Selective Antimicrobial Activities and Action ... - ACS Publications

Research Article www.acsami.org

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, People’s Republic of China ‡ Key Laboratory of Organic Solids, Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China S Supporting Information *

ABSTRACT: This work reports that cationic micelles formed by cationic trimeric, tetrameric, and hexameric surfactants bearing amide moieties in spacers can efficiently kill Gramnegative 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. First, 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. The inner membrane then 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

■

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, because of the degradation of proteases.19 The drawbacks of antimicrobial peptides could be overcome by bioinspired synthetic polymers.20,21 Recent studies found that, compared to individual polymer molecules, self-assembled cationic polymeric nanoparticles show better antimicrobial properties, because of 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 rod,26,27 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

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 ∼900 million severe infection cases and the deaths of 2 million children 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 must be developed with a different acting mechanism from that of conventional antibiotics. 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 © 2016 American Chemical Society

Received: December 27, 2015 Accepted: January 28, 2016 Published: January 28, 2016 4242

DOI: 10.1021/acsami.5b12688 ACS Appl. Mater. Interfaces 2016, 8, 4242−4249

Research Article

ACS Applied Materials & Interfaces

Medical Science, Chinese Academy of Sciences. Phosphate buffered saline (1× PBS, pH 7.4) was used throughout the work. 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 (at 7100 rpm for 2 min) and was washed with phosphate buffer saline (1× PBS, pH 7.4) 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 ∼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. Dynamic light scattering (DLS) measurements were conducted on an LLS spectrometer (Model ALV/SP-125) that was 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 using a traditional surface plating method.41 Certain concentrations of surfactants were separately added into E. coli PBS solution with the concentration of ∼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. One hundred microliters (100 μL) of 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 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 the equation42

possessing different molecular weights will show distinct toxicity and pharmacological activities.22,28 Small cationic amphiphilic molecules, especially their selfassemblies 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−bis-urea 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 PATC, 38 and hexameric surfactant PAHB39 bearing amide moieties (Scheme 1) were selected and synthesized to study their antibacterial

reduction ratio (%) =

Scheme 1. Chemical Structures of Cationic Ammonium Surfactants: Trimeric Surfactant (DTAD), Tetrameric Surfactant (PATC), and Hexameric Surfactant (PAHB)

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 three times. Cytotoxicity Assay. Hela cells were seeded into 96-well 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. DTAD, PATC, and PAHB with a series of concentrations below 10 μM then 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 of dimethylsulfoxide (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 using the equation

activities and mechanism to Gram-negative E. coli. The cytotoxicity of these compounds was also evaluated with mammalian cells. These oligomeric surfactants can selfassemble into cationic micelles with extremely low 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 (SEM), and zeta potential measurements.

■

A−B × 100 A

VR (%) =

A × 100 A0

where 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 six times. Scanning Electron Microscopy (SEM). The morphological changes of E. coli before and after the addition of 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. Two microliters (2 μL) of E. coli

EXPERIMENTAL SECTION

Materials. Cationic ammonium surfactants, trimeric DTAD,37 tetrameric PATC,38 and hexameric PAHB,39 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 4243


Research Article


∼2.0 and 60 nm. With increasing the surfactant concentration to twice 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 self-assemblies for DTAD, PATC, and PAHB above their CAC with the surfactants presenting a pyramid-like configuration.37−39 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 kill than Gram-positive ones,24,45 and half of the infections originate from Gram-negative E. coli.46 Therefore, we evaluated the activity of the three oligomeric surfactants (DTAD, PATC, and PAHB) against Gram-negative 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 Figures 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 ranks in the following order: PAHB > PATC > DTAD. This means that the antibacterial activity of the surfactants increases as the numbers of cationic headgroups and hydrophobic chains each increase. 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 in the Supporting Information), 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 to 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-dodecyl-N,N-dimethylglycine)-1,4-diaminobutane dihydrochloride (DABB) with a MIC value of ∼24 μM40 and the corresponding monomeric surfactant dodecyltrimethylammonium bromide (DTAB) with a MIC value of ∼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 type 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. 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

suspensions were deposited dropwise onto 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 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 a vacuum drying oven. Finally, the specimens were coated with platinum before SEM observation (Model S4800, Hitachi, Japan). Zeta Potential Measurements. E. coli was incubated by DTAD, PATC, and PAHB of different concentrations at 37 °C for 30 min, respectively. Unbound surfactants then 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 Model TAM 2277-201 microcalorimetric system (Thermometric AB, Järfälla, 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

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

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 DTAD,37 PATC,38 and PAHB,39 respectively). The reduction of CAC is caused by the salts in the buffer that screen the electrostatic repulsion between the quaternary ammonium head groups. The aggregates of these three oligomeric surfactants exhibit similar size-varying tendencies with increasing concentration above their CAC (Figure 2). Just beyond the CAC, DTAD, PATC, and PAHB present two types of aggregates with the hydrated radius Rh of 4244


Research Article


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

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. 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

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; and (d) antibacterial activity of DTAD, PATC, and PAHB toward E. coli, where error bars represent standard deviations of data for three separate measurements.

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 micelles (Figures 4b−d) are collapsed and

Figure 5. (a) 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 terms of 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.

Figure 4. SEM images of E. coli (a) before incubation and (b−d) after incubation with the micelles of (b) DTAD, (c) PATC, and (d) PAHB at a concentration of 2.0 μM. Arrows indicate lesions and collapses of the bacterial membrane. 4245


Research Article


surfactant molecules increases, 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 (see 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 approximately −15, −25, and −23 kJ/mol for trimeric DTAD, tetrameric PATC, and hexameric PAHB respectively, 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 addition of the micelles, the exothermic ΔHobs value 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 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 (see 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 (see Table 2). The corresponding surfactant number

exothermic and display a platform, then increase sharply to a maximum exothermic value. Upon further addition of 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 the 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 Scheme 2. Schematic Graph of Antibacterial Mechanism of the Surfactant Micelles to E. coli

Table 2. 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

envelope of E. coli consists of OM and IM, which are separated by the cell wall with a thin, intermittently cross-linked 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 following order: KPAHB > KPATC > KDATD (Table 1). This indicates that the interaction of the corresponding surfactant micelles with E. coli increases as the amount of cationic charges and hydrophobic chains in the

n0 (× 107)

nt (× 107)

surfactant

surface platinga

ITCb

surface platinga

ITCb

DTAD PATC PAHB

3.01 2.25 1.51

2.09 1.83 1.78

6.02 4.52 3.01

4.62 3.28 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, and CPAHB = 0.50 μM, as shown in Figure 3d), V is the total volume of mixed PBS solution with surfactant/E. coli, NA is Avogadro’s constant, and NE.coli is the number of E. coli. bFor ITC results, the value of n was determined according to the end points of two processes, as shown in the inset of Figure 5a. a

Table 1. Thermodynamic Parameters of the Binding between the Surfactant Micelles and E. coli Derived from ITC Curves in Figure 5a Binding Numbersa DTAD-E. coli PATC-E. coli PAHB-E. coli

Binding Constantsb

N1 (× 107)

N2 (× 107)

N (× 107)

K1 (× 106 M−1)

K2 (× 106 M−1)

K (× 1012 M−2)

1.88 ± 0.06 1.21 ± 0.04 1.30 ± 0.03

1.59 ± 0.02 1.43 ± 0.02 1.38 ± 0.01

3.47 ± 0.08 2.64 ± 0.06 2.68 ± 0.04

4.31 ± 0.59 8.35 ± 1.14 14.60 ± 1.92

357 ± 104 507 ± 143 989 ± 246

1538 ± 448 4233 ± 1194 14439 ± 3591

a

The binding number (N) is the number of surfactant molecules associated per E. coli. bK1 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. 4246


Research Article

ACS Applied Materials & Interfaces at the end of the first process of ITC curves is in 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 lacks direct and obvious bactericidal activity.50 In addition, the binding constant K1 values for the first interaction process of the surfactants with E. coli increase as the degree of oligomerization of the surfactants increases (i.e., K1,PAHB > K1,PATC > K1,DATD; see Table 1). This indicates that the electrostatic interaction between the surfactant micelles and E. coli are enhanced as the cationic headgroup numbers of the surfactants increase. Thus, the cationic surfactants with a greater degree of oligomerization have a stronger ability to disrupt 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 ultimately reaches saturation. 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 interaction plays a primary 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 completely killed, where the corresponding surfactant number at the end of the process of ITC curves is consistent 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 as the degree of oligomerization of the surfactants increases (see 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 almost two orders of magnitude larger than K1, indicating that the contribution of hydrophobic interaction to the antibacterial activity is more significant than that of electrostatic interaction (see Table 1). Cytotoxicity. In biomedical applications, it is very important that antimicrobial agents exhibit excellent antimicrobial activity but be 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 five 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 due to the fact that 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.

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

■

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 minimal inhibitory concentration (MIC) values for oligomeric surfactants are 1.70−0.93 μM, which are much lower than that of their corresponding dimeric and monomeric counterparts. In addition, the antibacterial activity of the oligomeric surfactants increases as the degree of oligomerization increases. On one hand, the increase in the degree of oligomerization 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 in the degree of oligomerization of the surfactants significantly improves the self-assembling 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 by 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 Gramnegative bacteria. Moreover, these high effective oligomeric surfactants hold great potential as novel antimicrobial agents for future applications.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b12688. The analysis method of MIC values and the ITC analysis process (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S. Wang). 4247


Research Article

ACS Applied Materials & Interfaces *E-mail: [email protected] (Y. Wang).

(17) Hoffmann, J. A.; Kafatos, F. C.; Janeway, C. A.; Ezekowitz, R. Phylogenetic Perspectives in Innate Immunity. Science 1999, 284, 1313−1318. (18) Marr, A. K.; Gooderham, W. J.; Hancock, R. E. Antibacterial Peptides for Therapeutic Use: Obstacles and Realistic Outlook. Curr. Opin. Pharmacol. 2006, 6, 468−472. (19) Hancock, R. E.; Sahl, H.-G. Antimicrobial and Host-Defense Peptides as New Anti-Infective Therapeutic Strategies. Nat. Biotechnol. 2006, 24, 1551−1557. (20) Engler, A. C.; Wiradharma, N.; Ong, Z. Y.; Coady, D. J.; Hedrick, J. L.; Yang, Y.-Y. Emerging Trends in Macromolecular Antimicrobials to Fight Multi-Drug-Resistant Infections. Nano Today 2012, 7, 201−222. (21) Palermo, E. F.; Kuroda, K. Structural Determinants of Antimicrobial Activity in Polymers Which Mimic Host Defense Peptides. Appl. Microbiol. Biotechnol. 2010, 87, 1605−1615. (22) Nederberg, F.; Zhang, Y.; Tan, J. P.; Xu, K.; Wang, H.; Yang, C.; Gao, S.; Guo, X. D.; Fukushima, K.; Li, L.; Hedrick, J. L.; Yang, Y.-Y. Biodegradable Nanostructures with Selective Lysis of Microbial Membranes. Nat. Chem. 2011, 3, 409−414. (23) Yuan, W.; Wei, J.; Lu, H.; Fan, L.; Du, J. Water-Dispersible and Biodegradable Polymer Micelles with Good Antibacterial Efficacy. Chem. Commun. 2012, 48, 6857−6859. (24) Qiao, Y.; Yang, C.; Coady, D. J.; Ong, Z. Y.; Hedrick, J. L.; Yang, Y.-Y. Highly Dynamic Biodegradable Micelles Capable of Lysing Gram-Positive and Gram-Negative Bacterial Membrane. Biomaterials 2012, 33, 1146−1153. (25) Zhang, C.; Zhu, Y.; Zhou, C.; Yuan, W.; Du, J. Antibacterial Vesicles by Direct Dissolution of a Block Copolymer in Water. Polym. Chem. 2013, 4, 255−259. (26) Fukushima, K.; Tan, J. P.; Korevaar, P. A.; Yang, Y. Y.; Pitera, J.; Nelson, A.; Maune, H.; Coady, D. J.; Frommer, J. E.; Engler, A. C.; Huang, Y.; Xu, K.; Ji, Z.; Qiao, Y.; Fan, W.; Li, L.; Wiradharma, N.; Meijer, E. W.; Hedrick, J. L. Broad-Spectrum Antimicrobial Supramolecular Assemblies with Distinctive Size and Shape. ACS Nano 2012, 6, 9191−9199. (27) Yao, D.; Guo, Y.; Chen, S.; Tang, J.; Chen, Y. Shaped Core/ Shell Polymer Nanoobjects with High Antibacterial Activities Via Block Copolymer Microphase Separation. Polymer 2013, 54, 3485− 3491. (28) Hunter, A. C. Molecular Hurdles in Polyfectin Design and Mechanistic Background to Polycation Induced Cytotoxicity. Adv. Drug Delivery Rev. 2006, 58, 1523−1531. (29) Hoque, J.; Konai, M. M.; Samaddar, S.; Gonuguntala, S.; Manjunath, G. B.; Ghosh, C.; Haldar, J. Selective and Broad Spectrum Amphiphilic Small Molecules to Combat Bacterial Resistance and Eradicate Biofilms. Chem. Commun. 2015, 51, 13670−13673. (30) Ghosh, C.; Manjunath, G. B.; Akkapeddi, P.; Yarlagadda, V.; Hoque, J.; Uppu, D. S.; Konai, M. M.; Haldar, J. Small Molecular Antibacterial Peptoid Mimics: The Simpler the Better! J. Med. Chem. 2014, 57, 1428−1436. (31) Liu, S. Q.; Venkataraman, S.; Ong, Z. Y.; Chan, J. M.; Yang, C.; Hedrick, J. L.; Yang, Y. Y. Overcoming Multidrug Resistance in Microbials Using Nanostructures Self-Assembled from Cationic BentCore Oligomers. Small 2014, 10, 4130−4135. (32) Fukushima, K.; Liu, S.; Wu, H.; Engler, A. C.; Coady, D. J.; Maune, H.; Pitera, J.; Nelson, A.; Wiradharma, N.; Venkataraman, S.; Huang, Y.; Fan, W.; Ying, J. Y.; Yang, Y. Y.; Hedrick, J. L. Supramolecular High-Aspect Ratio Assemblies with Strong Antifungal Activity. Nat. Commun. 2013, 4, 2861. (33) Ghosh, C.; Haldar, J. Membrane-Active Small Molecules: Designs Inspired by Antimicrobial Peptides. ChemMedChem 2015, 10, 1606−1624. (34) Nakata, K.; Tsuchido, T.; Matsumura, Y. Antimicrobial Cationic Surfactant, Cetyltrimethylammonium Bromide, Induces Superoxide Stress in Escherichia Coli Cells. J. Appl. Microbiol. 2011, 110, 568−579. (35) Haldar, J.; Kondaiah, P.; Bhattacharya, S. Synthesis and Antibacterial Properties of Novel Hydrolyzable Cationic Amphiphiles.

Author Contributions

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

The authors declare no competing financial interest.

■

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

■

REFERENCES

(1) Zhang, Q.; Lambert, G.; Liao, D.; Kim, H.; Robin, K.; Tung, C.k.; Pourmand, N.; Austin, R. H. Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments. Science 2011, 333, 1764−1767. (2) Martínez, J. L. Antibiotics and Antibiotic Resistance Genes in Natural Environments. Science 2008, 321, 365−367. (3) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad Bugs, No Drugs: No Eskape! An Update from the Infectious Diseases Society of America. Clin. Infect. Dis. 2009, 48, 1−12. (4) Harbottle, H.; Thakur, S.; Zhao, S.; White, D. Genetics of Antimicrobial Resistance. Anim. Biotechnol. 2006, 17, 111−124. (5) Ford, T. E.; Colwell, R. R. Global Decline in Microbiological Safety of Water: A Call for Action; American Academy of Microbiology: Washington, DC, 1996; pp 1−40. (6) Hoque, J.; Akkapeddi, P.; Yarlagadda, V.; Uppu, D. S.; Kumar, P.; Haldar, J. Cleavable Cationic Antibacterial Amphiphiles: Synthesis, Mechanism of Action, and Cytotoxicities. Langmuir 2012, 28, 12225− 12234. (7) Chen, J.; Wang, F.; Liu, Q.; Du, J. Antibacterial Polymeric Nanostructures for Biomedical Applications. Chem. Commun. 2014, 50, 14482−14493. (8) Jain, A.; Duvvuri, L. S.; Farah, S.; Beyth, N.; Domb, A. J.; Khan, W. Antimicrobial Polymers. Adv. Healthcare Mater. 2014, 3, 1969− 1985. (9) Walsh, C. Molecular Mechanisms That Confer Antibacterial Drug Resistance. Nature 2000, 406, 775−781. (10) Liu, L.; Xu, K.; Wang, H.; Tan, P. J.; Fan, W.; Venkatraman, S. S.; Li, L.; Yang, Y.-Y. Self-Assembled Cationic Peptide Nanoparticles as an Efficient Antimicrobial Agent. Nat. Nanotechnol. 2009, 4, 457− 463. (11) Fernandez-Lopez, S.; Kim, H.-S.; Choi, E. C.; Delgado, M.; Granja, J. R.; Khasanov, A.; Kraehenbuehl, K.; Long, G.; Weinberger, D. A.; Wilcoxen, K. M.; Ghadiri, M. R. Antibacterial Agents Based on the Cyclic D, L-A-Peptide Architecture. Nature 2001, 412, 452−455. (12) Oren, Z.; Shai, Y. Cyclization of a Cytolytic Amphipathic AHelical Peptide and Its Diastereomer: Effect on Structure, Interaction with Model Membranes, and Biological Function. Biochemistry 2000, 39, 6103−6114. (13) Qian, L.; Xiao, H.; Zhao, G.; He, B. Synthesis of Modified Guanidine-Based Polymers and Their Antimicrobial Activities Revealed by Afm and Clsm. ACS Appl. Mater. Interfaces 2011, 3, 1895−1901. (14) Yeaman, M. R.; Yount, N. Y. Mechanisms of Antimicrobial Peptide Action and Resistance. Pharmacol. Rev. 2003, 55, 27−55. (15) Kenawy, El-R.; Worley, S. D.; Broughton, R. The Chemistry and Applications of Antimicrobial Polymers: A State-of-the-Art Review. Biomacromolecules 2007, 8, 1359−1384. (16) Brogden, K. A. Antimicrobial Peptides: Pore Formers or Metabolic Inhibitors in Bacteria? Nat. Rev. Microbiol. 2005, 3, 238− 250. 4248


Research Article

ACS Applied Materials & Interfaces Incorporation of Multiple Head Groups Leads to Impressive Antibacterial Activity. J. Med. Chem. 2005, 48, 3823−3831. (36) Zhang, S.; Ding, S.; Yu, J.; Chen, X.; Lei, Q.; Fang, W. Antibacterial Activity, In Vitro Cytotoxicity and Cell Cycle Arrest of Gemini Quaternary Ammonium Surfactants. Langmuir 2015, 31, 12161−12169. (37) Wu, C.; Hou, Y.; Deng, M.; Huang, X.; Yu, D.; Xiang, J.; Liu, Y.; Li, Z.; Wang, Y. Molecular Conformation-Controlled Vesicle/Micelle Transition of Cationic Trimeric Surfactants in Aqueous Solution. Langmuir 2010, 26, 7922−7927. (38) Hou, Y.; Han, Y.; Deng, M.; Xiang, J.; Wang, Y. Aggregation Behavior of a Tetrameric Cationic Surfactant in Aqueous Solution. Langmuir 2010, 26, 28−33. (39) Fan, Y.; Hou, Y.; Xiang, J.; Yu, D.; Wu, C.; Tian, M.; Han, Y.; Wang, Y. Synthesis and Aggregation Behavior of a Hexameric Quaternary Ammonium Surfactant. Langmuir 2011, 27, 10570−10579. (40) Diz, M.; Manresa, A.; Pinazo, A.; Erra, P.; Infante, M. Synthesis, Surface Active Properties and Antimicrobial Activity of New Bis Quaternary Ammonium Compounds. J. Chem. Soc., Perkin Trans. 2 1994, 1871−1876. (41) Xing, C.; Xu, Q.; Tang, H.; Liu, L.; Wang, S. Conjugated Polymer/Porphyrin Complexes for Efficient Energy Transfer and Improving Light-Activated Antibacterial Activity. J. Am. Chem. Soc. 2009, 131, 13117−13124. (42) Chen, S.; Chen, S.; Jiang, S.; Xiong, M.; Luo, J.; Tang, J.; Ge, Z. Environmentally Friendly Antibacterial Cotton Textiles Finished with Siloxane Sulfopropylbetaine. ACS Appl. Mater. Interfaces 2011, 3, 1154−1162. (43) Freire, E.; Mayorga, O. L.; Straume, M. Isothermal Titration Calorimetry. Anal. Chem. 1990, 62, 950A−959A. (44) Freire, E.; Schön, A.; Velazquez-Campoy, A. Isothermal Titration Calorimetry: General Formalism Using Binding Polynomials. Methods Enzymol. 2009, 455, 127−155. (45) Yuan, H.; Liu, Z.; Liu, L.; Lv, F.; Wang, Y.; Wang, S. Cationic Conjugated Polymers for Discrimination of Microbial Pathogens. Adv. Mater. 2014, 26, 4333−4338. (46) Asensi, G. F.; dos Reis, E. M. F.; Del Aguila, E. M.; dos P. Rodrigues, D.; Silva, J. T.; Paschoalin, V. M. F. V. Detection of Escherichia Coli and Salmonella in Chicken Rinse Carcasses. Br. Food J. 2009, 111, 517−527. (47) LaDow, J. E.; Warnock, D. C.; Hamill, K. M.; Simmons, K. L.; Davis, R. W.; Schwantes, C. R.; Flaherty, D. C.; Willcox, J. A.; WilsonHenjum, K.; Caran, K. L.; Minbiole, K. P. C.; Seifert, K. Bicephalic Amphiphile Architecture Affects Antibacterial Activity. Eur. J. Med. Chem. 2011, 46, 4219−4226. (48) Wang, Y.; Corbitt, T. S.; Jett, S. D.; Tang, Y.; Schanze, K. S.; Chi, E. Y.; Whitten, D. G. Direct Visualization of Bactericidal Action of Cationic Conjugated Polyelectrolytes and Oligomers. Langmuir 2012, 28, 65−70. (49) Timofeeva, L.; Kleshcheva, N. Antimicrobial Polymers: Mechanism of Action, Factors of Activity, and Applications. Appl. Microbiol. Biotechnol. 2011, 89, 475−492. (50) Helander, I.; Nurmiaho-Lassila, E.-L.; Ahvenainen, R.; Rhoades, J.; Roller, S. Roller, S. Chitosan Disrupts the Barrier Properties of the Outer Membrane of Gram-Negative Bacteria. Int. J. Food Microbiol. 2001, 71, 235−244. (51) Som, A.; Tew, G. N. Influence of Lipid Composition on Membrane Activity of Antimicrobial Phenylene Ethynylene Oligomers. J. Phys. Chem. B 2008, 112, 3495−3502.

4249
