Antibacterial Activity, in Vitro Cytotoxicity, and Cell Cycle Arrest of

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Antibacterial activity, in vitro cytotoxicity and cell cycle arrest of Gemini quaternary ammonium surfactants Shanshan Zhang, Shiping Ding, Jing Yu, Xuerui Chen, Qunfang Lei, and Wenjun Fang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b01430 • Publication Date (Web): 16 Oct 2015 Downloaded from http://pubs.acs.org on October 23, 2015

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Antibacterial activity, in vitro cytotoxicity and cell cycle arrest of gemini quaternary ammonium surfactants Shanshan Zhang,† Shiping Ding,‡ Jing Yu,† Xuerui Chen,† Qunfang Lei,*,† Wenjun Fang*,† †

Department of Chemistry, Zhejiang University, Hangzhou 310027, China

‡

School of Medicine, Zhejiang University, Hangzhou 310058, China

KEYWORDS: Gemini quaternary ammonium surfactants; Antibacterial activity; Cytotoxicity; Cell cycle arrest

ABSTRACT: Twelve gemini quaternary ammonium surfactants have been employed to evaluate the antibacterial activity and in vitro cytotoxicity. The antibacterial effects of the gemini surfactants are performed on E. coli and S. aureus with minimum inhibitory concentrations (MIC) ranging from 2.8 to 167.7 µM. Scanning electron microscopy (SEM) analysis results show that these surfactants interact with the bacterial cell membrane, disrupt the integrity of the membrane, and consequently kill the bacteria. The data recorded on C6 glioma and HEK293 human kidney cell lines using an MTT assay exhibit low half inhibitory concentrations (IC50). The influences of the gemini surfactants on the cell morphology, the cell migration ability, and the cell cycle are observed through hematoxylin-eosin (HE) staining, cell wound healing assay, and flow cytometric analyses, respectively. Both the values of MIC and IC50 decrease against the growth of the alkyl chain length of the gemini surfactants with the same spacer group. In the

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case of surfactants 12-s-12, the MICs and IC50s are found to decrease slightly with the spacer chain length changing from 2 to 8 and again to increase at higher spacer length (s = 10 to 12). All of the gemini surfactants show great antibacterial activity and cytotoxicity, and they might exhibit potential applications in medical fields.

Introduction People nowadays are inevitably exposed to a variety of bacteria, fungi, and other microorganisms, and the resistance of pathogens to antibacterial agents is a growing concern in public health.1, 2 As important functional materials, the antimicrobial agents can kill or inhibit microbial growth and they have been widely applied in medicine, household articles, and food packaging, etc.3, 4 However, some frequently used medicines become less effective than before because of serious bacterial resistance. Therefore, new antibacterial materials are required and they are also necessitated to reduce the damage to environment. Surfactants are amphiphilic substances which are widely used as wetting agents, solubilizers, emulsifiers, etc. in many fields including food, pharmaceutical and petroleum industry.5 It has been found that the surfactants are bacteriostatic and bactericidal,6 and they can be applied as antimicrobial agents in biocatalysis and bioprocessing. Since one molecule of the gemini quaternary ammonium surfactants is composed of two cationic head groups connected with a spacer and two hydrophobic alkyl chains,

7-10

they usually have better interfacial

properties, such as lower critical micelle concentrations (CMC), more miscellaneous aggregate morphologies, greater solubilizing abilities, and enhanced surface activities than the corresponding single-chain surfactants at the same conditions.11-13 Moreover, quaternary ammoniums have been widely used as antibacterial agents for a long time and they are proved to


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be broad spectrum antimicrobial against bacteria, fungi, and viruses,14-16 because they kill microorganisms mainly through electrostatic and hydrophobic interactions with the cell surfaces.17 Usually, surfactants intercalate the cell membrane to cause changes of the molecular organization in the membrane and to increase membrane permeability, then cytoplasm diffusion and cell lysis occur.18-20 The advantageous characteristics of the gemini quaternary ammonium surfactants make them as a versatile class of compounds in biotechnology, medicine, food industry, cosmetic products and cleansers.21-23 For example, the gemini surfactants have been usually used as cell internalization promoters to replace the traditional cell-penetrating peptides (CPPs) in recent years, since they can improve the permeability of the cell membrane to enhance cellular uptake and to deliver drug efficiently. At the same time, they have better interfacial properties and lower cost than traditional CPPs.18 Cationic gemini surfactants are also suitable for the promising gene delivery system design,24 because they can interact with DNA molecules more intensely than the corresponding single-chain surfactants. They can also form mesomorphism to a large extent in aqueous solution, which is a remarkable feature for transfection competence. However, before used in the potential fields, the evaluation of cytotoxicity as well as environmental behavior of the gemini quaternary ammonium surfactants are of great importance.25-27 Although many reports have focused on the antimicrobial activities, the mechanism of action has not been yet fully understood.20, 23, 28, 29 Little attention has been paid to the cytotoxicity of these cationic gemini surfactants on mammalian or human cells. Besides, there are few reports on the selectivity towards bacterial cells and mammalian cells of gemini quaternary ammonium surfactants. In this work, a series of gemini quaternary ammonium surfactants with different length chains and methylene spacers were synthesized with reference to the previous work.13 The


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antibacterial activities of them against both E. coli and S. aureus were investigated by turbidity method with UV spectrophotometer and the mechanism of antibacterial action was explored by scanning electron microscopy (SEM). The cytotoxicity of these compounds was studied on the cell viability of C6 cell and HEK293 cell lines by means of MTT (3-(4, 5-dimethylthiazol-2-yl)2, 5-diphenyltetrazolium bromide) assay. HE staining and Cell wound healing assay were also performed on cells to explore the effects of these surfactants on cell proliferation and migration ability, respectively. Besides, the different species to induce cell cycle arrest were evaluated. By in-depth study of the action mechanism of gemini quaternary ammonium surfactants with different kinds of bacterial and cells, an insight into more efficient surfactants for biomedical applications was obtained, and selectivity of these surfactants toward bacteria and mammalian cells were also investigated.

Experimental Materials.

Phosphate

buffer

saline

(PBS),

sodium

dodecyl

sulfate

(SDS),

hexadecyltrimethylammonium bromide (CTAB), NaCl, Tryptone, Yeast Extract, Dulbecco's Modified Eagle's Medium (DMEM), trypsin/EDTA solution (170000 U/L of trypsin and 0.2 g/L of EDTA), fetal bovine serum (FBS), DMSO, and tetrazolium salt MTT were purchased from Sigma-Aldrich and used as received. Cell cycle and apoptosis analysis kit was obtained from Beyotime Institute of Biotechnology (China). The 75 cm2 flasks, 96-well plates, and 6-well plates were purchased from Corning Incorporated. E. coli (ATCC 22922), S. aureus (ATCC 25923), C6 rat glioma cell line, and HEK293 human embryonic kidney cell lines were provided


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by the Institute of Immunology, School of Medicine, Zhejiang University. Ultrapure water with a resistivity higher than 1.82·105 Ω·m at 25 °C was produced from the Millipore Q3 system. Synthesis. All of the gemini quaternary ammonium surfactants used in this work were synthesized with the previously reported method (Scheme 1).30, 31 They were prepared from alkyl dibromides and N, N-dimethyl alkyl tertiary amines in i-propanol under reflux for 12 h. The crude products were collected after i-propanol being evaporated. Excess alkyl bromides were removed by extraction with the acetone-methanol mixture, and the final products with high purity were then obtained. The synthesized surfactants were characterized with NMR (Bruker Advance 2B/400Hz) and elemental analysis (CarlaboEA1110) (Supporting Information). Antibacterial Activity. The obtained bacteria, E. coli and S. aureus, were freeze dried. They were cultured in Luria-Bertani (LB) broth (10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1000 mL water, pH=7) and stored at -80 °C in glycerol. This glycerol stock sample with an appropriate volume was added to each of 4 mL LB broth, and the bacteria were cultured at 37 °C for 6 h before use. Antibacterial activity was then determined via slight adjustments to the previous methods .32 The needed surfactant solutions were prepared by adding the surfactant compounds into autoclaved Millipore water. Then, 1mL tested surfactant solution at a given concentration and 10 µL bacterial broth (108 CFU/mL) were added to 1 mL LB broth with concentration doubled and spread in each tube. The tubes were cultured at 37 °C for 24 h, and the negative control sample was made up without surfactant. The minimum inhibitory concentration (MIC), which was defined as the lowest concentration of a surfactant inhibiting the development of visible bacteria growth (i.e. no turbidity) in the tubes after 24 h incubation at 37 °C, was obtained by analyzing


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the absorbance of bacteria at 600 nm. MIC tests were run with the third generation of bacteria, which was cultured for 6 h at 37 °C prior to the experiments. Scanning Electron Microscopy (SEM). E. coli and S. aureus were cultured for 6 h at 37 °C. The cells were then centrifuged and resuspended in LB broth at pH=7 (106 cells/mL). Afterwards, the bacteria were incubated in LB broth containing different concentrations of surfactants at 37 °C, 200 rpm shaking speed for 4 h. The solutions were centrifuged at 12000 rpm for 2 min, and the obtained cells were dehydrated for every 15 min sequentially with 30 %, 50 %, 70 %, 80 %, 90 % and 100 % ethanol. After that, the as-treated cells (5 µL) were dropped on the silicon wafer, dried at the room temperature, and then sputter coated.33-35 The SEM images were recorded on a scanning electron microscopy (ULTRO 55, Germany). Cytotoxicity Test. Culture of different cell lines. C6 glioma cell and HEK293 cell lines were maintained in DMEM supplemented with FBS (10% v/v) and antibiotics (penicillin, 50 U/mL; streptomycin, 50 U/mL). With the treatment of trypsin/EDTA, the cells were collected when 80% of the flask was confluent. Then, 200 µL suspension with a density of 1.0×104 cells/mL was seeded into each well of the 96-well plates. The plates were placed in the 5% CO2 incubator at 37 °C overnight to make cells adhere. Surfactant exposure. The surfactant solutions were prepared in sterile PBS with pH=7.4. Then a series of dilutions were performed by adding DMEM medium contained antibiotics to the stock surfactant solutions. After removal of the culture medium, the cells were treated with the DMEM medium-diluted surfactant solutions. The controls were independent of the surfactant treated samples. The blanks were prepared with cells cultured in sterile water. All of the samples were incubated in the 5 % CO2 incubator at 37 °C for 4 h.


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MTT Assay. The surfactant cytotoxicity against each cell was tested by the colorimetric method.36-38 After the culture medium was removed and MTT in PBS (5 mg/mL) was diluted to 0.5 mg/mL with DMEM medium, the as-prepared MTT solution (100 µL) was added to each well. The plates were then incubated in 5 % CO2 at 37 °C for 4 h. After that, the cells were washed with PBS to remove the medium, and the purple formazan product was dissolved by DMSO (100 µL per well). The absorbance of the resulting solutions after being shaken for 10 min at the room temperature was tested at 570 nm wavelength in the microplate reader (Thermo Labsysyem MK3). Cell viability was calculated according to the following equation:

(%) =

− × 100% −

where V is the cell viability, %; A, A0, and Ac are the absorbance of the treated cells, the blank in which the cells almost completely died, and the control, respectively. The value of half maximal inhibitory concentration (IC50) was obtained by fitting the cell viability (V) as a function of surfactant concentration (C) to the following equation:

(%) =

100 1+ IC50

where C is the concentration (µg/mL) of surfactant, and p defines the slope of the sigmoid curve. Hematoxylin-Eosin Staining. Cells were seeded on 12-well plates (1.0×105 cells/well, 1 mL/well) and incubated overnight. Surfactants with different concentrations were added after removing the culture medium and being washed twice with PBS. After incubated for 4 h, the cells were ﬁxed in 500 µL 5% formaldehyde solution for 10 min and stained with hematoxylin and eosin. The procedure of staining was as follows: The samples were incubated in the 0.5 mL


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hematoxylin for 40 min. Then, they were washed with PBS until the liquid became transparent. After that, 0.5 mL Eosin was added for 15 s and the repetition of the washing procedure was performed. Such prepared samples were dried and observed under the inverted phase contrast microscope (Olympus). Cell Migration Assay. To explore the migration ability of the cells, the wound healing assay was carried out. 3.0×105 cells per well were cultured in 6-well plates with 1 mL DMEM medium and incubated overnight. When the cells reached confluence, a scarification was made using a sterile pipette tip. After washing twice with PBS to remove the debris, cells were individually exposed to surfactants with different concentrations. The images of cells invading the wound at 0 and 24 h after scratching were obtained using a phase-contrast microscopy (Olympus). Cell Cycle Pattern Analysis. To evaluate the cell cycle arrest, around 1.0×106 cells were treated by surfactants in 6-well plates with approximately concentrations of 0.5 IC50 and IC50 for 24 h. Cells were resuspended in 1 mL PBS and then fixed with 1 mL precooled 70% ethanol (v/v) at 4 °C for 48 h. After that, cells were washed with prechilled PBS twice and the DNA was stained with cell cycle and apoptosis analysis kit (10 µg/mL PI, 0.1% RNase A) at 37 °C for 30 min and then analyzed by flow cytometry (BD FACSCalibur). Statistical Analysis. All of the presented data are given as the mean value with the standard deviation (SD) on the basis of at least three independent measurements. Multifactor analysis of variance (ANOVA) was performed to evaluate the statistical variability. Statistical significance was set at the level of p < 0.05.


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Results and Discussion Antibacterial Activity. The antibacterial activities of the gemini quaternary ammonium surfactants were determined. As the data listed in Table 1, the bacterial inhibition effects are found to be remarkable when the surfactant concentrations are in the range from 53.1 to 167.7 µM for E. coli (gram-negative bacteria) and from 2.7 to 3.8 µM for S. aureus (gram-positive bacteria), respectively. Generally, the cell membranes of gram-positive bacteria are composed of peptidoglycan layers, which allow surfactants to penetrate effortlessly. For gram-negative bacteria, the external layers of the membranes are mainly composed of lipopolysaccarides and proteins which restrict the entrance of biocides.39,

40

So, the gram-negative bacteria are less

sensitive than the gram-positive bacteria as their outer membranes are less permeable to such amphiphilic surfactants, which agrees well with the experimental results. It is also observed that the gemini surfactants exhibit stronger antimicrobial effect with the increase of alkyl chain length due to the more hydrophobic environment. The MIC values of all of the surfactants against both E. coli and S. aureus are smaller than the corresponding CMC values (Table 1) which were measured in LB broth by the surface tension method. At concentrations lower than CMCs, surfactant molecules are dissolved homogeneously in medium and can participate in the antibacterial activity effortlessly through electrostatic and hydrophobic interactions with bacterial cell surfaces. Hence, the monomeric species of these compounds are responsible for an antimicrobial response. It is known that the cell membranes are composed of lipids and the protein layers in bilayer fashion, which leads to the hydrophobicity. One of the most important functions for the lipoprotein membrane is permeability, which controls the biological reactions in the cell. Any changes of the selective permeability will bring damage to the cell system. Hydrocarbon tails of


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the quaternary ammonium surfactants can insert into the membranes of bilayers or liposomes, which increases the hydrophobicity of the bacterial cell membranes and leads to the formation of transient channels or pores on the membranes.41, 42 These channels or pores further increase the permeability of the cell membranes, leading to the leakage of cell contents and the disturbance in the biochemical reactions inside the cytoplasm.43 Besides, the cationic quaternary ammonium surfactants can make themselves adsorb on the interface of the negatively charged cell membrane. Because of the double charges and double alkyl chains, the gemini surfactants may facilitate more electrostatic and hydrophobic interactions with the cell membrane than the singlechain surfactants such as DTAB and CTAB. Hence, as shown in Table 1, the gemini surfactants exhibit more efficient biotical activities towards microorganisms in most cases. CTAB shows a little better antimicrobial effect than some of the gemini quaternary ammonium surfactants when investigated on E. coli. SEM Images of Bacteria Treated by Surfactants. To further understand the bacterial killing by gemini quaternary ammonium surfactants, scanning electron microscopy (SEM) observations were performed to display the interactions of both E. coli and S. aureus with 14-3-14. Images of bacteria untreated by surfactants (control) were also acquired (Figure 1). Both untreated bacteria showed the presence of normal cells with unspoiled cell membranes (Figure 1a for E. coli, and Figure 1c for S. aureus). However, the broken and probably dead bacteria were observed after being treated with 14-3-14 (Figure 1b for E. coli and Figure 1d for S. aureus). As known, quaternary ammonium compounds (QACs) are membrane active agents, which own target sites mainly at the cytoplasmic membrane in bacteria.44 Studies on cell wall porosity can also provide valuable information about intracellular entry of the QACs.45 Salton46 has proposed the steps of cationic agents invading bacteria. Firstly, the agents adsorb on the cell


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surfaces and penetrate into the cell walls. Secondly, the agents interact with the cytoplasmic membranes (lipid or protein) through the electrostatic and hydrophobic reactions, and then the membranes become disorganized. Thirdly, intracellular materials with low molecular weights leak out and proteins as well as nucleic acids degrade. Finally, the autolytic enzymes cause the cell wall lysis. So, the damaging effects including structural disorganization, cytoplasmic membrane incompletion on the bacterial cells occur. SEM as an intuitive mean to study the influence of gemini surfactants on bacteria in this work indicates the damage of the microorganism along with irregular shape and seemingly leakage of the contents. Combined with the previous opinion from literatures, it can be concluded that the antimicrobial activity of gemini quaternary ammonium surfactants is related to their ability to disrupt the integrity of bacterial membrane by electrostatic and hydrophobic adsorptions on the membrane surface. They interacted strongly with the bacterial cell membranes in which the phospholipids are negatively charged. Besides, lipopolysaccharides in gramnegative bacteria membranes as well as teichoic acids in gram-positive bacteria membranes are also negatively charged. Moreover, these surfactants can further interact with the hydrophobic lipid membrane because of the long hydrophobic alkyl chains and then disrupt the inner membrane, leading to the damage of cytoplasmic constituents and cell death. Toxicity. MTT assay as the indirect measurement to assess the cell viability had been performed, and the results are shown in Figures 2 and 3. All of the surfactants induce apparent doseresponse relationships, and the half inhibitory concentrations (IC50) are calculated as listed in Table 2. Noticeably, the cell activity for the treated cells with some surfactants at the lowest concentrations is slightly higher than that for the untreated cells (control). This so-called hormesis phenomenon can be explained by the stimulation effect of low concentration drug and


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inhibition effect of high concentration drug.47 The values of IC50 for the gemini surfactants with the same spacer chain length approximately go along with this trend: 12-s-12 > 14-s-14 > 16-s16. This is related to the hydrophobicity of monomeric species in the surfactants. The CMC values of these surfactants in pure water (Table 1) follow the order: 12-s-12 > 14-s-14 > 16-s-16, which indicates the increase of the hydrophobicity with the growth of alkyl chain length. Therefore, the higher toxicity of the longer chain-length gemini surfactant mainly results from the higher hydrophobicity which enables a better interaction of the surfactant with the plasma membrane. When the gemini surfactants possess the same alkyl chain like 12-s-12, values of IC50 are found to decrease slightly with the spacer chain length changing from 2 to 8 and again to rise at higher spacer length (s = 10 to 12) as seen from Table 2. This should be from the combined effects of the hydrophobicity and electrostatic interactions of the surfactants on the mammalian cells. On the one hand, the surfactants become more hydrophobic since the CMC values of them decrease with the spacer chain length changing from 2 to 12, which leads to the rise of the cytotoxicity. On the other hand, the degrees of counterion dissociation (α) of the micelles as reported previously13, 48, 49 increase with the growth of the spacer length (Table 2). This indicates that the cationic charge densities of the surfactants, which are inversely proportional to the change of α values, decrease with the rise of the spacer length. As a result, the electrostatic interactions between the gemini surfactants and cells are declined. Moreover, the IC50 values of all of the surfactants treated on C6 and HEK293 cells are smaller than the corresponding CMC values (Table 2) which were measured in DMEM medium by the surface tension method. This also indicates that the monomeric species of these compounds are responsible for the cytotoxicity. Obviously, cytotoxicity is significant at very low concentration for all of the surfactants,


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while the concentration at which the toxic effect occurs is just slightly higher than the MIC in the case of S. aureus and much lower than that of E. coli. With reference to these results, it is difficult to regulate the concentrations of the gemini surfactants which are expected to be used as antimicrobial agents without hurting human cells (HEK293) or as biopharmaceuticals with the requirements of pre-sterilization treatments for E. coli. Nevertheless, most of the gemini surfactants are more toxic than SDS and CTAB when they are treated on tumor cells (C6). This suggests that the gemini quaternary ammonium surfactants can be used as potential alternatives to the traditional surfactants in certain cases in medical and pharmaceutical fields18, 24 since less dosage is usually of supreme importance, along with considering that gemini surfactants have low CMC values, reduced surface tensions, and promoted adsorption ability in comparison with the corresponding single-chain surfactants. Effects of Surfactants on Cell Morphology and Migration. To evaluate the influence of surfactants on cell morphology, case studies in 14-3-14 were performed on C6 and HEK293 cell lines through HE staining, by which nuclei were stained distinctive blue with hematoxylin, and cytoplasm was stained red with eosin. As can be seen from Figures 4 and 5, morphological structures of two types of cells which were exposed to 14-3-14 for 4 h have changed a lot compared with that untreated (control). Meanwhile, cytoplasm of the cells cultured in 14-3-14 is significantly leaked and an amount of cell lysates can also be observed. In addition, the wound healing assay was also carried out to evaluate the cell migration ability after 24 h exposure to gemini surfactants by phase-contrast microscopy. As in the HE staining experiments, cells exposed to 14-3-14 with different concentrations were observed. Cells revealed a slower migration rate after they were treated with 14-3-14 in comparison to control cells (treated with FBS-free DMEM). Moreover, in the case of cells being exposed to 14-


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3-14 with the concentration higher than IC50 (10.0 µM for C6 and 6.0 µM for HEK293), not only the cells were unable to heal the wound, but also the cell number diminished significantly at the non-wounded area (Figure 6). Cell Cycle Arrest Induced by Surfactants. To further investigate the mechanism leading to the inhibition of cell proliferation and migration as well as the significant reduction of the viability of the exposed cells induced by gemini surfactant, the flow cytometery analysis was carried out on cells exposed to different surfactants. The cell cycle arrest can be determined by measuring the total amount of stained DNA in treated and untreated cells, considering the amount of DNA is doubled prior to mitosis. As an example, the cell cycle distributions of the C6 cells treated with 14-3-14 (0.5 IC50=3.7 µM and IC50=7.3 µM) and HEK293 cells treated with 12-4-12 (0.5 IC50=2.3 µM and IC50=4.7 µM) are shown in Figure 7. It is confirmed that the cell population increases with 11.69 % in the S-G2/M phase when treated with 3.7 µM of 14-3-14 as compared with control cells. When the surfactant concentration is increased to 7.3 µM, the cell population in the S-G2/M phase presents 13.52 % increase. The cell population decreases in the G0/G1 phase along with the population increases in the S-G2/M phase proves that 14-3-14 induces the cell cycle arrest at the S-G2/M phase. HEK293 cells treated with 12-4-12 at the low concentration behave analogously, whereas the cells exposed to 12-4-12 at the high concentration present higher increase of cell population in the sub-G1phase, suggesting the cells entering apoptosis. The influences of other surfactants on the cell cycle distribution were shown in the Supporting Information. Briefly, all of the gemini surfactants can induce cell cycle arrest even though the cell cycle patterns are significantly different. These results further reveal that the gemini surfactants are of great potential for biomedical applications.


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Conclusion Gemini quaternary ammonium surfactants with different spacer lengths and alkyl chains have been synthesized and characterized. All of these surfactants show higher antibacterial activity against S. aureus (gram-positive bacteria) than against E. coli (gram-negative bacteria) for the outer membranes of E. coli, which are less permeable. The interactions between surfactants and bacteria are confirmed by scanning electron microscopy, which shows that the amphiphiles interact strongly with the bacterial cell surfaces, thereby causing membrane disintegrated. The cytotoxicities of the gemini surfactants against C6 and HEK293 cells studied by MTT assay, HE staining, and cell migration assay witness an obvious reduction in cell viability with the increase of the surfactant concentration. The flow cytometery analysis results further show the mechanism of the inhibition of cell proliferation by a gemini surfactant, and the cell cycle arrests induced by surfactants can be evaluated. The gemini surfactants can be used as potential alternatives to the traditional surfactants in certain cases although the selectivities of them towards bacteria and mammalian cells should be further investigated.


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Figure 1. Scanning electron microscopy (SEM) images of E. coli and S. aureus after cultured 6 h at 37 °C, a, untreated E. coli; b, E. coli treated with surfactant 14-3-14 (43.9 µM); c, untreated S. aureus; d, S. aureus treated with surfactant 14-3-14 (1.5 µM).


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Figure 2. Concentration-viability curves of the C6 exposed to different surfactants for 4 h. Data are presented as mean ± SD of three independent results.


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Figure 3. Concentration-viability curves of the HEK293 exposed to different surfactants for 4 h. Data are presented as mean ± SD of three independent results.


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Figure 4. Fluorescence microscopic images of staining experiments in C6 cells. (a) untreated; (b) treated with FBS-free DMEM; (c) treated with surfactant 14-3-14 (3.0 µM); (d) treated with surfactant 14-3-14 (10.0 µM). The scale bar is 100 µm.


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Figure 5. Fluorescence microscopic images of staining experiments in HEK293 cells. (a) untreated; (b) treated with FBS-free DMEM; (c) treated with surfactant 14-3-14 (1.5 µM); (d) treated with surfactant 14-3-14 (6.0 µM). The scale bar is 100 µm.


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(a)

(b)

Figure 6. (a) Wound healing assay in C6 cells. A, exposed to FBS-free DMEM, B, exposed to 14-3-14 (3.0 µM), C, exposed to surfactant 14-3-14 (10.0 µM); (b) Wound healing assay in HEK293 cells. A, exposed to FBS-free DMEM, B, exposed to surfactant 14-3-14 (1.5 µM), C, exposed to surfactant 14-3-14 (6.0 µM).


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(a)

Control

(b)

0.5IC50

(c)

IC50

(d)

(e)

Control

(f)

0.5IC50

(g)

IC50

(h)

Figure 7. Flow cytometric analysis of the cell cycle progression on C6 cells [(a), untreated; (b), exposed to surfactant 14-3-14 (0.5 IC50=3.7 µM); (c), exposed to surfactant 14-3-14 (IC50=7.3 µM); (d), cell cycle distribution] and HEK293 cells [(e), untreated; (f), exposed to surfactant 124-12 (0.5 IC50=2.3 µM); (g), exposed to surfactant 12-4-12 (IC50=4.7 µM); (h), cell cycle distribution]. Data are presented as mean ± SD of three independent results. M1, M2, and M3 refer to G0/G1 phase, S phase, G2/M phase, respectively.

Scheme

2

1.

N n

n = 12, 14, 16

Synthesis

+

Scheme

of

Gemini

Quaternary

i-propanol

Br

N

Br s s = 3, 4

ref lux 12 h

n

Ammonium

s N

Surfactants.

2Br n

12-s-12: n = 12, s = 2, 3, 4, 5, 6, 8, 10, 12 14-s-14: n = 14, s = 3, 4 16-s-16: n = 16, s = 3, 4


22

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Table 1. Antibacterial Activities of Quaternary ammonium surfactants. Surfactants

CMC1 (mM)

CMC2 (mM)

MIC (µg/mL)

MIC (µM)

E. coli

S. aureus

E. coli

S. aureus

12-2-12

1.11a

0.392

80 ± 4

2.0 ± 0.2

130.1 ± 6.5

3.3 ± 0.3

12-3-12

1.00b

0.311

80 ± 3

2.0 ± 0.2

127.2 ± 4.8

3.2 ± 0.3

12-4-12

1.17b

0.264

80 ± 9

2.0 ± 0.2

124.5 ± 14.0

3.1 ± 0.3

12-5-12

1.18

0.257

58 ± 2

1.9 ± 0.1

87.9 ± 3.0

2.9 ± 0.2

12-6-12

1.19a

0.249

58 ± 3

1.9 ± 0.2

86.8 ± 4.5

2.8 ± 0.3

12-8-12

0.88a

0.250

57 ± 3

1.9 ± 0.2

82.1 ± 4.3

2.7 ± 0.3

12-10-12

0.65a

0.250

68 ± 5

2.4 ± 0.2

93.6 ± 6.9

3.3 ± 0.3

12-12-12

0.45a

0.202

127 ± 4

2.9 ± 0.3

167.7 ± 5.3

3.8 ± 0.4

14-3-14

0.175b

0.606

100 ± 1

2.0 ± 0.3

146.2 ± 1.5

2.9 ± 0.4

14-4-14

0.187b

0.599

100 ± 5

2.0 ± 0.5

143.3 ± 7.2

2.9 ± 0.7

16-3-16

0.024d

0.588

40 ± 8

2.0 ± 0.5

54.1 ± 10.8

2.7 ± 0.7

16-4-16

0.0272e

0.558

40 ± 5

4.0 ± 0.1

53.1 ± 6.6

2.8 ± 1.3

DTAB

15.34f

1.983

111 ± 1

13.8 ± 0.1

360.3 ± 3.2

44.8 ± 0.3

CTAB

0.98g

0.359

34 ± 1

1.8 ± 0.1

93.3 ± 2.7

4.9 ± 0.3

CMC1 for CMC values of Gemini surfactants measured in pure water. aref 48, bref 13, cref 50, d ref 51, eref 52, fref 53, gref 54; CMC2 for CMC values of gemini surfactants measured in LB broth from surface tension method. The plots of surface tension (γ) versus surfactant concentration (C) are shown in Figure S3.


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Table 2. Cytotoxic Effect (IC50) of Surfactants on C6 and HEK293 Cell Lines. Surfactants

CMC3 (mM)

α

IC50 (µg/mL)

IC50 (µM)

C6

HEK293

C6

HEK293

12-2-12

0.143

0.23a

7.9 ± 0.2

3.1 ± 0.3

12.0 ± 0.3

5.0 ± 0.5

12-3-12

0.118

0.262b

6.9 ± 0.4

3.1 ± 0.4

10.9 ± 0.6

4.9 ± 0.7

12-4-12

0.091

0.31a

5.4 ± 0.3

2.5 ± 0.2

8.4 ± 0.5

4.2 ± 0.3

12-5-12

0.089

3.5 ± 0.4

2.5 ± 0.1

5.3 ± 0.6

3.8 ± 0.2

12-6-12

0.088

0.46a

3.4 ± 0.3

2.2 ± 0.1

5.1 ± 0.3

3.3 ± 0.1

12-8-12

0.079

0.52a

3.5 ± 0.3

2.2 ± 0.1

5.0 ± 0.4

3.2 ± 0.1

12-10-12

0.075

0.58a

3.9 ± 0.2

2.6 ± 0.4

5.4 ± 0.3

3.6 ± 0.6

12-12-12

0.069

0.67a

5.4 ± 0.3

2.8 ± 0.2

7.2 ± 0.4

3.7 ± 0.2

14-3-14

0.271

0.287b

5.5 ± 0.1

3.0 ± 0.6

8.0 ± 0.1

4.4 ± 0.8

14-4-14

0.265

0.322b

5.1 ± 0.3

2.9 ± 0.4

7.4 ± 0.5

4.1 ± 0.6

16-3-16

0.340

0.36c

2.2 ± 0.2

3.8 ± 0.3

3.0 ± 0.3

4.2 ± 0.4

16-4-16

0.288

0.31c

2.7 ± 0.3

3.4 ± 0.2

3.5 ± 0.4

4.1 ± 0.3

CTAB

0.164

0.18c

3.7 ± 0.1

2.9 ± 0.1

10.3 ± 0.1

8.0 ± 0.1

SDS

0.335

18.2 ± 0.4

15.7 ± 0.1

63.2 ± 1.3

54.6 ± 0.3

CMC3 for CMC values of surfactants measured in DMEM medium from surface tension method. The plots of surface tension (γ) versus surfactant concentration (C) are shown in Figure S4. α is the degree of counterion dissociation of the surfactants in aqueous solutions, aref 48, bref 13. cref 55


24

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ASSOCIATED CONTENT Supporting Information. Analytical data for the gemini quaternary ammonium surfactants; Concentration-viability curves from 24 h exposure of E. coli and S. aureus to different surfactants (Figure S1- S2); Plots of surface tension (γ) versus surfactant concentration (C) for different surfactants in LB broth and DMEM medium at temperature T = 37 °C and pressure p = 0.1 MPa (Figure S3- S4); Effects of gemini surfactants on cell cycle distribution in C6 cells and HEK293 cells (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]. Tel: +86 571 88981416. Fax: +86-571-88981416. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No.21073164, 21473157). REFERENCES 1.

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Antibacterial activity, in vitro cytotoxicity and cell cycle arrest of gemini quaternary ammonium surfactants Shanshan Zhang,† Shiping Ding,‡ Jing Yu,† Xuerui Chen,† Qunfang Lei,*,† Wenjun Fang*,†

The graphic is “For Table of Contents Only”.


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Antibacterial Activity, in Vitro Cytotoxicity, and Cell Cycle Arrest of

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