Antibacterial Activity of Geminized Amphiphilic Cationic

Nov 25, 2015 - State Key Laboratory of Offshore Oil Exploitation; CNOOC Research ... Shengli Oilfield Branch Company, SINOPEC, Dongying 257000, ...
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Antibacterial Activity of Geminized Amphiphilic Cationic Homopolymers Hui Wang,† Xuefeng Shi,† Danfeng Yu,† Jian Zhang,‡ Guang Yang,‡ Yingxian Cui,‡ Keji Sun,§ Jinben Wang,*,† and Haike Yan† †

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ‡ State Key Laboratory of Offshore Oil Exploitation; CNOOC Research Institute, Beijing 100027, People’s Republic of China § Oil Production Technology Research Institute, Shengli Oilfield Branch Company, SINOPEC, Dongying 257000, People’s Republic of China S Supporting Information *

ABSTRACT: The current study is aimed at investigating the effect of cationic charge density and hydrophobicity on the antibacterial and hemolytic activities. Two kinds of cationic surfmers, containing single or double hydrophobic tails (octyl chains or benzyl groups), and the corresponding homopolymers were synthesized. The antimicrobial activity of these candidate antibacterials was studied by microbial growth inhibition assays against Escherichia coli, and hemolysis activity was carried out using human red blood cells. It was interestingly found that the homopolymers were much more effective in antibacterial property than their corresponding monomers. Furthermore, the geminized homopolymers had significantly higher antibacterial activity than that of their counterparts but with single amphiphilic side chains in each repeated unit. Geminized homopolymers, with high positive charge density and moderate hydrophobicity (such as benzyl groups), combine both advantages of efficient antibacterial property and prominently high selectivity. To further explain the antibacterial performance of the novel polymer series, the molecular interaction mechanism is proposed according to experimental data which shows that these specimens are likely to kill microbes by disrupting bacterial membranes, leading them unlikely to induce resistance. groups,20,21 and chemical composition.22 Ikeda and coworkers23,24 investigated the molecular weight influence of antibacterial activity of polymethacrylates with pendant groups of biguanide and homopolymers of polyacrylates and their copolymers with acrylamide. Eren et al.25 prepared a series of pyridinium functionalized polynorbornenes with different lengths of carbon side chain and tested these polymers for antibacterial and hemolytic activity. Chen et al.26 synthesized the quaternary ammonium dendrimers and studied the counterion dependence of the antimicrobial activity. Krumm et al.21 prepared a polymer which could control both antibacterial activity and cell toxicity by attaching a biocleavable satellite group. Mizutani et al.22 synthesized a series of copolymers with different structural characteristics to test the influence of polymer properties on their antibacterial and hemolytic activities. Up to now, investigations on antibacterial activity with geminized amphiphilic cationic homopolymers, containing both double hydrophilic groups and two hydrophobic moieties in each structural unit, have rarely been reported. This kind of homopolymers not only has ultrahigh positive charge density

1. INTRODUCTION Since Fleming discovered penicillin more than 80 years ago, antimicrobials have played an important role in medical fields and healthcare. The cationic surfactants, especially amphiphilic quaternary ammonium salts, are widely studied for their excellent antibacterial effects.1−3 Compared to monomeric and small organic antimicrobials, the use of cationic polymers provides promise for increasing their efficiency and selectivity, prolonging their lifetime, and reducing the environmental problems.4 The membrane surface of bacteria is usually negatively charged because of the lipid headgroups of their membranes, which is provided by lipoteichoic acids and teichoic acids of Gram-positive bacteria and lipopolysaccharides of Gramnegative bacteria.5 Thus, cationic antimicrobial polymers first adsorb on the surface of the negatively charged cell membrane and then insert their hydrophobic groups into the bacterial cell membranes, thereby disrupting them.6 Because of the physical way of destroying the cell membrane,7−9 these cationic antimicrobial polymers are postulated to reduce the likelihood of pathogens developing resistance. There are many factors for antibacterial agents that can affect their antimicrobial activity such as molecular weight,10−12 spacer length between polymer backbone and active site,13,14 hydrophilic−hydrophobic balance,15−17 nature of counterions,18,19 biologically active end © XXXX American Chemical Society

Received: February 16, 2015 Revised: November 16, 2015

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revealed that antibacterial selectivity toward a wide range of pathogenic bacteria over mammalian cells can significantly be enhanced by adjusting the charge density and hydrophobicity of the candidate antimicrobials. Cryo-transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to visualize the bacterial morphological changes caused by the homopolymers. Further, biophysical study such as homopolymer-induced dye leakage from model liposomes was carried out. On the basis of the experimental results, the antibacterial mechanism was proposed.

which is conducive to adsorption on the cell membrane but also has strong hydrophobic interaction between the long hydrophobic chains of the homopolymer and the liposomes of the bacterial membrane, which destroy the membrane much more efficiently. Thus, the novel class of geminized amphiphilic cationic homopolymers should be a promising candidate of antibiotics with excellent antibacterial effects. In the present work, two geminized amphiphilic homopolymers, poly-1,3-bis(N,N-dimethyl-N-octylammonium)-2-propyl acrylate dibromide (referred as PAGC8) and poly-1,3-bis(N,Ndimethyl-N-benzylammonium)-2-propyl acrylate dibromide (referred as PAGBn), and their counterparts, polyacryloyloxyethyl-N,N-dimethyl-N-octylammonium bromide (referred as PASC8) and polyacryloyloxyethyl-N,N-dimethyl-N-benzylammonium bromide (referred as PASBn), comprising a single hydrophilic group and one hydrophobic moiety in each structural unit, were synthesized. Their antibacterial activity as well as their hemolysis of human red blood cells was tested in order to study the charge density and hydrophobic effects on antibacterial activity. Their corresponding monomers were investigated as well for comparison (Scheme 1). Results

2. EXPERIMENTAL SECTION Materials. PAGC8, PAGBn, PASC8, and PASBn as well as their monomers were prepared according to our previous protocol,27 and their synthetic routes are shown in Scheme 2. Deuterium oxide (99.9%) and chloroform-d (99.9%) were purchased from Acros, and soybean lecithin (98%) was obtained from Aladdin. Acryloyl chloride, 1-bromine octane, benzyl bromine, ammonium persulfate, ammonium iron(II) sulfate hexahydrate, and solvents were purchased from Beijing Chemical Co. (analytical reagent (AR) grade). All reagents except those especially mentioned were used as received. The ampicillin resistant Gram-negative bacteria (Escherichia coli, TOP10) were donated by Prof. Shu Wang from Institute of Chemistry, Chinese Academy of Sciences, China. They purchased the E. coli from Beijing Bio-Med Technology Development Co., Ltd., and transfected it with ampicillin resistant plasmids (pcDNA3, Invitrogen).28 The optical density (OD) of the E. coli cell suspension was regulated on a Jasco V550 spectrometer. Gel Permeation Chromatography (GPC). The homopolymer molecular weights and polydispersities were determined by Waters Breeze 1515 GPC analysis system (dimethylformamide, DMF). The chromatographic column was Styragel HT3.4.5 7.8 × 300 mm long, and the molecular weights were estimated against polystyrenes. The homopolymers were dissolved in DMF and were filtered with 0.45 μm organic membrane aperture prior to analysis. Steady-State Fluorescence. Pyrene was employed as a fluorescence probe at the concentration of 1 μM, and the phosphate-buffered saline (PBS) solutions of the samples were stirred at room temperature overnight before measurement. Steady-state fluorescence spectra were obtained with a Hitachi F-4500 spectrofluorometer at 25.0 ± 0.5 °C. The emission spectra scanned from 350 to 550 nm using a 335 nm excitation wavelength. The width of the slit was 2.5 nm. The critical

Scheme 1. Chemical Structures of Polymerizable Surfactants (I) and the Corresponding Homopolymers (II)

Scheme 2. Synthetic Routes for Polymerizable Cationic Gemini Surfactants and the According Homopolymers

B

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in PBS. Samples dissolved in PBS were then added to 1 mL of a solution of the stock hRBC in PBS to reach a final volume of 2 mL (final erythrocyte concentration, 5% v/v). The resulting suspension was incubated for 30 min at 37 °C. The samples were then centrifuged at 8000 r/min for 3 min. Release of hemoglobin was monitored by measuring the absorbance of the supernatant at 540 nm. Controls for zero hemolysis (blank) and 100% hemolysis consisted of hRBC suspended in PBS and Triton 1%, respectively. Each assay was performed in three replicates. The percentage of hemolysis was defined as

aggregation concentration (CAC) was taken as a point where a distinct change in the decrease of I1/I3 occurred.29 Electrical Conductivity. The conductivity of the homopolymer solutions was measured as a function of concentration using a JENWAY model 4320 conductivity meter in a temperature-controlled (25.0 ± 0.1 °C), double-walled glass container with a circulation of water. Sufficient time was allowed for equilibrium between successive additions. Scanning Electron Microscope (SEM). The samples were fixed in 2.5% glutaraldehyde for 2 h at 4 °C, then were washed three times with cacodylate buffer, and were dehydrated through a series of graded ethanol solutions (25, 50, 75, 95, and 100%). The samples were subsequently freeze-dried and coated with platinum before examination in SEM. Then, the images were taken on Hitachi S-4300 scanning electron microscopy at a operating voltage of 10 kV. Cryogenic Transmission Electron Microscopy (CryoTEM). The sample was embedded in a thin layer of vitreous ice on freshly carbon-coated holey TEM grids by blotting the grids with filter paper and then by plunging them into liquid ethane cooled by liquid nitrogen. Frozen hydrated specimens were imaged by using an FEI Tecnai 20 electron microscope (LaB6) operated at 200 kV with the low-dose mode (about 2000 e/ nm2) and the nominal magnification of 50 000. For each specimen area, the defocus was set to 1−2 μm. Images were recorded on Kodak SO163 films and then were digitized by Nikon 9000 with a scanning step 2000 dpi corresponding to 2.54 Å/pixel. Synthesis. The synthetic route and the structure of gemini surfmers and the corresponding homopolymers are shown in Scheme 2, and the synthetic method was reported in the previous paper.27 Bacterial Killing Experiments. A single colony of E. coli on a solid Luria−Bertani (LB) agar plate was transferred to 10 mL liquid LB culture medium in the presence of 50 μg/mL ampicillin and was incubated overnight with constant shaking of 120 rpm at 37 °C. Bacteria were harvested by centrifuging (9000 rpm for 2 min) and were washed by PBS three times. The supernatant was discarded, and the remaining E. coli was resuspended in PBS. The optical density (OD600) of the bacterial suspension was adjusted to 1.0 and subsequently was diluted 5 fold by PBS. A certain volume and concentration of antimicrobial PBS solutions was placed into a sterile centrifuge tube, and then a certain quantity of PBS was added to make the total solution volume 400 μL, and the next 100 μL of prepared bacterial suspension was mixed into the solution. Each sample of a given concentration was placed for 30 min and then was serially diluted (10 000 fold) in PBS. A 100 μL portion of the diluted bacterial E. coli was spread on the solid LB agar plate, and the colonies formed after 12−16 h incubation at 37 °C were counted. Each test was carried out in three replicates. The bacterial inhibition ratio was calculated by

hemolysis (%) =

OD(test) − OD(negative control) OD(positive control) − OD(negative control) × 100%

Liposome Preparation. Buffer A (10 mM Na2HPO4, pH 7.0) and buffer B (10 mM Na2HPO4, 90 mM NaCl, pH 7.0) were used. Calcein dye was dissolved in buffer A to achieve a concentration of 5 mM. An amount of 250 mg lecithin was dissolved into 25 mL chloroform in a round-bottomed flask. Then, the organic solvent was evaporated by a rotary evaporator (Yalong Instrument Co. Ltd., Shanghai, China) under vacuum, leaving a uniform thin film on the wall of the flask, which was then hydrated by 50 mL calcein solution. Thereafter, the mixture of calcein solution was sonicated in a bath-type sonicator with the temperature under 10 °C until it became clear, after which it was subjected to five freeze−thaw cycles (using liquid nitrogen to freeze and warm water to thaw). Unincorporated calcein was removed from the liposomes by repeated centrifugal washes in buffer B using a supercentrifuge (16 000g) (TGL-16B, Shanghai Anting Scientific Instrument Factory, Shanghai, China). Finally, the vesicle suspension was diluted with buffer B to prepare aliquots with a final lipid concentration of about 3 g/L. Homopolymers were added to the lipid solutions to make the final concentration up to their minimum inhibitory concentration (MIC) values which were measured by bacterial killing experiments. The kinetics of leakage was monitored by following the increase of calcein fluorescence intensity at 515 nm (excitation at 490 nm, slit width 2.5 nm) because of the inhibition of self-quenching. Complete leakage was achieved by addition of 10 μL of 0.2% Triton X-100 to the 2 mL solution, and the corresponding fluorescence intensity was used as 100% leakage for the calculation of the leakage fraction. The calcein release percentage was calculated by R% =

F − Fini × 100% Ffin − Fini

where R(%) is the release percentage, Fini and F are the fluorescence intensity of calcein in PC liposome solutions measured before and after adding homopolymers, respectively, and Ffin is the final intensity when the calcein−liposomes are completely destroyed by adding 0.2% Triton X-100 (w/w).

bacterial inhibition ratio (%) = ⎛ ⎞ the colony forming units of the sample ⎜1 − ⎟ the colony forming units of the controlled sample ⎠ ⎝

3. RESULTS AND DISCUSSION The cell wall of bacteria usually has negative net charges because of the lipid headgroups of their membranes, at which the cationic antibacterials are attracted. If the cationic antimicrobials have amphiphilic groups, they can adsorb and then disrupt the bacterial membranes, leading to cell death.30 To research the influence of positive charge density and

× 100%

Hemolysis Assay. The samples were tested for their hemolytic activities against human red blood cells (hRBC). Fresh hRBC with EDTA were rinsed four times with PBS by centrifugation for 3 min at 8000 r/min and were resuspended C

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Mn

PAGC8 PASC8 PAGBn PASBn AGC8 ASC8 AGBn ASBn

× × × ×

9.9 9.8 6.2 6.1 586 336 542 314

3

10 103 103 103

PD

fluorescence

1.14 1.12 1.39 1.3

4.8 5.6 13.7 39 13.8 195 35 414

± ± ± ± ± ± ± ±

0.2 0.2 0.5 1 0.5 5 1 10

CAC (μg/mL) in water fluorescence 130 70 1200 1110 1460 2400 1510 3600

± ± ± ± ± ± ± ±

5 2 50 30 50 100 50 100

conductivity 120 60 850 510 550 1580 630 1810

± ± ± ± ± ± ± ±

5 2 30 30 10 50 10 50

α 0.92 0.76 0.92 0.83 0.88 0.88 0.89 0.88

a Molecular weights of the samples were measured by GPC, and critical aggregation concentration (CAC) was measured by steady-state fluorescence in PBS and water. The conductivity experiment was carried out in water.

Figure 1. Fluorescence intensity ratio I1/I3 curves of monomers (A in PBS and C in water) and homopolymers (B in PBS and D in water) and electrical conductivity (κ) curves of (E) monomers and (F) homopolymers in water at 25 °C.

low molecular weight. Ikeda et al.23 found that the antibacterial properties decreased sharply when the molecular weights of materials were more than 1.2 × 105 Da, and they considered that the antibacterial activity of the samples was on the basis of the permeability through the cell membrane. The molecular weights of the homopolymers studied are less than 1 × 104 Da as shown in Table 1; thus, it should be little obstacle for them to permeate through the cytomembrane.

hydrophobic chain on antibacterial and hemolytic activity, four homopolymers and their corresponding monomers were synthesized. Characterization. The molecular weight of antibacterial agent has an important effect on antimicrobial activity, which can be illuminated according to the permeability through the cell wall.31 Chen et al.26 pointed out that the diffusion of antibacterial agents with high molecular weight into the cytoplasmic membrane was lower than that of the ones with D

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Figure 2. Dead percentage of E. coli versus concentrations of (A) homopolymers and (B) monomers; (C) MIC and HC50 data for amphiphilic homopolymers and the corresponding monomers.

Table 2. MIC and HC50 of the Tested Compounds

a

antimicrobials

PAGC8

PASC8

PAGBn

PASBn

AGC8

ASC8

AGBn

ASBn

MIC (μg/mL) HC50(μg/mL) antibacterial selectivitya

10 40 4

20 60 3

50 3000 60

200 >10 000 >50

500 3000 6

800 >10 000 >12.5

1200 ≫10 000

>1500 ≫10 000

The antibacterial selectivity is the ratio of the HC50 to the MIC.

The relative intensity of the first vibronic band I1 and the third I3 of pyrene is commonly used to measure the CAC.32 The variations of I1/I3 versus the concentration of the candidate antibacterials in water and PBS solution are shown in Figure 1 (A−D). When the samples are at the lowest concentration (C) and the I1/I3 ratios are almost constant with C increasing, it can be inferred that the vast majority of pyrene molecules exit in an aqueous environment. As C is increased, the I1/I3 ratios decrease rapidly at a certain range, manifesting that the intermolecular aggregation begins to form. Thus, CAC values of the amphiphilic materials can be determined by the turning points. The CAC values of the samples in PBS are about 100 times lower than those in water, suggesting that inorganic ions contribute to the hydrophobic groups gathered to each other, which is partly due to the decrease in electrostatic repulsive interactions. Further, the CAC values of the monomers are much higher than those of the corresponding homopolymers, demonstrating that the associative ability of monomers is weaker than that of the homologous homopolymers. For the same reason, the aggregation ability of PAGC8 and PASC8 is better than that of PAGBn and PASBn, respectively, which indicates that the aggregation capacity of the long alkyl chain is stronger than that of the benzyl group. It can be speculated that the interaction between lipids of bacterial membrane and alkyl chains of the specimens is stronger than that between lipids of bacterial membrane and benzyl groups.

Positive charge density is very important to antibacterial activity for cationic biocides. To compare the charge density of the samples in water solution, the conductivity was measured. The aggregation ionization degrees (α) of the homopolymers have been calculated from the electrical conductivity curve, which are taken from α = (dκ/dC)C>Cm/(dκ/dC)C 10 000 μg/mL). Mammalian cell membranes and bacterial cell membranes have a fundamental difference. In a mammalian cell, the outer membrane is usually electrically neutral, while in a bacteria cell, the outer membrane is normally negatively charged.39 The selectivity is always from the different electrostatic interactions between antibacterial agents and mammalian cells compared to microbial cells.40 The bacterial agents first absorb on the membrane and then insert the hydrophobic moieties into the lipid bilayer of bacteria, damaging the membrane structure.7,8 Thus, we speculate that the interaction between lipid bilayer and benzyl groups is much weaker than that between lipid bilayer and octyl group. It is well-known that charge density and hydrophobicity have strong effects on the antibacterial activity for cationic amphiphilic polymers. In the case of our experiment, the MIC of PAGC8 and PAGBn is significantly lower than that of PASC8 and PASBn, respectively, indicating that high charge density and higher density of hydrophobic groups are beneficial to antimicrobial activity. The higher the charge density of the cationic agent, the stronger is the adsorption on the surface of bacteria. Therefore, the adsorption of PAGC8 and PAGBn onto bacterial membranes is likely to be stronger than that of PASC8 and PASBn. Furthermore, the hydrophobic groups of the samples insert into the lipid bilayer of the cell wall and induce it to reassemble, destroying the intact cell membranes and eventually leading bacteria to death. The double hydrophobes in each structure unit in PAGC8 and PAGBn show a synergistic effect in the process of aggregation;27 thus, the interaction with the lipid bilayer is stronger than that of the hydrophobic moieties in PASC8 and PASBn. As a result, the geminized amphiphilic homopolymers are more active than the corresponding single ones in antibacterial property.

It was also found that the MIC of the monomers is much higher than that of the homologous homopolymers, manifesting that the antimicrobial activity of the monomers is much lower than that of the homopolymers. The formation of homopolymers causes the local concentration of the pendant groups to increase, which augments the charge density and hydrophobicity and enhances the hydrophobic synergies between the homopolymers and the lipid bilayer of bacterial membranes. As a result, the antibacterial effects of homopolymers are much greater than their homologue monomers. The charge density and hydrophobicity are the key factors to increase the antibacterial activity of specimens, but a hydrophobic chain could interact with the mammalian cell, resulting in high hemolytic activity and toxicity to human beings. Therefore, moderate hydrophobicity should be considered in designing antibacterials. According to the earlier discussion, PAGBn, with fair antibacterial activity and very low hemolytic activity, can be an ideal candidate for antimicrobials. It was reasonably envisaged that the balance between charge density and hydrophobicity is the key in optimizing antibiosis. Antimicrobial Mechanism. The E. coli morphologies studied by cryo-TEM and SEM are shown in Figure 3. The

Figure 3. (I) Cryo-TEM images of E. coli bacteria (E, F) before and (A, B) after 0.5 h treatment with PAGC8 and PASC8 and (C, D) PAGBn and PASBn at 2 × MIC; (II) SEM images of E. coli bacteria (K) before and (G, H, I, J) after 0.5 h treatment with PAGC8 and PASC8 at 2 × MIC.

microbial cells treated with homopolymers exhibit obvious differences compared to the controlled ones. The surfaces of bacteria used for comparison are smooth and intact with clear edges (E, F), but those exposed to the environment of homopolymers appear to have a rough surface and some blebs (A, B), demonstrating that the cell surfaces of bacteria were severely damaged, and some bacteria even were totally broken. It was also found that there are some particles absorbed on the destroyed membranes after the treatment with homopolymers (C, D), which is attributed to the fact that the aggregates of the lipid bilayer of the bacterial membrane were induced by the homopolymers. SEM observation results showed that the surfaces of control bacteria are smooth and intact (K), while the bacteria have a rough surface with some protrusions or broken cells after the treatment by the homopolymers (G, H, I, J), F

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Figure 4. (A) The fluorescence spectra of calcein in PC liposome solutions with different specimens; (B) extent of calcein efflux in PC liposomes after treatment with the homopolymers.

which is in accordance with the images of cryo-TEM. This indicates that the homopolymers can severely damage and even lysis the cell membrane, which is similar to antibacterial peptides.41,42 Liposomes formed by lecithin (PC) can be used to model membrane cells when macromolecular antimicrobial agents were studied for the destructive potential to the cell membrane.43 To evaluate the role of the hydrophobic moieties in the homopolymer molecules in antimicrobial activity, the release of the encapsulated calcein from large unilamellar liposomes formed by PC was carried out, and the morphology of the liposomes upon the homopolymer treatment was observed. It was found from Figure 4A that before adding the homopolymers, the fluorescence intensity of the PC solution was weak as the calcein, being encapsulated in the liposomes, had a self-quenching concentration, and the intensity increased after being treated with the homopolymers, indicating that the liposomes broke and the calcein effluxed into the buffer solution which had a lower and nonself-quenching concentration. On the other hand, the calcein leakage ratios of PAGC8 and PASC8 were much higher than those of PAGBn and PASBn as shown in Figure 4B. Both PAGC8 and PASC8 exhibited significant activity against PC liposomes with the release percentage of calcein more than 80%, while PAGBn and PASBn showed weak disruption against the lipids with the release percentage of calcein less than 40%, which is attributed to the fact that long alkyl chains are prone to aggregate with the lipid bilayer, and then they destroy the liposomes more effectively than benzyl groups. These results give a better explanation that homopolymers with alkyl chains are more active in antibacterials and hemolysis than those with benzyl groups. To further observe the interaction between PC and the homopolymers, TEM experiments were carried out, and the images are shown in Figure 5. PC formed liposomes with a diameter of 100−300 nm in buffer A, but the liposomes disappeared and irregular bilayer tubules formed after adding PAGC8 (as model homopolymer), demonstrating that the homopolymer could interact with PC and break the liposomes aggregate structures. Similarly, the homopolymers could interact with the bacterial membrane and damage it. According to the earlier experiments, the antibacterial mechanism of the antimicrobial homopolymers is proposed as shown in Figure 6. As the cell walls of bacteria have negatively charged species, such as teichoic acids and lipopolysaccharides, cationic homopolymers can absorb on

Figure 5. (A) TEM images of PC liposomes in buffer A; (B) TEM images of PC after interaction with homopolymer PAGC8 at 2 × MIC.

Figure 6. Schematic representation of the antibacterial mechanism of geminized homopolymers.

the bacterial membrane by electrostatic interaction. The ionic interactions between the homopolymers and the bacterial cell wall are strengthened with the increasing of homopolymer charge density. Furthermore, the hydrophobic groups of the homopolymers insert into the lipid bilayer of the cell wall and induce it to reassemble, disrupting membrane structure. Thus, the geminized homopolymers have higher antibacterial activity than the corresponding single-chained homopolymers.

4. CONCLUSIONS Two kinds of geminized amphiphilic cationic homopolymers, as well as their corresponding counterpart with a single amphiphilic group in its repeat units, were synthesized, and the antibacterial and hemolytic activity was investigated. Geminized amphiphilic homopolymers, with higher charge density and stronger hydrophobicity than their corresponding counterparts with a single amphiphilic group in its repeat units, are much more effective in antibacterial activity. The MIC values of the antibacterial materials are higher than the CAC, demonstrating that self-association is needed for antibacterial activity. The homopolymers are more effective than their corresponding monomers in antibacterial property. Analysis of the experimental results enables us to conclude that the agents with long alkyl chains are more active in antibiosis and G

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(10) Engler, A. C.; Tan, J. P. K.; Ong, Z. Y.; Coady, D. J.; Ng, V. W. L.; Yang, Y. Y.; Hedrick, J. L. Antimicrobial Polycarbonates: Investigating the Impact of Balancing Charge and Hydrophobicity Using a Same-Centered Polymer Approach. Biomacromolecules 2013, 14, 4331−4339. (11) Huang, J. Y.; Murata, H.; Koepsel, R. R.; Russell, A. J.; Matyjaszewski, K. Antibacterial Polypropylene via Surface-Initiated Atom Transfer Radical Polymerization. Biomacromolecules 2007, 8, 1396−1399. (12) Timofeeva, L. M.; Kleshcheva, N. A.; Moroz, A. F.; Didenko, L. V. Secondary and Tertiary Polydiallylammonium Salts: Novel Polymers with High Antimicrobial Activity. Biomacromolecules 2009, 10, 2976−2986. (13) Palermo, E. F.; Vemparala, S.; Kuroda, K. Cationic Spacer Arm Design Strategy for Control of Antimicrobial Activity and Conformation of Amphiphilic Methacrylate Random Copolymers. Biomacromolecules 2012, 13, 1632−1641. (14) Punia, A.; He, E.; Lee, K.; Banerjee, P.; Yang, N. L. Cationic amphiphilic non-hemolytic polyacrylates with superior antibacterial activity. Chem. Commun. 2014, 50, 7071−7074. (15) Al-Badri, Z. M.; Som, A.; Lyon, S.; Nelson, C. F.; Nusslein, K.; Tew, G. N. Investigating the Effect of Increasing Charge Density on the Hemolytic Activity of Synthetic Antimicrobial Polymers. Biomacromolecules 2008, 9, 2805−2810. (16) Chin, W.; Yang, C. A.; Ng, V. W. L.; Huang, Y.; Cheng, J. C.; Tong, Y. W.; Coady, D. J.; Fan, W. M.; Hedrick, J. L.; Yang, Y. Y. Biodegradable Broad-Spectrum Antimicrobial Polycarbonates: Investigating the Role of Chemical Structure on Activity and Selectivity. Macromolecules 2013, 46, 8797−8807. (17) Punia, A.; Mancuso, A.; Banerjee, P.; Yang, N. L. Nonhemolytic and Antibacterial Acrylic Copolymers with Hexamethyleneamine and Poly(ethylene glycol) Side Chains. ACS Macro Lett. 2015, 4, 426−430. (18) Waschinski, C. J.; Barnert, S.; Theobald, A.; Schubert, R.; Kleinschmidt, F.; Hoffmann, A.; Saalwachter, K.; Tiller, J. C. Insights in the Antibacterial Action of Poly(Methyloxazoline)s with a Blocidal End Goup and Varying Satellite Groups. Biomacromolecules 2008, 9, 1764−1771. (19) Waschinski, C. J.; Zimmermann, J.; Salz, U.; Hutzler, R.; Sadowski, G.; Tiller, J. C. Design of Contact-Active Antimicrobial Acrylate-Based Materials Using Biocidal Macromers. Adv. Mater. 2008, 20, 104−108. (20) Waschinski, C. J.; Herdes, V.; Schueler, F.; Tiller, J. C. Influence of Satellite Groups on Telechelic Antimicrobial Functions of Polyoxazolines. Macromol. Biosci. 2005, 5, 149−156. (21) Krumm, C.; Harmuth, S.; Hijazi, M.; Neugebauer, B.; Kampmann, A. L.; Geltenpoth, H.; Sickmann, A.; Tiller, J. C. Antimicrobial Poly(2-Methyloxazoline)s with Bioswitchable Activity through Satellite Group Modification. Angew. Chem., Int. Ed. 2014, 53, 3830−3834. (22) Mizutani, M.; Palermo, E. F.; Thoma, L. M.; Satoh, K.; Kamigaito, M.; Kuroda, K. Design and Synthesis of Self-Degradable Antibacterial Polymers by Simultaneous Chain- and Step-Growth Radical Copolymerization. Biomacromolecules 2012, 13, 1554−1563. (23) Ikeda, T.; Hirayama, H.; Yamaguchi, H.; Tazuke, S.; Watanabe, M. Polycationic Biocides with Pendant Active Groups - MolecularWeight Dependence of Antibacterial Activity. Antimicrob. Agents Chemother. 1986, 30, 132−136. (24) Ikeda, T.; Yamaguchi, H.; Tazuke, S. New Polycationic Biocides - Synthesis and Antibacterial Activities of Polycations with Pendant Biguanide Groups. Antimicrob. Agents Chemother. 1984, 26, 139−144. (25) Eren, T.; Som, A.; Rennie, J. R.; Nelson, C. F.; Urgina, Y.; Nusslein, K.; Coughlin, E. B.; Tew, G. N. Antibacterial and Hemolytic Activities of Quaternary Pyridinium Functionalized Polynorbornenes. Macromol. Chem. Phys. 2008, 209, 516−524. (26) Chen, C. Z. S.; Beck-Tan, N. C.; Dhurjati, P.; van Dyk, T. K.; LaRossa, R. A.; Cooper, S. L. Quaternary Ammonium Functionalized Poly(Propylene Imine) Dendrimers as Effective Antimicrobials: Structure-Activity Studies. Biomacromolecules 2000, 1, 473−480.

hemolysis than the ones with benzyl groups, which is mainly attribute to the fact that the long alkyl chains are prone to induce the lipid bilayer of bacterial membrane to aggregate than the benzyl group to destroy the membrane. Through a series of mechanistic studies, the homopolymers are demonstrated to be expeditious sterilization in physical nature of membrane disruption mechanism, which makes bacteria unlikely to induce resistance. It is envisaged that geminized amphiphilic cationic polymers with moderate hydrophobicity hold great potential for use as promising antibacterial agents in combating challenging pathogenic infection.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b03182. Electrical conductivity curves (κ) of monomers and homopolymers in water at 25 °C (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 86-10-62523395. Fax: 8610-62523395. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful for the Important National Science and Technology Specific Project of China (2011ZX05024-00403) and CNOOC Comprehensive Scientific Research Project (2013-YXZHKY-013, CCL2013RCPS0184RSN) for supporting this research.

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DOI: 10.1021/acs.langmuir.5b03182 Langmuir XXXX, XXX, XXX−XXX