Selective Antimicrobial Activities and Action Mechanism of Micelles

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Selective Antimicrobial Activities and Action Mechanism of Micelles Self-assembled by Cationic Oligomeric Surfactants Chengcheng Zhou, Fengyan Wang, Hui Chen, Meng Li, Fulin Qiao, Zhang Liu, Yanbo Hou, Chunxian Wu, Yaxun Fan, Libing Liu, Shu Wang, and Yilin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12688 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on January 29, 2016

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ACS Applied Materials & Interfaces

Selective Antimicrobial Activities and Action Mechanism of Micelles Self-assembled by Cationic Oligomeric Surfactants Chengcheng Zhou,† Fengyan Wang,‡ Hui Chen,‡ Meng Li,‡ Fulin Qiao,† Zhang Liu,† Yanbo Hou,† Chunxian Wu,† Yaxun Fan,† Libing Liu,‡ Shu Wang,*,‡ and Yilin Wang*,† †

Key Laboratory of Colloid and Interface Science, Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡ Key Laboratory of Organic Solids, Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China

ABSTRACT: This work reports that cationic micelles formed by cationic trimeric, tetrameric and hexameric surfactants bearing amide moieties in spacers can efficiently kill Gram-negative E. coli with a very low minimum inhibitory concentration (1.70–0.93 μM), and do not cause obvious toxicity to mammalian cells at the concentrations used. With the increase of the oligomerization degree, the antibacterial activity of the oligomeric surfactants increases, i.e, hexameric surfactant > tetrameric surfactant > trimeric surfactant. Isothermal titration microcalorimetry, scanning electron microscopy and zeta potential results reveal that the cationic micelles interact with the cell membrane of E. coli through two processes. Firstly, the integrity of outer membrane of E. coli is disrupted by the electrostatic interaction of the cationic ammonium groups of the surfactants with anionic groups of E. coli, resulting in loss of the barrier function of the outer membrane. Then the inner membrane is disintegrated by the hydrophobic interaction of the surfactant hydrocarbon chains with the hydrophobic domains of the inner membrane, leading to the cytoplast leakage. The formation of micelles of these cationic oligomeric surfactants at very low concentration enables more efficient interaction with bacterial cell membrane, which endows the oligomeric surfactants with high antibacterial activity. KEYWORDS: cationic micelle, hexameric surfactant, tetrameric surfactant, trimeric surfactant, antimicrobial activity, action mechanism

INTRODUCTION The growing emergence of antibiotic-resistant microorganisms, especially multidrug-resistant bacterial strains, has become a great threat to public health.1-4 Currently, pathogenic bacteria cause around 900 million severe infection cases and 2 million children death every year.5, 6 To address the challenges associated with bacterial infections, extensive efforts have been made to develop highly efficient antimicrobial agents with a lower propensity to bacterial resistance.7, 8 Generally, bacterial resistance largely stems from the action mechanism of antibiotics by acting on specific intracellular targets, where even point mutations of bacteria can render antibiotics inactive.9 Thus in order to slow the development of bacterial resistance to antibiotics, new antibacterial agents need to be developed with different acting mechanism from that of conventional antibiotics.

Up to date, much work has been devoted to designing various peptides and polymers as antimicrobials.8, 10-28 These compounds have cationic and amphiphilic features. Unlike in the case of conventional antibiotics, where the bacterial cell morphology is preserved, they act primarily by targeting and disintegrating the entire bacterial cell membrane through electrostatic attraction and insertion into the lipid domains of cell membrane.14, 15 It is difficult for bacteria to repair a physically damaged cell membrane, hence curbing the potential development of bacterial resistance.10, 16, 17 Despite superiorities in bacterial resistance over conventional antibiotics, both peptides and polymers are still limited in practical applications because of several inherent problems.18 Antimicrobial peptides are mainly limited by their short half-lives in vivo due to proteases degradation.19 The drawbacks of antimicrobial peptides could be overcome by bio-inspired synthetic polymers.20, 21 Recent studies found that compared to individual poly1

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mer molecules, self-assembled cationic polymeric nanoparticles show better antimicrobial properties due to the increased local mass and cationic charges, which are important factors in cell membrane lysis.22-24 So far, the reported antibacterial polymeric nanoparticles have covered micelle,22-24 vesicle,25 sphere,26 rod26, 27 and so on. However, synthetic polymers exhibit wide molecular weight distributions, which must be overcome to ultimately ensure their monodispersity in future applications because polymers possessing different molecular weights will show distinct toxicity and pharmacological activities.22, 28 Small cationic amphiphilic molecules, especially their self-assemblies with increased local cationic charge concentration and aggregate mass, have been proposed to be a strategy to counter the problems faced by synthetic polymers as antibacterial agents.29-33 For example, the nanofibers from amphiphilic terephthalamide-bisurea derivatives were verified to be effective against drug-resistant fungi C. neoformans, and could prevent the development of drug resistance.32 On the other hand, conventional monomeric cationic surfactants with quaternary ammonium headgroups show strong bactericidal potency by directly disrupting the bacterial cell membrane and are widely used for disinfection and sanitation in various fields, such as hospitals and food industry.34 Haldar et al. revealed that with the increase of cationic headgroup numbers, the antibacterial activity of surfactants increases.35 As reported, dimeric surfactants with two cationic headgroups and two hydrophobic chains show higher antibacterial activity over their corresponding monomeric surfactants.6, 29, 36 Thus cationic oligomeric surfactants, consisting of three or more amphiphilic moieties chemically connected by spacer groups and showing lower critical aggregation concentration (CAC) and multiple aggregate structures,37-39 are expected to exhibit excellent antibacterial activity. In addition, recent studies also suggested that the incorporation of amide linkages is favorable for the improvement of antibacterial activity and biocompatibility of surfactants.6, 36, 40 Herein, cationic ammonium oligomeric surfactants, trimeric surfactant DTAD,37 tetrameric surfactant PATC38 and hexameric surfactant PAHB39 bearing amide moieties (Scheme 1) were selected and synthesized to study their antibacterial activities and mechanism to Gram-negative E. coli. The cytotoxicity of these compounds was also evaluated with mammalian cells. These oligomeric surfactants can self-assemble into cationic micelles with extremely low critical aggregation concentration (CAC) and selectively and efficiently kill E. coli over mammalian cells. The action activity follows the order of hexameric surfactant > tetrameric surfactant > trimeric surfactant. The related antibacterial mechanism has also been studied by isothermal titration microcalorimetry, scanning electron microscopy and zeta potential measurements. EXPERIMENTAL SECTION

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Materials. Cationic ammonium surfactants, trimeric DTAD37, tetrameric PATC38 and hexameric PAHB39, were synthesized and purified as we reported previously. The Ampr Escherichia coli (E. coli) was purchased from Beijing Bio-Med Technology Development Co., Ltd. Hela cells were obtained from Center for Cell, Institute of Basic Medical Science, Chinese Academy of Sciences. Phosphate buffered saline (1× PBS, pH 7.4) was used throughout the work.

Scheme 1. Chemical structures of cationic ammonium surfactants: trimeric surfactant (DTAD), tetrameric surfactant (PATC) and hexameric surfactant (PAHB).

Preparation of E. coli Solutions. A single colony of Ampr E. coli on a solid Luria Broth (LB) agar plate was transferred to 10 mL liquid LB culture medium with 100 μg/mL ampicillin and then was grown for 6–8 h at 37 °C under constant shaking of 180 rpm. E. coli was harvested by centrifuging (7100 rpm for 2 min) and was washed with phosphate buffer saline (1× PBS, pH 7.4) for two times. The supernatant was removed and the remaining E. coli was suspended in PBS, and then diluted to the optical density of 1.0 at 600 nm (OD600 = 1.0). There was about 108 CFU/ml E. coli in the solution (OD600 = 1.0). Surface Tension Measurements. The surface tension measurements were conducted with a Pt/Ir plate method on a DCAT21 tensiometer (Dataphysics Co., Germany) at 25.00 ± 0.01 °C. The tensiometer was calibrated by measuring pure water before each set of measurements. The tests were repeated at least two times. Dynamic Light Scattering (DLS) Measurements. DLS measurements were conducted on LLS spectrometer (ALV/SP-125) equipped with a 22 mW He-Ne laser (632.8 nm wavelength) with a refractive index matching bath of filtered toluene surrounding the cylindrical scattering cell. The samples were filtered by 450 nm filters. The scattering angle was 90°. Assessment of Antibacterial Activity. The antimicrobial activity of DTAD, PATC and PAHB to E. coli was evaluated by traditional surface plating method.41 Certain concentrations of surfactants were separately added into E. coli PBS solution with the concentration of approximately 2 × 107 CFU/ml, and the mixtures were incubated for 30 min at 37 °C. Next, the E. coli suspensions were serially diluted by 104-fold with PBS. 100 μL diluted E. coli was spread on the solid agar plate (LB) with 100 μg/mL ampicillin and then incubated for 14–16 h at 37 °C. All the experiments were performed in triplicate. The effects of the surfactants on the bacteria were assessed based on the 2


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reduction ratio of E. coli colonies in the batch culture. E. coli colonies on the agar plates were counted and the reduction ratio was calculated according to equation [(A − B)/A] ×100%,42 where A is the mean number of E. coli colonies in the control sample (without surfactants), and B is the mean number of E. coli after treated with the surfactants. The diameter of the solid agar plates was 90 mm. The results were repeated for three times. Cytotoxicity Assay. Hela cells were seeded into 96well culture plates at a density of 8 × 103 cells/well and were grown for 12–24 h until adherent at 37 °C and in a humified atmosphere containing 5% CO2. Then DTAD, PATC and PAHB with a series of concentrations below 10 μM were added into 96-well culture plates, respectively. After 24 h incubation at 37 °C, the supernatant was removed and MTT (0.5 mg/mL in medium, 100 μL/well) was added to the wells followed by incubation at 37 °C for 4 h. Subsequently, after removing the supernatant, 100 μL DMSO per well was added to sufficiently dissolve the produced formazan. After shaking the plates for 5 min, absorbance values per wells were read with a microplate reader at 520 nm. The cell viability rate (VR) was calculated by the equation of VR = A/A0 × 100% (A is the absorbance of the experimental groups with surfactants, and A0 is the absorbance of the control group without surfactants.). Each assay was repeated for six times. Scanning Electron Microscopy (SEM). The morphological changes of E. coli before and after adding different surfactant micelles were observed by SEM. After the treatment described in the antibacterial experiments above, E. coli was immediately fixed with 0.5% glutaraldehyde PBS solution for 30 min at room temperature. The E. coli was centrifuged (7100 rpm for 5 min) at 4 °C and the supernatant was removed, and then the E. coli pellets were resuspended in sterile water. 2 μL E. coli suspensions were dropped on clean silicon slices followed by naturally drying in the super clean bench. After the specimens became dried, 0.1% glutaraldehyde was added for further fixation for 1 h and then 0.5% glutaraldehyde for another 1 h. Next, the specimens were washed with sterile water for three times, dehydrated by adding ethanol in a graded series (70% for 6 min, 90% for 6 min, and 100% for 6 min) and then dried in vacuum drying oven. Finally, the specimens were coated with platinum before SEM observation (Hitachi S4800, Japan). Zeta Potential Measurements. E. coli was incubated by DTAD, PATC and PAHB of different concentrations at 37 °C for 30 min, respectively. And then unbound surfactants were removed by centrifugation (7100 rpm, 5 min) at 4 °C. The pellets obtained were suspended in PBS and the suspensions were placed on ice for zeta potential measurements. Untreated E. coli (without surfactants) was also incubated under the same conditions as the control. Isothermal Titration Microcalorimetry (ITC). The calorimetric measurements were conducted on a TAM 2277-201 microcalorimetric system (Thermometric AB, Järfȧlla, Sweden) with a stainless steel sample cell of 1 mL

at 25.00 ± 0.01 °C. The sample cells were initially loaded with 750 μL PBS or E. coli PBS solution (OD600 = 1.0), and then the surfactant solution (40 μM DTAD, 30 μM PATC and 25 μM PAHB, respectively) was injected consecutively into the stirred sample cell in portions of 10 μL via a 500 μL Hamilton syringe controlled by a 612 Thermometric Lund pump until the interaction progress was completed. The system was stirred at 60 rpm with a gold propeller. Each ITC curve was repeated at least twice with deviation within ±4%. The dilution enthalpies of the surfactants were subtracted from the corresponding observed enthalpy curve of the surfactants with E. coli. The binding parameters of the surfactants and E. coli were obtained by fitting the enthalpy curves with the model of the two sets of binding sites provided in Origin® scientific plotting software v7.0.43,44

RESULTS AND DISCUSSION Aggregation with Extremely Low CAC. The CAC values of oligomeric surfactants DTAD, PATC and PAHB in PBS were determined from the clear breakpoints of the surface tension curves shown in Figure 1. DTAD, PATC and PAHB in PBS have extremely low CAC values (0.87, 0.68 and 0.52 μM, respectively), which are significantly lower than those in water (200, 80 and 50 μM for DTAD37, PATC38 and PAHB39 respectively). The reduction of CAC is caused by the salts in the buffer which screen the electrostatic repulsion between the quaternary ammonium head groups. The aggregates of these three oligomeric surfactants exhibit similar size varying tendency with the increase of concentration above their CAC (Figure 2). Just beyond the CAC, DTAD, PATC and PAHB present two kinds of aggregates with the hydrated radius Rh of about 2.0 and 60 nm. With increasing the surfactant concentration to two times of their CAC, the large size distribution disappears and completely transforms to small micelles of 2.0–3.0 nm. Considering that the scattering intensity of the aggregates is proportional to the sixth power of their hydrated radius Rh, the small micelles are the major selfassemblies for DTAD, PATC and PAHB above their CAC with the surfactants presenting a pyramid-like configuration.37-39 72 64

γ (mN/m)

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DTAD PATC PAHB

56 48 40 32 0.1

1

C (µM) Figure 1. Variations of surface tension of DTAD, PATC and PAHB with the concentration in PBS of pH 7.4 at 25.00 ± 0.01 °C. 3



a

1.50 µM

2.00 µM 1.50 µM

1.00 µM

1.00 µM 0.1

1

10

100

1000

2.00 µM

Relative Intensity

2.00 µM

c

b Relative Intensity

2.50 µM

Relative Intensity

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1.00 µM

0.75 µM 0.50 µM

0.75 µM 0.1

1

10

100

1000

Rh (nm)

Rh (nm)

0.1

1

10

100

1000

Rh (nm)

Figure 2. The size distribution of DTAD (a), PATC (b) and PAHB (c) with different concentrations in PBS solution at 25.0 ± 0.1 °C.

Antibacterial Activities of Cationic Micelles to E. coli. It is widely accepted that Gram-negative bacteria with the barrier function of outer membrane are more difficult to be killed than Gram-positive ones,24,45 and half of infections originate from Gram-negative E. coli.46 Therefore, we evaluated the activity of the three oligomeric surfactants DTAD, PATC and PAHB against Gramnegative E. coli by the surface plating method. It can be found that DTAD, PATC and PAHB have a strong effect against the growth of E. coli (Figure 3). As shown in Figure 3a-c, dense bacterial colonies are observed in the control sample without surfactant treatment and sporadic bacterial colonies are observed in the samples treated with the surfactants. The killing efficacy calculated by colony counting (Figure 3d) indicates that the antibacterial activity of the surfactants follows the order of PAHB > PATC > DTAD. This means that the antibacterial activity of the surfactants increases with increasing the number of cationic headgroups and hydrophobic chains. In addition, minimal inhibitory concentration (MIC), i.e., the minimum concentration of an antimicrobial agent required for inhibiting bacterial growth, is an important parameter used to evaluate the activity of antimicrobial agents.22 For the present oligomeric surfactants ， their MIC values against E. coli are 1.70, 1.15 and 0.93 μM for DTAD, PATC and PAHB, respectively (derived from Figure S1), which are larger than their respective CACs in PBS (0.87, 0.68 and 0.52 μM). As shown in Figure 3d, the monomers of these surfactants are not potent enough to inhibit microbial growth whereas self-assembled micelles exhibit very high antibacterial activity, suggesting that the formation of micelles is necessary for the antimicrobial activity. This may be attributed that the formation of micelles enhances local surfactant concentration and cationic charge concentration, leading to strong interactions between the surfactants and bacterial cell membrane and in turn converting into effective antimicrobial activities. Obviously, oligomeric surfactants DTAD, PATC and PAHB exhibit much higher activity against E. coli compared to their dimeric counterpart N,N'-bis(N-dodecylN,N-dimethylglycine)1,4-diaminobutane dihydrochloride (DABB) with a MIC value of about 24 μM40 and corresponding monomeric surfactant dodecyltrimethylammonium bromide (DTAB) with a MIC value of about 250 μM.47 On one hand, the larger cationic charge number

and multiple hydrophobic chains of oligomeric surfactants make them strongly interact with the cell membrane. On the other hand, this kind of molecular structure endows the oligomeric surfactants with lower CAC. The formation of micelles enables more efficient interaction with the cell membrane, which further enhances the antibacterial activity of the oligomeric surfactants. The mechanism will be further discussed in the following text.

Figure 3. Number of colony forming units (CFU) of E. coli before (control) and after adding oligomeric surfactants with different concentrations on LB agar plate. (a) CFU of E. coli suspension incubated with DTAD. (b) CFU of E. coli suspension incubated with PATC. (c) CFU of E. coli suspension incubated with PAHB. (d) Antibacterial activity of DTAD, PATC and PAHB toward E. coli, where error bars represent standard deviations of data for three separate measurements.

Action Mechanism of Cationic Micelles to E. coli. To gain visual insights into the antimicrobial activities of the surfactant micelles, the morphological changes of E. coli in response to exposure to the different surfactant micelles were observed by SEM and the images are shown in Figure 4. For the control groups without surfactants (Figure 4a), the E. coli structures are intact and display clear edges and smooth bodies. In sharp contrast, the E. coli structures treated with DTAD, PATC and PAHB mi4


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celles (Figure 4b-d) are collapsed and merged. Moreover, the leakage of cytoplast also appears (Figure 4c). Therefore, we hypothesize that the cationic micelles target the negatively charged cell membrane of E. coli and then disrupt the cell membrane, which results in the leakage of cytoplast and finally leads to cell death.

Figure 4. SEM images of E. coli before (a) and after incubation with the micelles of DTAD (b), PATC (c) and PAHB (d) at the concentration of 2.0 μM. Arrows indicate lesions and collapses of bacterial membrane.

To further study the interactions between E. coli and the surfactant micelles, ITC was employed to investigate the thermodynamic changes in the binding process of E. coli with the surfactant micelles upon adding the micelles. In Figure 5a, when the surfactants micelles were titrated into E. coli solutions, the observed enthalpy (ΔHobs) values are initially less exothermic and display a platform, then increase sharply to a maximum exothermic value. Upon further adding the surfactant micelles, ΔHobs becomes smaller and finally returns to zero. The variation situation of the ΔHobs curves indicates that the interaction between E. coli and the micelles involves two processes. This means that E. coli has two different domains to bind with the surfactant micelles. According to literature,48 one may be located in its outer membrane (OM) possessing the barrier properties, while another may be located in its inner membrane (IM). As shown in Scheme 2, the outer envelope of E. coli consists of OM and IM which are separated by the cell wall with a thin, intermittently crosslinked peptidoglycan network structure.48 The surface of E. coli is negatively charged, which is mainly provided by the lipopolysaccharides and anionic phospholipids of OM.49 The ITC curves of the surfactants with E. coli are analyzed by the model of the two binding-site sets described in the Supporting Information. The obtained binding parameters of the surfactant molecules with E. coli are shown in Table 1. The binding number (N) is the number of the surfactant molecules with one E. coli, K1 and K2 are the stepwise binding constants, and K is the overall binding constant of the two interaction processes of the surfactant with E. coli. The overall binding constant (K) and the stepwise binding constants (K1 and K2) follow the relationship of K = K1 × K2.44 Obviously, the K values for the three surfactants decrease in the order of KPAHB > KPATC > KDATD (Table 1). This indicates that the interaction of the corresponding surfactant micelles with E. coli increases with the increase of the amount of cationic charges and hydrophobic chains in the surfactant molecules,

which is responsible for the different antibacterial activity of the surfactants. In particular, the ITC results also suggest the action mechanism of the surfactant micelles to E. coli in detail (Scheme 2). During the first interaction process between the micelles and E. coli, the cationic micelles primarily target the negatively charged outer membrane of E. coli by electrostatic interaction. As shown in Figure 5a, with the addition of the cationic micelles into E. coli solution, the ΔHobs are initially about −15, −25 and −23 kJ/mol for trimrtic DTAD, tetrameric PATC and hexameric PAHB, which indicates that the interaction between these surfactant micelles and the OM of E. coli is an exothermic process. The exothermic enthalpy is mainly contributed by the electrostatic binding between the cationic ammonium groups of the surfactants with the anionic groups of OM of E. coli. Upon further adding the micelles, the exothermic ΔHobs sharply increases to a maximum, suggesting the saturation of the electrostatic interaction between the cationic micelles and OM. In this process, the number (N1) of the surfactant molecules associated with a single E. coli is 1.88 × 107, 1.21 × 107 and 1.30 × 107 for DTAD, PATC and PAHB, respectively (Table 1). On the other hand, with the addition of the surfactant micelles, the zeta potentials of E. coli do not change distinctly (Figure 5b), which suggests that the micelles bound on OM insert into the lipid domains.45 As a result, the integrity of OM is disrupted, leading to the loss of the barrier function. In order to know if E. coli has been killed when the barrier function of OM was destroyed during the first interaction process, the antibacterial results attained by the surface plating method are compared with those from ITC (Table 2). The corresponding surfactant number at the end of the first process of ITC curves is in a good agreement with the lowest surfactant number (n0) required to exhibit antibacterial activity from the surface plating method. Therefore, by this point, the number of added cationic micelles is potent enough to disrupt the barrier function of the OM but lack direct and obvious bactericidal activity.50 Additionally, the binding constant K1 values for the first interaction process of the surfactants with E. coli increase with the increase of the oligomerization degree of the surfactants, i.e, K1, PAHB > K1, PATC > K1, DATD (Table 1). This indicates that the electrostatic interaction between the surfactant micelles and E. coli are enhanced with increasing the cationic headgroup numbers of the surfactants. Thus the cationic surfactants with greater oligomerization degree have stronger ability in disrupting the barrier function of the OM of E. coli. Next, the second interaction process between the surfactant micelles and E. coli shows a gradual decreasing exothermic ΔHobs value and finally returns to zero (Figure 5a), indicating that the interaction between the cationic micelles and IM reaches the saturation in final. The corresponding number (N2) of the surfactant molecules associated with a single E. coli is 1.59 × 107, 1.43 × 107 and 1.38 × 107 for DTAD, PATC and PAHB, respectively (Table 1). In this process, the electrical attraction is no longer the dominant force, while hydrophobic interac5


ACS Applied Materials & Interfaces tion plays a main role. The micelles can diffuse through the poriferous cell wall and insert into the lipophilic domain of IM by the hydrophobic interaction of the hydrophobic chains of the surfactants with the lipid domain. This process disintegrates the IM of cells followed by the leakage of cytoplast. By the end of the second interaction process, E. coli have been fully killed, where the corresponding surfactant number at the end of the process of ITC curves is in line with that required to completely kill E. coli (nt) obtained by surface plating method (Table 2). That is to say, the disintegration of the IM of E. coli eventually leads to cell death. Similarly, the binding constant K2 values of the second process also increase with the increase of the oligomerization degree of the surfactants (Table 1), which accounts for the enhancement of antibacterial activity by increasing the amount of hydrophobic chains. Beside, for these cationic oligomeric surfactants, the binding constants K2 are at least two times higher than K1, indicating that the contribution of hydrophobic interaction to the antibacterial activity is more significant than that of electrostatic interaction (Table 1). 0

-50 -100 0

-150

∆ Hobs (kJ/mol)

∆ Hobs (kJ/mol)

a

DTAD-E.coli PATC-E.coli PAHB-E.coli

-200

nt

-30 -60 -90

-120 -150

n0 0

-250 -1

0

1

2

1

2

3

4

5

nsurfactant/nE.coli (× 10-7)

3

4

-7

5

6

6

nsurfactant/nE.coli (× 10 ) -12

Scheme 2. The schematic graph of antibacterial mechanism of the surfactant micelles to E. coli.

Cytotoxicity. In biomedical applications, it is very important that antimicrobial agents exhibit excellent antimicrobial activity but are nontoxic to mammalian cells. Therefore, the toxicity of these oligomeric surfactants toward mammalian cells was evaluated by the MTT assay. As shown in Figure 6, the oligomeric surfactants do not exhibit obvious cytotoxicity on Hela cells even when the concentrations used are 5 times more than their corresponding MIC values. That is to say, the oligomeric surfactants of very low concentrations show very high antimicrobial activity to E. coli but do not have obvious cytotoxicity on Hela cells at the concentrations used. This selectivity is possibly because the surface of bacteria possess more negative charges than that of mammalian cells.51 Thus the electrostatic interaction between E. coli and the cationic micelles is much stronger than that between Hela cells and the cationic micelles, leading to excellent antimicrobial activity, but insignificant cytotoxicity activity. 120

-15

Cell Viability (%)

b ζ potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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DTAD PATC PAHB

100

-18

DTAD-E.coli PATC-E.coli PAHB-E.coli

-21

-24 0.0

0.5

1.0

1.5

2.0

2.5

Csurfactant (µM)

80 60 40 20 0

Figure 5. (a) The variation of observed enthalpy changes (ΔHobs) against the surfactant/E.coli molar ratio by titrating 40 μM DTAD, 30 μM PATC and 25 μM PAHB into E. coli PBS solution (OD600 = 1.0), respectively. ΔHobs values are expressed in kJ/mol of surfactant. The dilution enthalpy of the surfactants has been deducted. (b) Zeta potential results of E. coli in the absence and presence of DTAD, PATC and PAHB, respectively.

0.5

1.0

2.0

3.0

5.0

10.0

Csurfactant (µM) Figure 6. Cell viability of Hela cells after incubation with the aqueous solutions of DTAD, PATC and PAHB at different concentrations.

6


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TABLE 1. Thermodynamic Parameters of the Binding between the Surfactant Micelles and E. coli Derived from ITC Curves in Figure 5a. The Binding Number (N) is the Number of Surfactant Molecules Associated with Per E. coli. K1 and K2 are the Stepwise Binding Constants, and K is the Overall Binding Constant of the Two Interaction Processes of the Surfactants with E. coli. N1

N2 7

N 7

K1 7

6

K2 -1

6

K -1

12

-2

(× 10 )

(× 10 )

(× 10 )

(× 10 M )

(× 10 M )

(× 10 M )

DTAD-E. coli

1.88 ± 0.06

1.59 ± 0.02

3.47 ± 0.08

4.31 ± 0.59

357 ± 104

1538 ± 448

PATC-E. coli

1.21 ± 0.04

1.43 ± 0.02

2.64 ± 0.06

8.35 ± 1.14

507 ± 143

4233 ± 1194

PAHB-E. coli

1.30 ± 0.03

1.38 ± 0.01

2.68 ± 0.04

14.60 ± 1.92

989 ± 246

14439 ± 3591

TABLE 2. The Lowest Surfactant Number Required to Show Antibacterial Activity (n0) and the Surfactant Number Required to Completely Kill E. coli (nt) Obtained by Surface Plating Method and ITC. 7

Surfactant

7

n0 (× 10 ) a

nt (× 10 )

Surface Plating

ITC

DTAD

3.01

PATC

2.25

b

a

b

Surface Plating

ITC

2.09

6.02

4.62

1.83

4.52

3.28

PAHB 1.51 1.78 3.01 3.20 For the surface plating method, n = C × V × NA/NE. coli, where C is the minimum concentration of surfactant showing the activity against E. coli (CDTAD = 1.00 μM, CPATC = 0.75 μM, CPAHB = 0.50 μM as shown in Figure 3d). V is the total volume of mixed PBS b solution with surfactant/E. coli, NA is avogadro constant, and NE. coli is the number of E. coli. For ITC results, n value was determined according to the end points of two processes as shown in the inset of Figure 5a. a

CONCLUSION In summary, the cationic micelles self-assembled by cationic oligomeric surfactants bearing amide linkages are highly active against Gram-negative E. coli but are nontoxic to mammalian cells at the concentrations used. The MIC values for oligomeric surfactants are 1.70–0.93 μM, which are much lower than that of their corresponding dimeric and monomeric counterparts. And the antibacterial activity of the oligomeric surfactants increases upon increasing their oligomerization degree. On one hand, the increase of oligomerization degree of the surfactants brings about more cationic charges and hydrocarbon chains, allowing stronger interaction with both the outer membrane and the inner membrane of the bacterial cell. On the other hand, the increase of oligomerization degree of the surfactants significantly improves the selfassembling abilities into cationic micelles, which further enhances the interactions with the cell membrane. It is also revealed that these cationic micelles kill E. coli based on two interaction processes: (1) disrupting the integrity of outer membrane by the electrostatic interaction between the cationic micelles and the anionic surface of E. coli, accompanied with the loss of the barrier function; (2) disintegrating the cell inner membrane through the hydrophobic interaction between the hydrocarbon chains of the surfactants and the lipid of the E. coli membrane, followed by the cytoplast leakage, which eventually leads to the death of bacteria. Our studies may advance a better

understanding of action mechanism of amphiphilic assemblies against Gram-negative bacteria. Moreover, these high effective oligomeric surfactants hold great potential as novel antimicrobial agents for future applications.

ASSOCIATED CONTENT Supporting Information The analysis method of MIC values and the ITC analysis process. This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Note The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by Chinese Academy of Sciences and National Natural Science Foundation of China (21025313, 21321063)

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