/1, 30/1, 60/1, 70/1 and 80/1 to yield a theoretical degree of polymerization (DP) of 4, 8, 15, 30, 60, 70 and 80, respectively. The mixture was stirred at 60 °C for 8 h under a nitrogen atmosphere. The reaction was stopped by exposing it to air and dried under vacuum at 60 °C (approximately 70% yield). The catalyst was removed by neutral alumina column chromatography with acetone as the eluent. For convenience, the products were named PDMAEMA-n, and n represents the DP of PDMAEMA. 1 H NMR (400 MHz, D2O, δ, ppm): 0.97 (s, CeCH3), 1.98 (s, CeCH2eC), 2.37 (s, N(CH3)2), 2.77 (t, NeCH2eCH2), 4.20 (t, OeCH2eCH2). FT-IR (KBr, cm− 1): 2863–2947 (νCH), 1732 (νC]O), 1020,1047 (νCeN). PDMAEMA-BC was synthesized in a quaternization reaction between PDMAEMA and benzyl chloride (BC), as briefly stated in the following: 6.0 g of PDMAEMA and 4.8 g of BC were dissolved in 20 g of an ethyl alcohol/methylbenzene (1/1, m/m) mixture in a 100 mL Schlenk flask. The mixture was subjected to three freeze-pump-thaw cycles and then stirred at 70 °C under a nitrogen atmosphere for 24 h; subsequently, the mixture was precipitated with anhydrous diethyl ether, and the precipitate was washed with anhydrous diethyl ether three times and dried under vacuum at 40 °C overnight (approximately 90% yield). For convenience, the products were named PDMAEMA-BCn, and n represents the DP of PDMAEMA-BC. 1 H NMR (400 MHz, D2O, δ, ppm): 1.06 (s, CeCH3), 3.14 (s, N+(CH3)2), 3.87(t, CH2eCH2eN+), 4.35–4.76 (m, N+eCH2eΦ, OeCH2eCH2), 7.59 (s, ΦeH). FT-IR (KBr, cm− 1): 2870–2979 (νCH), 1726 (νC]O), 1041 (νCeN), 768 (γΦeH).
plays an important role in determining the antimicrobial properties. Homopolymers of quaternary ammonium salts exhibited far better antimicrobial activities than the corresponding monomers [12,13]. Additionally, Ikeda and his co-workers investigated the antimicrobial activity of polymethacrylate containing pendant biguanide units and found that polymer samples with low and high molecular weights exhibited lower bactericidal activity against S. aureus than those in the intermediate range [14]. Chen and co-workers determined that the antimicrobial properties of quaternary ammonium functionalized poly (propyleneimine) dendrimers have a parabolic dependence on molecular weight [3]. Huang and co-workers prepared polypropylene (PP) coated with a non-leachable biocide by chemically attaching poly (quaternary ammonium) (PQA) to the surface of PP and found that polymers with relatively high molecular weight (Mn > 10 kDa) showed almost 100% killing efficiency (against E. coli), while shorter PQA chains (Mn = 1.5 kDa) demonstrated less activity with the same grafting density [15]. The difference in cell structure between fungi and bacteria leads to differences in the antimicrobial activities of quaternary ammonium salts against bacteria and fungi. Therefore, it is very essential to conduct research on the relationship between the antifungal activity and molecular weight of poly quaternary ammonium salts. To this end, in this paper, we synthesized a series of acrylate polymers containing quaternary ammonium salts (PDMAEMA-BC) with tunable molecular weights via ATRP and systematically assessed the antimicrobial activities against two typical phytopathogenic fungi (Rhizoctonia solani (R. solani), whose main morphology is a mycelium and Fusarium oxysporum f. sp. cubense race 4 (Foc4), whose main morphology is a spore), Gramnegative bacteria (Escherichia coli (E. coli)), Gram-positive bacteria (Staphylococcus albus (S. albus)) and pathogenic fungi (Candida albicans (C. albicans)) using DMAEMA-BC and PDMAEMA-BC with various molecular weights. 2. Experimental 2.1. Materials 2-(dimethylamino)ethyl methacrylate (DMAEMA, 99%), ethyl 2bromoisobutyrate (EtBriB, 98%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 98%) and copper(I) bromide (CuBr, 99%) were supplied by Shanghai Macklin Biochemical Technology Co. Ltd. (Shanghai, China). Benzyl chloride (BC, 99%) was supplied by Aladdin Reagent Co. Ltd. (Shanghai, China). Beef extract was supplied by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Peptone was supplied by Guangdong Ring Kay Microbial Technology Co., Ltd. (Guangzhou, China). Agar was supplied by MYM Biological Technology Company (Shanghai, China). RPMI-1640 liquid medium was supplied by HyClone Company of America (Utah, USA). All microorganisms were kindly supplied by the Fungus Laboratory, Department of Plant Pathology, South China Agricultural University.
2.3. Synthesis of an acrylate quaternary ammonium salt monomer (DMAEMA-BC) An acrylate quaternary ammonium salt monomer (DMAEMA-BC) was synthesized via a quaternization reaction between DMAEMA and BC. 6.0 g of DMAEMA and 4.8 g of BC were dissolved in 20 g of an ethyl alcohol/methylbenzene (1/1, m/m) mixture in a 100 mL Schlenk flask. The mixture was subjected to three freeze-pump-thaw cycles and then stirred at 40 °C under a nitrogen atmosphere for 24 h; subsequently, the mixture was precipitated with anhydrous diethyl ether, and the precipitate was washed with anhydrous diethyl ether three times and dried under vacuum at 40 °C overnight (approximately 90% yield). 1 H NMR (400 MHz, D2O, δ, ppm): 1.98 (s, CeCH3), 3.18 (s, N+(CH3)2), 3.83–3.86 (t, CH2eCH2eN+), 4.65(s, N+eCH2eΦ), 4.73–4.75 (t, OeCH2eCH2), 5.82, 6.21 (d, CH2]C(CH3)), 7.62 (s, ΦeH). FT-IR (KBr, ν, cm− 1): 2853–2970 (νCH), 1722 (νC]O),1635 (νC]C), 1041 (νCeN), 768 (γΦeH).
2.2. Synthesis of poly(2-(dimethylamino)ethyl methacrylate) containing quaternary ammonium salts (PDMAEMA-BC) Poly(2-(dimethylamino)ethyl methacrylate) containing quaternary ammonium salts (PDMAEMA-BC) was prepared in a two-step process, i.e., first, the controlled synthesis of PDMAEMA, followed by
Scheme 1. Synthetic route to PDMAEMA and PDMAEMA-BC.
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100 μL of the polymer solutions with different concentrations were transferred to a 96-well plate. Medium was added instead of polymer solution as a control plate. Then, the mixtures were incubated at 28 °C (R. solani, Foc4 and C. albicans) for 48 h or 37 °C (E. coli and S. albus) for 24 h, after which 50 μL of a 0.5 wt% 2,3,5-triphenyl tetrazolium chloride (TTC) solution was added into the 96-well plate, and the contents of each well were mixed by vortex shaking prior to incubation in the dark at 28 °C (R. solani, Foc4 and C. albicans) or 37 °C (E. coli and S. albus) for 24 h. The MIC value was the lowest concentration at which there was no visible growth, i.e., no red color due to the reduction of TTC [17]. The MFC or MBC test is the most common estimation of fungicidal or bactericidal activity and is defined as the lowest concentration of antimicrobial agent needed to kill 99.9% of the initial inoculums after incubation [18]. To determine the MFC or MBC values, 100 μL of the mixtures in the wells that were at or above the MIC value were plated on medium and incubated at 28 °C (R. solani, Foc4 and C. albicans) for 48 h or 37 °C (E. coli and S. albus) for 24 h. The MFC or MBC value was the lowest concentration at which fewer than 5 colony-forming units were observed. Additionally, the antimicrobial agent was classified as a bactericide or fungicide based on its MFC/MIC or MBC/MIC ratio. If this ratio was above 4, the antimicrobial agent was classified as bacteriostatic or fungistatic, otherwise they would be classified as bactericide or fungicide, respectively [19,20].
2.4. Characterization FT-IR spectra were collected on a VERTEX-70 spectrometer (Bruker Instrument Corp., Germany) using KBr pellets. 1H NMR spectra were obtained using a Bruker Avance III-400 (Bruker Instrument Corp., Germany) spectrometer with CDCl3 or D2O as the solvent. Size exclusion chromatography (SEC) was performed on a Waters system (Waters Corp., USA) equipped with a Waters 515 pump, a Waters 2414 differential refractive index detector, and three μ-Styragel columns. The samples were analyzed at 30 °C using THF (1 mL/min) as the eluent. The instrument was calibrated using polystyrene standards. Critical micelle concentrations (CMC) were determined using a DDS-11A conductivity meter (Shanghai Hongyi Instrument Co. Ltd., China). Zeta potentials and particle size distributions were analyzed using a Brookhaven BI-90 Plus particle size analyzer (Brookhaven Instrument Corp., USA) using ultrapure water as the solvent. The morphologies of the micelles were analyzed with a JEM-2100 transmission electron microscope (JEOL Ltd., Japan). 2.5. Antimicrobial tests 2.5.1. Test microorganisms and media The microorganisms included two typical phytopathogenic fungi (Rhizoctonia solani (R. solani) and Fusarium oxysporum f. sp. cubense race 4 (Foc4)), Gram-negative bacteria (Escherichia coli (E. coli)), Gram-positive bacteria (Staphylococcus albus (S. albus)) and pathogenic fungi (Candida albicans (C. albicans)). The media were prepared according to the literature [13,16].
3. Results and discussion 3.1. Design and synthesis of DMAEMA-BC and PDMAEMA-BC
2.5.2. Determination of mycelial growth inhibition (MGI) The antifungal activities of the samples were tested using the inhibition of mycelial growth. To prepare polymer solutions with different concentration, the samples were dissolved in sterilized water. Next, 45 mL of PDA and 5 mL of polymer solution were poured into sterile Petri dishes (diameter of 90 mm). Sterilized water was added instead of a polymer solution as control Petri dish. Then, a 6-mm disc containing mycelia was transferred to the center of the Petri dishes and incubated at 28 °C. Each experiment was conducted in triplicate. The diameter of the fungal colony was measured using a cross method three times and averaged. MGI was calculated using Eq. (1).
MGI = (R C − RT)/R C × 100%
By designing and synthesizing a series of PDMAEMA-BC polymers with tunable molecular weights, we could then examine the relationship between DP and antimicrobial activity. The chemical structures of the products were confirmed by FT-IR and 1H NMR spectroscopies. As a multifunctional monomer containing double bonds and tertiary amino groups, 2-(dimethylamino)ethyl methacrylate (DMAEMA) could be polymerized by ATRP and then quaternized by haloalkanes for the preparation of antibacterial materials [9]. Quaternized PDMAEMA is considered a promising antimicrobial agent [21]. By adjusting the mole feeding ratio of [DMAEMA]/[EtBriB] from 4/1 to 80/1, a series of PDMAEMA polymers with various molecular weights were synthesized by ATRP. The molecular weights of PDMAEMA, determined by SEC, are shown in Table 1. As shown in Figs. 1 and 2, the absorption peaks at 2863–2947, 1732, 1047 and 1020 cm− 1 represented the infrared absorption bands of saturated CeH, C]O and CeN, respectively, and the signals for the CeCH3, CeCH2eC, N(CH3)2, NeCH2eCH2 and OeCH2eCH2 protons appeared at 0.97, 1.98, 2.37, 2.77 and 4.20 ppm, respectively. A series of PDMAEMA-BC polymers were obtained from a quaternization reaction between PDMAEMA and benzyl chloride (BC). As
(1)
whereby RC and RT were the radial growth of the fungi colonies in the control plate and the treatment plate, respectively. The radial growth of the fungi colony was calculated by Eq. (2):
Radial growth (mm) = Average diameter of fungi colony (mm) − 6 (mm) (2) 2.5.3. Determination of effective concentration (EC) The antimicrobial activities of DMAEMA-BC and PDMAEMA-BC against R. solani and Foc4 were evaluated using the EC50 and EC90 values. The regression equation was obtained by using the natural logarithm of the polymer solution concentration as the abscissa and the biological statistical probability, converted to MGI, as the ordinate. The EC50 and EC90 values were calculated using the regression equation when the MGI values were 50% and 90%, respectively.
Table 1 Molecular weights of the PDMAEMA polymers prepared with different [DMAEMA]/ [EtBriB] feeding ratios.
2.5.4. Determination of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and minimum bactericidal concentration (MBC) The antimicrobial properties of DMAEMA-BC and PDMAEMA-BC against R. solani, Foc4, E. coli, S. albus and C. albicans were evaluated by the TTC coloration method [17]. Bacterial suspensions (108 CFU/mL) were incubated in medium and diluted 1000 times to obtain a concentration of 105 CFU/mL. Next, 100 μL of the bacterial suspension and
Samples
[DMAEMA]/[EtBriB] feeding ratio, mol/mol
Mna (g/mol)
Mwa (g/mol)
PDI (Mw/ Mn)a
PDMAEMA-4 PDMAEMA-8 PDMAEMA-16 PDMAEMA-30 PDMAEMA-60 PDMAEMA-70 PDMAEMA-80
4/1 8/1 16/1 30/1 60/1 70/1 80/1
0.71 × 103 1.41 × 103 2.99 × 103 5.16 × 103 9.25 × 103 11.6 × 103 14.7 × 103
0.84 × 103 1.58 × 103 4.52 × 103 6.97 × 103 1.09 × 104 13.7 × 103 23.3 × 103
1.18 1.12 1.51 1.35 1.18 1.18 1.59
a
3
Based on SEC results.
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Fig. 1. FT-IR spectra of PDMAEMA, DMAEMA-BC and PDMAEMA-BC. Fig. 3. CMC values of DMAEMA-BC and the PDMAEMA-BC polymers with different molecular weights.
shown in Figs. 1 and 2, the absorption peaks at 2870–2979, 1726, 1041 and 768 cm− 1 represented the infrared absorption bands of saturated CeH, C]O, CeN and Φ-H, respectively, and the signals for the CeCH3, N+(CH3)2, CH2eCH2eN+, N+eCH2eΦ, OeCH2eCH2 and ΦeH protons appeared at 1.06, 3.14, 3.87, 4.35–4.76 and 7.59 ppm, respectively. After the quaternization reaction, the N(CH3)2 proton signals shifted from approximately 2.37 to 3.14 ppm and the characteristic protons signals of BC (4.35–4.76 and 7.59 ppm) appeared, which indicated that the expected structure of PDMAEMA-BC were formed. DMAEMA-BC was obtained from a quaternization reaction between DMAEMA and benzyl chloride (BC). As shown in Figs. 1 and 2, the absorption peaks at 2853–2970, 1722, 1635, 1041 and 768 cm− 1 represented the infrared absorption bands of saturated CeH, C]O, C]C, CeN and ΦeH, respectively, and the signals form the CeCH3, N+(CH3)2, CH2eCH2eN+, N+eCH2eΦ, OeCH2eCH2, CH2]C(CH3) and ΦeH protons appeared at 1.98, 3.18, 3.83–3.86, 4.65, 4.73–4.75,
5.82, 6.21 and 7.62 ppm, respectively. 3.2. Critical micelle concentrations (CMC) of DMAEMA-BC and PDMAEMA-BC in water The concentration above which micelles form is called the critical micelle concentration (CMC) [22]. We measured the CMC values of DMAEMA-BC and PDMAEMA-BC by the electrical conductivity method. Fig. S1 (Supporting information) shows the conductivity plots for DMAEMA-BC and the PDMAEMA-BC polymers with different molecular weights, and Fig. 3 shows the CMC values calculated from the curves in Fig. S1. The test results showed that the CMC value of PDMAEMA-BC with low DP is higher than those of the other DMAEMA-BC and PDMAEMA-BC samples with higher DP. It was reasonable to assume
Fig. 2. 1H NMR spectra of PDMAEMA, PDMAEMA-BC and DMAEMA-BC.
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which were consistent with the results of zeta potentials and particle sizes. 3.4. Antimicrobial activity against bacteria and pathogenic fungi Screening of the antimicrobial activities of DMAEMA-BC and PDMAEMA-BC against bacteria and pathogenic fungi was performed using E. coli, S. albus and C. albicans as test organisms because they represent Gram-negative, Gram-positive bacteria and pathogenic fungi, respectively. The MIC and MBC or MFC values of DMAEMA-BC and PDMAEMA-BC were determined using the TTC coloration method. Fig. 7 is a typical photograph from the determination of the MIC values for DMAEMA-BC and PDMAEMA-BC-4 against E. coli, S. albus, C. albicans, R. solani and Foc4. The MIC and MBC or MFC values for DMAEMA-BC and PDMAEMA-BC against E. coli, S. albus and C. albicans are shown in Fig. 8. As shown in Fig. 8, the results indicated that the MIC and MBC or MFC values of the homopolymers were far lower than those of the monomers against both E. coli, S. albus and C. albicans, which meant that the activity of the polymers was much higher than that of the corresponding monomers. The CMC values of DMAEMA-BC and PDMAEMA-BC were lower than the MIC and MBC or MFC; thus, DMAEMA-BC and PDMAEMA-BC are able to form micelles. However, in the particle size distribution test, we found that DMAEMA-BC is not capable of forming large micelles. Furthermore, although the formation of micelles in a real environment is difficult, the linkages formed by covalent bonds guarantee the aggregation of charge. It was reasonable to assume that the change from a monomer to a polymer could increase the local charge density; thus, adsorption on bacterial cell surfaces was enhanced for the polymer compared to the monomer, and binding to the cytoplasmic membrane was also facilitated by the polymer because of the presence of negatively charged species in the membrane [13]. Besides, for DMAEMA-BC and the PDMAEMA-BC polymers with different molecular weights, the best antimicrobial activities against animal pathogenic bacteria and fungi were not observed for the high and low molecular weights. The optimal antimicrobial activity against E. coli, S. albus and C. albicans were achieved when the DP of PDMAEMA-BC was about 8. It is noticed that the MIC and MBC values for PDMEMA-BC-8 against E. coli were lower than the CMC, which meant that PDMEMA-BC-8 exhibited the best antimicrobial activity against E. coli without forming micelles. Without considering the formation of micelles, the processes of adsorbing onto the bacterial cell surface and disrupting the cytoplasmic membrane are likely enhanced by increasing the molecular weight of the antimicrobial agents, whereas the process of diffusing through the cell wall is inhibited by increasing the molecular weight [14]. Peptidoglycan is a major part of bacteria cell walls with open network structures. Bacteria cell walls generally do not act as significant permeability barriers against compounds with molecular masses below 50 kDa [23]. Additionally, it was observed that the DMAEMA-BC and PDMAEMA-BC with high chain lengths gave higher zeta potentials, which led to a decrease in the particle size. Thus, the largest particle size of PDMAEMA-BC was obtained from a moderate chain length which means that the degree of aggregation was the highest when the PDMAEMA-BC chain length was moderate. In conclusion, the observed optimal molecular weight for antibacterial activity can be explained based on the combined effect of those two kinds of controlling factors. Additionally, compared with the antimicrobial activities of the PDMAEMA-BC against E. coli, the test results indicated that the antimicrobial activities of the PDMAEMA-BC against S. albus were minimally dependent on the molecular weight when the molecular weight of PDMAEMA-BC was too high. This finding can be explained using the fact that Gram-positive bacteria tend to have a loose cell wall, whereas Gram-negative bacteria have an outer membrane structure in the cell wall that forms an additional barrier to foreign molecules [3,8]. This allowed the PDMAEMA-BC polymers with high molecular weight to
Fig. 4. Zeta potentials of DMAEMA-BC and the PDMAEMA-BC polymers with different molecular weights.
that the change from a monomer to a polymer with low DP increase the degree of branch, and the hydrophobicity of the backbone was increased with increase of the chain length. However, the environment in which bacteria live is more complex than pure water. The formation of micelles by DMAEMA-BC and PDMAEMA-BC is more difficult in real environment that contains salt, leaked cytoplasmic contents, and so on. 3.3. Zeta potentials and particle size distributions of DMAEMA-BC and PDMAEMA-BC in water The zeta potentials and particle sizes of DMAEMA-BC and the PDMAEMA-BC polymers displayed in Figs. 4 and 5 showed that: (1) PDMAEMA-BC with low DP exhibited the low zeta potential, which meant it easier to aggregates; (2) PDMAEMA-BC with low DP exhibited the larger particle sizes than the samples with higher DP; (3) the particle size of PDMAEMA-BC was decreasing due to the increase of absolute value of zeta potential of PDMAEMA-BC; (4) the particle size of DMAEMA-BC was below the minimum diameter that can be measured, which meant that DMAEMA-BC was unable to form large micelles. As shown in Fig. 6, the TEM study of the micellar conformation of DMAEMA-BC and PDMAEMA-BC shows that the particle sizes of PDMAEMA-BC with low DP were larger than those of the other samples,
Fig. 5. Particle size distributions of PDMAEMA-BCs with different molecular weights. The particle size distribution of DMAEMA-BC could not be determined because the diameters were below the minimum diameter that can be measured.
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Fig. 6. Transmission electron microscope of (A) DMAEMA-BC, (B) PDMAEMA-BC-4, (C) PDMAEMA-BC-8, (D) PDMAEMA-BC-16, (E) PDMAEMA-BC-30 and (F) PDMAEMA-BC-80.
Fig. 7. Typical photograph of the MIC value determination for DMAEMA-BC and the PDMAEMA-BC-16 polymers against E. coli, S. albus, C. albicans, R. solani and Foc4.
and the PDMAEMA-BC polymers with different molecular weights, the optimal antimicrobial activity against R. solani and Foc4 were achieved when the DP of PDMAEMA-BC were 4 and 16, respectively. Similar trends were observed in the antimicrobial activities of DMAEMA-BC and PDMAEMA-BC against bacteria and pathogenic fungi. It is noticed that most of the MIC and MFC values for PDMEMA-BC against R. solani and Foc4 were lower than the CMC, which meant that the factor of
diffuse through the cell walls of the Gram-positive bacteria more easily. 3.5. Antifungal activity against phytopathogenic fungi (R. solani and Foc4) Screening of the antimicrobial activities of DMAEMA-BC and PDMAEMA-BC against phytopathogenic fungi was performed using R. solani and Foc4 as test organisms. As shown in Fig. 8, for DMAEMA-BC 6
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Fig. 8. MIC and MBC (for bacterial) or MFC (for fungi) values of DMAEMA-BC and PDMAEMA-BC against (A) E. coli, (B) S. albus, (C) C. albicans, (D) R. solani and (E) Foc4.
DMAEMA-BC and PDMAEMA-BC-16 against R. solani and Foc4. The mycelial growth inhibition by DMAEMA-BC and PDMAEMA-BC-16 against R. solani and Foc4 in PDA medium is shown in Fig. 10, and the EC50 and EC90 values were calculated using the regression equation when the MGI values were 50% and 90%, respectively. As shown in Fig. 11, PDMAEMA-BC-4 and PDMAEMA-BC-16 exhibited the best antimicrobial activity against R. solani and Foc4 respectively, and the
chain length played a dominant role compared to the factor of the formation of micelles. With an increase in the chain length of PDMAEMA-BC, the aggregated charge was more stable in the real environment due to the covalent bond linkages. However, diffusion through the cell wall became more difficult with increasing PDMAEMABC chain length, which led to a decrease in the antimicrobial activity. Fig. 9 is a typical photograph displaying the antifungal effects of
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Fig. 9. Typical mycelial growth of R. solani and Foc4 in PDA medium after application of DMAEMA-BC and PDMAEMA-BC-16. The data were based on three replicates, and the test was repeated twice with similar results. Representative results are shown.
Fig. 10. Mycelial growth inhibition by DMAEMA-BC and PDMAEMA-BC-16 against R. solani and Foc4 in PDA medium. The growth of R. solani and Foc4 was measured after 48 h of incubation at 28 °C.
Fig. 11. EC50 and EC90 values of DMAEMA-BC and PDMAEMA-BC against (A) R. solani and (B) Foc4. (*: The calculated EC50 and EC90 values of DMAEMA-BC against Foc4 are much larger than 20.)
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References
antimicrobial activities were lower for the higher and lower molecular weights. For PDMAEMA-BC, the optimal antimicrobial activity against animal pathogenic bacteria and fungi was achieved with an intermediate chain length, which was consistent with the results of MIC and MFC values. Compared to bacteria, the structures of fungi cells are much more complicated. From the studies of numerous fungi, the cell wall has been shown to be primarily composed of chitin, glucans, mannans and glycoproteins, and the wall composition frequently varies markedly between species of fungi [24,25]. In addition, fungi cells contain organelles, such as mitochondria, endoplasmic reticuli and Golgi bodies. Therefore, the ability of antimicrobial agents to diffuse through the cell wall and disrupt the cellular structure is likely different between fungi and bacteria and even between various fungi. Most of the MIC and MFC values for PDMAEMA-BC against R. solani and Foc4 were lower than those against E. coli, S. albus and C. albicans, which meant the PDMAEMA-BC polymers were more suitable for inhibiting phytopathogenic fungi. Additionally, the antifungal activities of the PDMAEMA-BC polymers against R. solani were shown to have a greater molecular weight dependence than those against Foc4.
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4. Conclusions In this paper, DMAEMA-BC and a series of PDMAEMA-BC homopolymers with tunable molecular weights were synthesized to quantify the relationship between their antimicrobial properties and chain length. From the present results, optimal antimicrobial (E. coli, S. albus, C. albicans, R. solani, and Foc4) activities were achieved for an intermediate DP of PDMAEMA-BC. To properly explain the relationship between antimicrobial activity and molecular weight, the bacteria structure and the ability of the antimicrobial agent to diffuse through the cell wall and disrupt the cellular structure were considered. PDMAEMA-BC with low DP exhibited the low zeta potential, which meant it easier aggregates and form micelles with large particle size. On the other hand, the factor of chain length played a dominant role and the optimal antimicrobial activity was achieved with an intermediate chain length by taking account of the influence of both aggregated charge due to the covalent bond linkages and diffusion through the cell wall. The systematic approach to examining the antimicrobial activities of PDMAEMA-BC homopolymers with varying DP is expected to guide the future design of antimicrobial polymers as potential fungicide agents for controlling plant disease. Acknowledgments This work was supported by the Science and Technology Program of Guangzhou, China, under grant 201704020084; the Science and Technology Planning Project of Guangdong Province, China under grant 2016A020210105; and the National Natural Science Foundation of China under grant 31201552. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.reactfunctpolym.2017.10.010.
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