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Assessing the Biocidal Activity and Investigating the Mechanism of Oligo-p-phenylene-ethynylenes Jing Wang, Lian-Gang Zhuo, Wei Liao, Xia Yang, Zhenghua Tang, Yue Chen, Shunzhong Luo, and Zhijun Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16243 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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ACS Applied Materials & Interfaces

Assessing the Biocidal Activity and Investigating the Mechanism of Oligo-p-phenylene-ethynylenes Jing Wang1, Liangang Zhuo1, Wei Liao1, Xia Yang1, Zhenghua Tang3,4*, Yue Chen2, Shunzhong Luo1, Zhijun Zhou1* 1

Institute of Nuclear Physics and Chemistry, China Academy of Engineering Physics, 64

Mianshan Road, Mianyang, Sichuan, 621900, P. R. China. Email: [email protected] 2

Department of Nuclear Medicine, The Affiliated Hospital of Southwest Medical University,

Luzhou, Sichuan, 646000, P. R. China. 3

New Energy Research Institute, School of Environment and Energy, South China University of

Technology, Guangzhou Higher Education Mega Centre, Guangzhou, 510006, P. R. China. Email: [email protected] 4

Guangdong Provincial Key Laboratory of Atmospheric Environment and Pollution Control,

Guangdong Provincial Engineering and Technology Research Center for Environmental Risk Prevention and Emergency Disposal, South China University of Technology, Guangzhou Higher Education Mega Centre, Guangzhou, 510006, P. R. China. KEYWORDS: oligo-p-phenylene-ethynylene, light-activated biocide, singlet oxygen, OPE internalization, membrane perturbation ABSTRACT A number of oligo-p-phenylene-ethynylenes (OPEs) have exhibited excellent biocidal activity against both Gram-negative and Gram-positive bacteria. While cell death may occur in the dark, these biocidal compounds are far more effective in the light as a result of their abilities to generate cell-damaging reactive oxygen species. In this study, the interactions of four OPEs with E.coli and S.aureus have been investigated. Compared to the OPEs with quaternary

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ammonium salts (Q-OPE), the OPEs with tertiary ammonium (T-OPE) effectively kills many more bacterial cells under light irradiation, presumably by severe perturbations of the bacterial cell wall and cytoplasmic membrane. According to the findings from this study, such intriguing light-induced antibacterial behavior is probably attributed to the combination of bacterial membrane disruption and the interfacial or intracellular generation of singlet oxygen or other ROS. Singlet oxygen was proved to be formed from irradiation of the OPEs while the varying cell membrane perturbation abilities of OPEs enhance antibacterial activity. INTRODUCTION Bacterial contamination can cause a variety of maladies, including superficial skin/ wound infections associated with implanted medical devices, food poisoning, sepsis, and other lifethreatening illness.1-4 In the past few decades, the introduction of antibiotics had been continuously making a striking impact on the treatments of infectious diseases and dramatically decreased the mortality.5-9 However, the rapid emergence and spread of multiple antibioticsresistant bacteria have posed a new threat to human health.10-15 Photodynamic therapy (PDT) has been suggested to be an alternative antibiotic treatment of some infections. Generally, the process of PDT begins with a photosensitizer absorbing visible or ultraviolet light to generate a triplet state of the compound leading to the formation of reactive oxygen species (ROS), in particular, singlet oxygen (1O2), via an energy or electron transfer process, 16-17 then a phototoxic effect occurs to bacteria through an oxidative reaction.18-20 Cationic poly-phenylene-ethynylenes (PPE) and corresponding oligomers (OPE) have attracted extensive research interests due to the ability of efficient 1O2 generation and the ensuing lethal biological effect.21-27 OPEs with controlled conjugation levels and functional side chains have recently been studied by the Whitten and Schanze groups and UV light-induced biocidal activity


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was observed.28-32 Interestingly, a recent study indicates that the highly effective biocidal nature of the EO-OPE(Th,C2) stems from rapidly inducing germination of Bacillus spores, rendering the bacteria highly vulnerable to ROS and ultraviolet irradiation.33 Therefore, OPEs have shown broad spectrum toxicity to microbes. Mechanistic studies regarding the biocidal activities of OPEs reveal that dramatic changes on the surface and internal organization of bacterial cells occurred.20 Researchers believe that positive charged OPEs allow themselves to interact and perturb strongly with the anionic bacterial membrane, so the cytotoxic 1O2 generated by OPEs can destroy the bacteria more effectively when it stays close to or in the cell membrane or even in the cytoplasm. Another reason for modifying OPEs with quaternary ammonium groups is to increase their hydrophilicity, as most reported OPEs are sparingly soluble or even insoluble in water, which decreases the concentrations of OPEs in water, thus weakens their biocidal effects. It is worth noting that, recent computational results indicate that assemblies such as dimers or trimers of OPEs may lead to enhanced biocidal activity in contrast to OPE monomers,34 which, in turn, suggests that poor solubility of OPEs may contribute to biocidal effects. Previous studies, for the light-activated killing, the structure-activity relationship between S-OPE-n (H) and OPEn series is obviously different.29 The proposed light-induced antibacterial mechanism has been attributed to the combination of bacterial membrane disruption and the generation of 1O2 or other ROS. Given that the association of OPEs with negatively charged bacterial membrane is not only driven by electrostatic interaction, but hydrophobic interaction exerts an important influence on it, thus the hydrophilicity of OPEs increased by methylation may not conducive to the antibacterial effects.35-36 Meanwhile, the toxic reagent methyl iodide is essential for conversion from tertiary ammonium to quaternary ammonium, which limits the scope of their application.


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Moreover, in the previous studies, most researchers have focused their studies on the 1O2 generation ability of OPEs, the structure-activity relationship between their hydrophobicity and their biocidal activity has not been evaluated and established. Herein, to investigate the structureactivity relationship and find a balance between electrostatic and hydrophobic interactions, we report the synthesis, internalization, and photochemical properties of OPEs with pendant groupstertiary ammonium (T-OPE) or quaternary ammonium salts (Q-OPE), as shown in Scheme 1. In addition, the Gram-positive bacterium (Staphylococcus aureus) and Gram-negative bacterium (Escherichia coli) were used to investigate the antimicrobial activity of OPEs under ultraviolet irradiation. Additionally, the abilities of bacterial membrane perturbations and the morphological damages were studied. Scheme 1. Structures of OPEs

T-OPEs

Q-OPEs

MATERIALS AND METHODS 1, 2-Dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt) (DOPG), E.coli total lipid, and cholesterol were purchased from Avanti Polar Lipids and used as received. 5(6)Carboxyfluorescein (hereafter referred to as fluorescein) was purchased from Sigma-Aldrich.


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Superfine Sephadex G-25 was obtained from GE Healthcare Bio-Science. All other chemicals and culture media were purchased from Sigma-Aldrich or Alfa Aesar. All of the solvents were purchased from Honeywell with HPLC grade and used without purification. E.coli strains (ATCC 25922) and S.aureus (ATCC 25923) were obtained from the Institute of Microbiology Chinese Academy of Sciences. Ultrapure water was used throughout study (Milli-Q, 18.2 ΩM cm-1 resistivity). UV-vis absorption spectra were record on a Perkin Elmer Lambda 35 spectrophotometer. The bacterial killing under irradiation was carried out using a Mejiro Genossen MVL-210 fiber light and the emission spectrum is attached in the Supporting Information. The details of all experimental methods, including synthesis of OPEs, bacterial strains & growth conditions, bacterial killing experiments, singlet oxygen measurements,37 preparation of fluorescein-loaded vesicles & vesicle leakage assays,38-39 monolayer insertion assay40 and observation of cell morphology,20 are all given in Supporting Information. RESULTS AND DISCUSSIONS Light-Induced Antibacterial Activity OPEs against S.aureus. The biocidal effects of all OPEs against Gram-positive S.aureus was studied under fiber light irradiation for 60 min and 120 min. After 60 min irradiation duration as indicated in Figure S1, there were no significant killings resulted for Q-OPEs with concentrations up to 0.9 µg/mL, in contrast, 75.8% and 94.4% of S.aureus were killed by OPE(C2,H) and OPE(C3,H) at 0.9 µg/mL, respectively. When increasing the irradiation time to 120 min, the antibacterial activity is enhanced for all of oligomers (Figure 1) except for OPE(C2+,H). Compared with the killing rate of 3.2% (OPE(C2+,H) ) and 32.5% (OPE(C3+,H)), much higher killing rates were obtained from OPE(C2,H) (95.6%) and OPE(C3,H) (100%) at 0.3


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µg/mL. The order of antibacterial effects of the OPEs series against S.aureus is thus summarized as follows: OPE(C3,H)>OPE(C2,H)>OPE(C3+,H)>OPE(C2+,H), and the killing ability increased with the increasing of OPE concentrations and extension of irradiation time.

Figure 1. S.aureus against OPEs upon exposure to fiber light at various concentrations for 120 min. The error bars represent the standard deviations of three parallel measurements. OPEs against E.coli. Next, the biocidal activity of all OPEs against Gram-negative E.coli was studied using the same procedure as the S.aureus experiments. Generally, it has been found that Gram-negative bacteria are more difficult to be killed by antibiotics than Gram-positive bacteria.4, 22,29 Similar trends were also found in this study as higher concentrations were required for killing Gram-negative bacteria E.coli. As shown in Figure 2, OPE(C2+,H) is also the least effective oligomer in killing E.coli, and no killing was observed at 9.0 µg/mL after 120 min irradiation. OPE(C3+,H) induced 37.7 % bacteria dead under the same conditions. Again, the highest biocidal activity was observed from the T-OPE series, which was similar to the result obtained from S.aureus. Significant antibacterial activities could even be observed with T-OPEs upon exposed to the fiber light for 60 min (Figure S3). After irradiation for 120 min, more than 75.0% of the E.coli cells were killed by OPE(C2,H) and OPE(C3,H) at 9 µg/mL. Meanwhile, dark antibacterial activity of OPEs against E.coli was not observed (Figure S4).


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Figure 2. E.coli against OPEs upon exposure to fiber light at various concentrations for 120 min. The error bars represent the standard deviations of three parallel measurements. Photogeneration of 1O2 by OPEs In the previous reports, it was documented that 1O2 generation is responsible for the light-induced biocidal effect.32, 41-43 Therefore, the 1O2 generation ability of OPEs was also investigated in this study. 1,3-Diphenylisobenzofuran (DPBF) was selected as the probe to detect the singlet oxygen generation because DPBF readily undergoes a 1,4-cycloadditon on reaction with singlet oxygen to form endoperoxides which irreversibly yield 1,2-dibenzoylbenzene. When DPBF reacts with 1

O2, the cycloaddition will result in loss of the π-system of isobenzofuran and the absorbance

peak of DPBF at 412 nm will disappear, which is commonly employed to detect the presence of singlet oxygen. The time dependence of the DPBF photobleaching in the absence and presence of OPEs is shown in Figure 3. Multiple measurements were conducted and very small standard deviations can be observed, indicating a reliable reproducibility. From the control experiment without OPEs, we can tell that DPBF is unstable and easily photoquenched, however, more absorbance decrease can be seen in the presence of OPEs. OPE(C2,H) and OPE(C2+,H) yield larger photoquenching


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effects than that of OPE(C3,H) and OPE(C3+,H), although all OPEs absorbance at 412 nm was adjusted for excluding the varying absorbance of OPEs (see Figure S6 for absorbance of all OPEs). Meanwhile, the 1O2 yields of OPE(C2,H) and OPE(C3,H) are similar to their positive charged counterparts.

Figure 3. Time dependence of the photobleaching of the DPBF absorbance at 412 nm, A412 (DPBF), upon fiber light irradiation, in the absence and presence of OPEs. All the results were averaged from 3 individual experiments, after subtraction of the OPE absorbance and normalization to 1 at time zero. From the results above, the 1O2 yields are not significantly different among OPEs with tertiary ammonium terminals to those with quaternary ammonium terminals with the same length of carbon chains, yet, the length of carbon chain affects the 1O2 generation yields. According to the results of light-induced antibacterial activity, T-OPE exhibited much more potential in killing bacteria than did Q-OPE, it clearly indicated that singlet oxygen was not the key factor responsible for the big differences in T-OPEs and Q-OPEs biocidal abilities. Disruption of Bacterial Membrane Mimicking Liposomes


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Biological membranes, consisting largely of a lipid bilayer, are vital components of all living systems, forming the outer boundary of living cells or internal cell compartments and acting as important filters to regulate complex processes. The structural complexity of biological membranes and their sophisticated interactions with intra- and extracellular networks make direct investigations extremely difficult. Therefore, artificial model membranes have played an important role in unraveling the physical and chemical characteristics during their interactions with extracellular substance. Liposomes have been widely utilized as an experimental cell-surface model and allow us to gain insight into chemical adsorption and its internalization.44-45 Interactions between antimicrobial molecules and liposomes have been extensively studied to mimic how biocides interact with biomembranes and to fundamentally understand the mechanism of their biocidal activities.38, 46-47 In this study, on the basis of the differences in lipid composition between Gram-positive S.aureus and Gram-negative E.coli, two model anionic liposomes were utilized to simulate bacterial cell membranes (Table S1). The membrane perturbation activities of the OPEs were evaluated by fluorescein release assays (Figure 4). For the OPEs dissolved in DMSO, the same amount of DMSO is added in DMSO-control group to eliminate the effects from DMSO in OPEs groups. No significant effects induced by DMSO were observed. The lipid composition of L-1 is DOPG, which was used as a model of S.aureus.48 All OPEs show activity against L-1 (Figure 4A). The dye released from L-1 as time went on even when there was no OPEs or DMSO, however, adding OPEs to the system accelerated the fluorescein releasing progress. As shown in Figure 4A, the order of releasing effects of the OPEs series against L-1 is as follows: OPE(C2,H)> OPE(C3,H)> OPE(C3+,H)> OPE(C2+,H). Interestingly, this order is consistent with that of biocidal ability against S.aureus of OPEs (Figure 1). And L-2,


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made from E.coli total lipid, was used as a model of E.coli. The order of releasing effects of the OPEs series against L-2 (Figure 4B) is also consistent with the order of OPEs biocidal abilities against E.coli (Figure 2). The OPEs used in this study possess structural and 1O2 generating diversity, while the biocidal effects keep consistent with the membrane perturbation abilities. The results from dye-release assays show that the membrane disruption ability is highly dependent on the pendant groups that modify the electrostatic and hydrophobic interactions. For the oligomers tested, the methylation decreased their ability to incorporate or perturb lipid membrane, which led to the leakage of dye molecules from inside the liposomes to the bulk phase (Figure 4).


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Figure 4. Fluorescein leakage profile from DOPG liposome (A) and E.coli total lipid liposome (B) with the addition of OPE in PBS buffer at room temperature under dark condition (ex. 495 nm, em. 510 nm). Fluorescence from liposomes incubated alone was subtracted. Comparison of the Insertion of the OPEs into Lipid Monolayers at the Air-Water Interface. Lipid monolayer at the air-water interface has been used to study interactions between polymers or peptides and lipid monolayer, where polymer insertion events can be directly detected and quantified.49-52 In the current study, insertion of OPEs into lipid monolayer at the air-water interface composed of DOPG or E.coli total lipid were carried out at constant surface area. To evaluate the electrostatic and hydrophobic interaction effects on membrane insertion ability, the extent of the insertion of the four OPEs into lipid monolayer was measured and the results are compared in Figure 5. Each of OPEs was injected into DOPG and E.coli total lipid monolayer holding at a constant surface area with a surface pressure around 39 mN/m and 33 mN/m, respectively, where either DOPG or E.coli total lipid was in a well packed liquid condensed phase (data not shown). From the results shown in Figure 5, the injection of OPEs into the aqueous sub-phase resulted in increases of the monolayer surface pressure when the surface area was held constant. The results indicate that the OPEs can insert into both the DOPG and E.coli total lipid monolayer, and confirm that the OPEs exhibit affinity to the negatively charged lipid membrane and their polymer-membrane interactions lead to the insertion of OPEs into the lipid membrane. The results further corroborate our conclusions drawn from the liposome fluorescein leakage studies. At a constant surface area, T-OPEs insert into the DOPG monolayer faster and get higher pressures compared to Q-OPEs (Figure 5A), when the surface area was held constant (~100 cm2). The surface pressure increases from ca. 30 mN/m to ca. 38 mN/m over 18000 seconds


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after the injection of OPE(C2,H). After the injection of OPE(C3,H), the surface pressure also increases from ca. 30 mN/m to ca. 40 mN/m. However, after the injection of Q-OPE, the surface pressure does not show significant increase. As shown in Figure 5B, in the case of the E.coli total lipid monolayer, T-OPEs also induce a higher pressure increase (from ca. 31.5 mN/m to ca. 38 mN/m) compared to that of Q-OPEs (from ca. 31.5 mN/m to ca. 34 mN/m). The insertion results provide additional evidence for our earlier findings, where T-OPEs appear to associate and become incorporated into the DOPG liposome more rapidly and more efficiently. And consistent with the results obtained from dye-leakage assays, the OPEs show pendantgroup-dependent monolayer insertion abilities. Specifically, increasing the hydrophilicity by methylation of OPEs decreases the extent of insertion of OPE oligomers. The results from the interactions of OPEs with two different membrane systems consistently show that T-OPEs exhibit a higher affinity and stronger interactions with lipid membranes and can more readily and efficiently insert into lipid membranes. The differences in the interactions between the two kinds of OPEs with lipid membrane may stem from their structural difference, note that T-OPEs have tertiary ammonium terminals compared to the quaternary ammonium terminals in Q-OPEs. We propose that the different interactions between the two kinds of OPEs with negatively charged lipid membrane may account for the differences in their biocidal activity. The stronger membrane interactions displayed by T-OPEs render them more effective to internalize into the cell membrane, thus induce more effective biocidal effects by 1O2. More detailed investigation to elucidate the structure-activity relationship of OPEs is currently underway for a number of OPEs with different structures. Taken together, the results obtained from lipid monolayer insertion assays provide additional evidence for the pendant-group-dependent membrane perturbation ability of OPEs.


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Figure 5. Constant area measurements of DOPG (A) and E.coli total lipid (B) monolayer after OPE injection on a water sub-phase at room temperature. Visualization of Light-Enhanced Antimicrobial Actions against Gram-Negative Bacteria. Although interactions with the plasma membrane are necessary for the bactericidal actions of OPE compounds, interactions of these compounds with the bacterial cell envelope are also crucial since the cell envelope serves as the first point-of-contact for exogenous materials. As


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described earlier, the cell envelopes of Gram-positive and Gram-negative bacteria are compositionally and structurally different. And the resistance of E.coli to antibacterial agent is much stronger than that of S.aureus. Thus, we focus on the interactions of OPEs with Gramnegative E.coli surface, which will provide a deep insight into the toxicity mechanism. The complexities of the cell envelopes make such biological entities difficult to mimic with model system. In this study, the interactions of OPEs with Gram-negative bacteria E.coli under fiber light irradiation were studied by visualizing cell morphology using SEM (Figure 6). SEM was used to image morphological damages to bacterial cells upon incubation with OPEs either in the dark or under the fiber light irradiation. The morphological damages were not observed in the dark with incubation of OPEs, nor did the E.coli under the fiber light irradiation without OPEs or incubated with OPE(C2+,H). Meanwhile, some morphological damages can be observed in the E.coli incubated with OPE(C3+,H) and significant damages were found in the E.coli with either OPE(C2,H) or OPE(C3,H). The cell morphology collapsed, which is probably induced by cytoplasm leakage. The higher resolution SEM for the damages of E.coli, incubating with OPE(C2,H) or OPE(C3,H), was shown in Figure S7. The morphological damages to E.coli were consistent with both the OPEs-induced antibacterial activity and the perturbation of bacterial membrane liposome.


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Figure 6. SEM images of E.coli cells (ATCC 25922) (1×108 CFU/mL) alone (A) and incubated with 10 µg/mL OPE (C2+, H) (B) or OPE (C3+,H) (C) or OPE (C2, H) (D) or OPE (C3, H) (E) under fiber light irradiation (9 mw/cm2) for 120 min. Together with all the results obtained, it is clear that the biocidal effects are closely related to the structure of oligomers, concentrations, incubation time, and the strains of bacteria. Particularly, the threshold effective concentrations of different OPEs for different bacteria are various. In contrast to Gram-negative bacteria, all OPEs showed greater biocidal activity against Grampositive bacteria, which probably derived from the more complicated cell envelopes and protective mechanisms of the Gram-negative organisms. Most interestingly, the light-induced biocidal effects of T-OPEs on both bacteria are much more efficient than Q-OPEs even though they share very similar structural scaffolds. The tertiary or quaternary ammonium groups on OPEs remarkably modify the electrostatic properties and therefore lead to the great changes in hydrophobicity, which, in turn, affect the interactions between OPEs and bacterial membrane. Subsequently, the enhanced interactions may destroy or perturb cytoplasmic membrane and cause the leakage of cell components.4, 56-57


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As proposed by Ikeda and co-worker, two key steps for a dark killing process involved are biocide adsorption onto a bacterial surface and then diffusion through the cell wall.55 The bacterial membrane presents an overall negative charge. T-OPEs, which possess an estimated pKa of 10~11, can be protonated in aqueous solution easily (pH 7.4), thus present as positive charged state, and Q-OPEs used in this research are all undergone methylation and cationic oligomers. Thus two kinds of OPEs may adsorb onto the bacterial surface by electrostatic interaction and/or internalize into the membrane of bacteria by hydrophobic effects. Besides, the results from interactions between OPEs and bilayer or monolayer model have demonstrated that T-OPEs can much more easily induce fluorescein leakage and insertion of lipid monolayer. Therefore, probably the enhanced interactions between T-OPEs and bacterial membrane play a key role in their biocidal activities. Under fiber light irradiation, it becomes evident from our results that light irradiation causes great damages to both E.coli cells and S.aureus cells in the presence of OPEs due to reactive singlet oxygen. Singlet oxygen has a relative long lifetime (10-6-10-5 s) and diffusion distance in pure water. However, in cells, both lifetime and diffusion range of 1O2 must be significantly reduced due to its high reactivity toward biomolecules in the cytoplasm.53-54 For the effective distance of 1O2 within nanometers, the damage caused by 1O2 is likely related to the positions of OPEs in the cells. Hence, if the OPE can associate with the cell membrane and further cross the membrane or even internalize into the cytoplasm, the antibacterial activity of OPEs will be probably remarkably enhanced. Upon irradiation in the presence of OPEs, the relative position of OPE to the bacteria will play a key role in inactivating bacteria. There seems to be a synergic effect between singlet oxygen generation and adsorption and/or internalization of OPE to produce antibacterial activity, while the latter is determined by a combination of electrostatic and


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hydrophobic interactions, and loss of the balance between electrostatic and hydrophobic interactions will cause a great change of the biocidal effect of OPEs. Based on light-induced biocidal activity and mechanistic study, the possible mechanism for TOPE inactivating bacteria is proposed in this manuscript (Scheme 2). The tertiary ammonium of T-OPE is protonated in aqueous solution (pH 7.4), present as a positively charged molecule, which promotes its rapid adsorption onto a bacterial surface, since protonation process is reversible, the hydrophobicity will increase once proton dissociates and cause the increase of entropy by the release of interfacial water through the binding of T-OPEs to membranes,58 thus drive T-OPEs internalization in the outer envelopes of bacteria. Upon irradiation of UV light, the interfacial generated singlet oxygen will produce great antibacterial activity. Scheme 2. The presumed mechanism of T-OPEs against bacteria.

CONCLUSIONS We have studied the antibacterial activity of four OPEs against Gram-positive and Gramnegative bacteria both in the dark and under fiber light irradiation. The light-induced biocidal activities of OPEs were enhanced with concentration and irradiation time, while the dark killing


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abilities were not observed. Gram-negative bacteria were found to be less susceptible to OPEs than Gram-positive bacteria. To the best of our knowledge, we firstly observed the phenomenon that in each case under the ultraviolet irradiation, the T-OPEs showed much more powerful biocidal activities than Q-OPEs. Mechanistic studies indicate that the essential factor for killing bacteria under light irradiation is 1O2 and/or ROS, however, for a group of OPEs with similar singlet oxygen yields, the varying cell envelop perturbation abilities of OPEs eventually tune and determine the biocidal abilities of these OPEs, and the biocidal activity has exhibited a synergic effect of 1O2 and OPE internalization. The results also indicate that OPE internalization is determined by a combination of electrostatic and hydrophobic interactions, which likely promotes a favorable entropy increase by the release of interfacial water through the binding of OPEs to membranes. There is a balance between electrostatic and hydrophobic interactions, and loss of the balance will lead to the reduction of biocidal activities of OPEs. All new knowledge of the mechanism will be used in future synergistic experimental and computational studies to design antimicrobial agents based on the phenylene-ethynylene motif with higher efficacy. ASSOCIATED CONTENT Supporting Information Detailed description of the synthesis and characterization data of four OPEs compound mentioned in this research, details of bacterial growth conditions and bacterial killing experiments, preparation of fluorescein-loaded vesicles and vesicle leakage assays, monolayer insertion assay, observation of E.coli morphology, singlet oxygen detection, additional fiber light and dark activated biocidal data. AUTHOR INFORMATION *Phone: (86) 0816-2485484. Emails: [email protected]; [email protected]


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ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (Grant No. 21401176, 21571164 and 21171153), Science and Technology Development of Foundation of China Academy of Engineering Physics (2014A0301011), Institute of Nuclear Physics and Chemistry Foundation (Grant No. 2013CX03 and 2012CX01); Z. H. T acknowledges Guangdong Innovative and Entrepreneurial Research Team Program (No. 2014ZT05N200). REFERENCES (1) Welden, C. W.; Hossler, R. A. Evolution in the Lab: Biocide Resistance in E-coli. Am. Biol. Teach. 2003, 65, 56-61. (2) O'Hanlon, S. J.; Enright, M. C. A Novel Bactericidal Fabric Coating with Potent in Vitro Activity against Meticillin-resistant Staphylococcus aureus (MRSA). Int. J. Antimicrob. Agents 2009, 33, 427-431. (3) Liu, Y.; Strauss, J.; Camesano, T. A. Adhesion Forces between Staphylococcus epidermidis and Surfaces Bearing Self-Assembled Monolayers in the Presence of Model Proteins. Biomaterials 2008, 29, 4374-4382. (4) Rawlinson, L. A. B.; Ryan, S. M.; Mantovani, G.; Syrett, J. A.; Haddleton, D. M.; Brayden, D. J. Antibacterial Effects of Poly(2-(dimethylamino ethyl)methacrylate) against Selected Gram-Positive and Gram-Negative Bacteria. Biomacromolecules 2010, 11, 443-453. (5) Palermo, E. F.; Sovadinova, I.; Kuroda, K. Structural Determinants of Antimicrobial Activity and Biocompatibility in Membrane-Disrupting Methacrylamide Random Copolymers. Biomacromolecules 2009, 10, 3098-3107.


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