Tuning Antibacterial Activity of Cyclodextrin-Attached Cationic

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Tuning Antibacterial Activity of Cyclodextrin-Attached Cationic Ammonium Surfactants by A Supramolecular Approach Chengcheng Zhou, Hua Wang, Haotian Bai, Pengbo Zhang, Libing Liu, Shu Wang, and Yilin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11528 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on August 31, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

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

Tuning

ACS Applied Materials & Interfaces

Antibacterial

Cationic

Ammonium

Activity

of

Surfactants

Cyclodextrin-Attached by

A

Supramolecular

Approach Chengcheng Zhou,† Hua Wang,† Haotian Bai,‡ Pengbo Zhang,‡ Libing Liu,‡ Shu Wang,‡, §,* and Yilin Wang†,§,* †

Key Laboratory of Colloid and Interface Science, Beijing National Laboratory for Molecular Sciences (BNLMS), CAS

Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡

Key Laboratory of Organic Solids, Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Research/Education

Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China; §

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

KEYWORDS: cyclodextrin-based cationic surfactant, aggregates, host-guest interaction, antimicrobial activity, cytotoxicity

ABSTRACT: Two β-Cyclodextrin-attached cationic ammonium surfactants bearing a dodecyl chain (APDB) and a hexadecyl chain (APCB) were synthesized to reduce the cytotoxicity of cationic surfactants to mammalian cells and endow the surfactants with host-guest recognition sites, and three kinds of guest molecules were utilized to improve the antibacterial ability of APDB and APCB via host-guest interaction by regulating the electrostatic or hydrophobic interaction of APDB or APCB with 1

ACS Paragon Plus Environment


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

Page 2 of 31

bacteria. The guest molecules include AD-NH3+ carrying one positive charge, DB with a benzene ring group and a dodecyl chain, and single chain cationic ammonium surfactant DTAB or CTAB. Either AD-NH3+ or DB increases the killing efficacy of APCB against S. aureus at 50 µM from 59% to about 75%, while DTAB or CTAB improves the killing efficacy of APCB to more than 90%. In particular, only a very small amount CTAB can improve the antibacterial activity of APCB to a very high level, but keeps very low cytotoxicity. However the mixtures of the guest molecules with APDB are devoid of any activity against S. aureus. This is mainly attributed that APCB and its mixtures with the guest molecules form 100–200 nm spherical aggregates, while the mixtures of APDB with the guest molecules cannot form aggregates at lower concentration. It is revealed that the three kinds of guest molecules trapped in the APCB spherical aggregates lead to diverse interaction modes of the APCB spherical aggregates with S. aureus, accounting for the different killing efficacy of the APCB/guest molecule mixtures. This supramolecular strategy provides an effective approach for the construction of highly efficient antibacterial agents with low cytotoxicity. INTRODUCTION Cationic surfactants with cationic and amphiphilic features are widely used as antibacterial agents in various fields, such as food industry and hospital.1-3 However, the widespread use of cationic surfactants has caused their build-up in the environment, hence produces selective pressure on bacteria and triggers the emergence of bacterial resistance.1,3-5 In order to reduce the exposure of bacteria to cationic surfactants, much work has been devoted to designing various cationic surfactants so that the desired highly efficient antimicrobial activity can be realized at lower doses.2,6-10 Considering that cationic 2


Page 3 of 31

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


surfactants show antibacterial activity mainly by targeting and disintegrating bacterial cell membranes via electrostatic and hydrophobic interactions,2,7,9,11 it has been a common strategy to increase the cationic charge density and hydrophobicity of cationic surfactants by covalent linkage. However, this strategy requires complicated molecular synthesis. Moreover, the antibacterial activity and cytotoxicity of synthesized cationic surfactants are difficult to be predicted,8,12-13 and consequently they are often limited in invalid synthesis and strong cytotoxicity. Recently, supramolecular strategy has been proposed to fabricate antibacterial agents due to its simple and modular characteristic.14-20 For example, our previous work achieved mild and highly efficient antimicrobial agents by noncovalently incorporating cyclodextrins into cationic ammonium trimeric surfactant.16 Cyclodextrins (CDs) are a well-known class of donut-shape macrocyclic hosts with a hydrophilic exterior and a hydrophobic cavity.21-24 This feature endows CDs with the ability to encapsulate a large range of hydrophobic guest molecules, and thus control the properties of surfactants by a simple host-guest approach.25-27 Moreover, it has been found that the incorporation of CD is favorable for improving biocompatibility of surfactants.16,28-30 Thus, in this work, we attempt to covalently attach β-CD to cationic surfactants so as to improve the biocompatibility of cationic surfactants and endow them with host-guest recognition sites,29 and then tune the antibacterial activity of β-CD-attached cationic surfactants on demand by simply adding appropriate guest molecules. It is anticipated to achieve highly efficient antibacterial agents with low cytotoxicity. For this purpose, two β-CD-attached cationic ammonium surfactants separately bearing a dodecyl chain (APDB) and a hexadecyl chain (APCB) were synthesized (Scheme 1). Three kinds of model guest molecules (Scheme 2), including AD-NH3+ carrying one positive charge, DB carrying a 3



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

Page 4 of 31

benzene ring group and a dodecyl chain, and single chain cationic ammonium surfactant DTAB or CTAB, were chosen to adjust the number of cationic charges and alkyl chains of APDB or APCB. The inclusion of the guest molecules by APDB and APCB via the host-guest interaction regulates the electrostatic or hydrophobic interaction of APDB or APCB with bacteria, and thus controls the antibacterial potency of APDB and APCB.

Scheme

1.

Synthesis

procedure

of

β-cyclodextrin-based

quaternary

ammonium

surfactant

β-CD-N-APDB (abbreviated as APDB) and β-CD-N-APCB (abbreviated as APCB).

AD-NH3+ DB DTAB CTAB

Scheme 2. Chemical structures of the chosen guest molecules.

4


Page 5 of 31

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


EXPERIMENTAL SECTION Materials. Mono-(6-amino-6-deoxy)-β-cyclodextrin (≥ 98%) was purchased from Shandong Binzhou Zhiyuan Bio-Technology Co. Methyl acrylate (AR) and all organic solvents were obtained from Beijing Chemical Co. 3-(Dimethylamino)-1-propylamine (99%) and dodecyl bromide (98%) were purchased from Alfa Aesar. Hexadecyl bromide (98%), superdry N,N-dimethylformamide (99.8%) and 1-adamantanamine hydrochloride (99%) were obtained from J&K Scientific Ltd. Dodecylbenzene (> 98%)

was

purchased

from

TCI.

Dodecyltrimethylammonium

bromide

(99%)

and

hexadecyltrimethylammonium bromide (≥ 99%) were obtained from Sigma-Aldrich Co. S. aureus (ATCC6538) was purchased from Beijing Bio-Med Technology Development Co., Ltd. HaCaT cells were obtained from Center for Cell, Institute of Basic Medical Science, Chinese Academy of Sciences. Ultrapure water and phosphate buffered saline (1× PBS, pH 7.4) were used during the experiments. Synthsis.

Mono-6(N-(3-(3-aminopropanamido)propyl)-N,N-dimethyldodecan-1-aminium

bromide)-6-deoxy-β-cyclodextrin

(β-CD-N-APDB,

abbreviated as

APDB)

and

Mono-6(N-(3-(3-aminopropanamido)propyl)-N,N-dimethylhexadecan-1-aminium bromide)-6-deoxy-β-cyclodextrin (β-CD-N-APCB, abbreviated as APCB) were synthesized according to Scheme 1 and characterized by 1H NMR, MS and elemental analysis. Mono-6(methyl 3-aminopropanoate)-6-deoxy-β-cyclodextrin in Step 1. The solution of mono-(6-amino-6-deoxy)-β-cyclodextrin (0.500 g, 0.440 mmol) and methyl acrylate (0.125 g, 1.45 mmol) in 14 mL superdry DMF/methanol with v/v ratio of 4 : 3 was stirred at room temperature for 24 h. Then DMF/methanol and excessive methyl acrylate were removed under vacuum, and a bright brown substance was obtained with almost 100% yield. 1H NMR (D2O, 400 MHz): δ 2.55 (2H, t, COCH2), 2.86 (2H, t, NHCH2), 3.05 (1H, m, NHCH2CD), 3.37 (1H, m, NHCH2CD), 3.65 (3H, s, OCH3), 3.51–3.59 (14H, m, H2, H4 of CD), 3.80–3.88 (26H, m, H3, H5, H6 of CD), 5.00 (7H, s, H1 of CD). MALDI-TOF 5



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

Page 6 of 31

(m/z): 1220.6 (M+H), 1242.7 (M+Na). Mono-6(3-amino-N-(3-(dimethylamino)propyl)propanamide)-6-deoxy-β-cyclodextrin in Step 2. The above product of step 1 (0.538 g, 0.441 mmol) in 6 mL 3-(dimethylamino)-1-propylamine was stirred overnight at 80.0 °C. Excessive 3-(dimethylamino)-1-propylamine was removed under vacuo. The obtained residue was repeatedly dissolved by a little water, and then excess ethanol was added to precipitate a light brown powder, which was dried to obtain the desired compound with almost 66.1% yield. 1H NMR (D2O, 400 MHz): δ 1.10 (5H, m, CH2, NCH3), 1.84 (2H, m, NCH2), 2.53 (2H, m, NHCH2CD), 2.81 (3H, s, NCH3), 3.07 (2H, t, COCH2), 3.22 (2H, t, NHCH2), 3.51–3.57 (18H, m, H2, H4 of CD, CONHCH2), 3.81–3.89 (24H, m,H3, H5, H6 of CD), 5.00 (7H, s, H1 of CD). MALDI-TOF (m/z): 1290.5 (M+H), 1312.9 (M+Na). APDB and APCB. The solution of dodecyl bromide (0.068 g, 0.27 mmol) or hexadecyl bromide (0.082 g, 0.27 mmol) and the product of step 2 (0.107, 0.08 mmol) in dry DMF (5 mL) was stirred for 36 h at 45 °C. Solvent was removed in vacuo, and the crude product was repeatedly dissolved by a little water, and then excess acetone was added to precipitate a light brown powder, which was dried to obtain the desired compound in almost 65.0% yield. For APDB, 1H NMR (DMSO-d6, 400 MHz): δ 0.88 (3H, m, CH3), 1.25 (16H, m, (CH2)8), 1.50 (2H, m, CH2), 1.63 (2H, m, CH2), 1.79 (1H, br, NH), 2.00 (2H, m, CH2), 2.09 (3H, s, CH3N+), 2.16 (3H, s, CH3N+), 2.23 (2H, m, COCH2), 2.68–2.74 (4H, m, NHCH2CD, NHCH2), 2.98 (2H, m,CH2N+), 3.33–3.57 (44H, m, H3, H5, H6, H2, H4 of CD, CH2N+, CONHCH2), 4.48 (6H, br, 6-OH of CD), 4.84 (7H, d, H1 of CD), 5.71 (14H, br, 2-OH, 3-OH of CD), 8.00 (1H, br, CONH); MALDI-TOF (m/z): 1458.3; elemental analysis calcd (%) for C62H112BrN3O35·9H2O: C, 43.76; H, 7.70; N, 2.47; Found: C, 43.66; H, 7.03; N, 2.44. For APCB, 1H NMR (DMSO-d6, 400 MHz): δ 0.86 (3H, t, CH3), 1.24 (26H, m, (CH2)13), 1.64 (2H, m, CH2), 1.79 (1H, br, NH), 1.99 (2H, m, CH2), 2.33 (2H, m, COCH2), 2.98 (3H, s, CH3N+), 3.10 (2H, m, NHCH2CD), 3.22 (2H, m, NHCH2), 3.33 (27H, m, H2, H4, 6


Page 7 of 31

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


H6 of CD, 2CH2N+, CH3N+), 3.64 (22H, m, H3, H5, H6 of CD, CONHCH2), 4.48 (6H, br, 6-OH of CD), 4.84 (7H, d, H1 of CD), 5.68 (14H, br, 2-OH, 3-OH of CD), 8.14 (1H, br, CONH); MALDI-TOF (m/z): 1514.2; elemental analysiscalcd (%) for C66H120BrN3O35·8H2O: C, 45.57; H, 7.88; N, 2.42; Found: C, 45.69; H, 7.46; N, 2.34. Preparation of S. aureus Solutions. A single colony of S. aureus on a solid NB agar plate was transferred into 10 mL liquid NB medium and was grown for about 8 h at 37 °C under the shaking of 180 rpm. To remove the NB medium, S. aureus was harvested by centrifuging (7100 rpm for 2 min) and was washed with PBS for two times. After removing the supernatant, the remained S. aureus was suspended with PBS, and then diluted to the optical density of 1.0 at 600 nm (OD600 = 1.0). There was about 5 × 108 CFU/mL S. aureus in the solution with OD600 = 1.0. Cytotoxicity Assay. HaCaT cells at a density of 6 × 103 cells/well were seeded into 96-well culture plates and were grown for 12–24 h until adherence in a humified atmosphere containing 5% CO2 and at 37 °C. APDB, APCB and APCB/guest molecule mixtures with a series of concentrations were added into 96-well culture plates, respectively. After incubated for 24 h at 37 °C, the supernatant was removed and 100 µL 0.5 mg/mL MTT was added, and then incubated for 4 h at 37 °C. Subsequently, after removing the supernatant, 100 µL DMSO was added into well to dissolve the produced formazan. After shaking the plates for 2 min, absorbance values per wells at 520 nm were read by a microplate reader. The cell viability rate (VR) was calculated by the equation of VR = A/A0 × 100% (A is the absorbance of the experimental groups, and A0 is the absorbance of the control group.). Each assay was repeated for six times. Assessment of Antibacterial Activity. The antimicrobial activity of APDB, APCB, APDB/guest molecule mixtures and APCB/guest molecule mixtures against S. aureus was evaluated by surface plating method. Certain concentrations of APDB, APCB, APDB/guest molecule mixtures or APCB/guest 7



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

Page 8 of 31

molecule mixtures were separately added into S. aureus PBS solution (~ 108 CFU/mL). After incubated at 37 °C for 30 min, the S. aureus suspensions were serially diluted by 104-fold with PBS. Then 100 µL diluted S. aureus was spread on the solid NB agar plate, and incubated at 37 °C for 14–16 h. All the experiments were performed in triplicate. The activity of APDB, APCB, APDB/guest molecule mixtures or APCB/guest molecule mixtures against S. aureus was assessed by [(A − B)/A] × 100%, where A is the mean number of S. aureus colonies in the control sample without surfactant or surfactant/guest molecule mixture, and B is the mean number of S. aureus with surfactant or surfactant/guest molecule mixture. The results were repeated three times. Surface Tension Measurements. Surface tension values of APDB in PBS and APCB in water were measured by the drop volume method at 25.00 ± 0.01 °C. Every drop was kept for about half an hour, and each surface tension value (γ) was determined from at least five consistent measured values. The surface tension measurements of APCB in PBS were conducted by using a Pt/Ir plate method on a DCAT21 tensiometer (Dataphysics Co., Germany). Each surface tension curve was repeated at least twice. Dynamic Light Scattering (DLS). The size distributions of APCB, APDB/guest molecule mixtures and APCB/guest molecule mixtures were measured at 25 °C on an LLS spectrometer (Model ALV/SP-125), which was equipped with a 22 mW He-Ne laser (λ = 632.8 nm). The scattering angle was set to 90°. The samples were not filtered before measurements. ζ-Potential Measurements. The surface charge property of the aggregates of APCB with and without the guest molecules in PBS was studied by ζ-potential measurement on Nano ZS (Malvern Instruments) equipped with a 4 mW He-Ne laser (λ = 632.8 nm) and a thermostatted chamber at 25 °C. Cryogenic Transmission Electron Microscopy (Cryo-TEM). APCB and the APCB/guest molecule mixtures were dropped on freshly carbon-coated holey TEM grids, followed by blotting the grids with 8


Page 9 of 31

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


filter paper. Then the TEM grids were plugged into liquid nitrogen to make the samples embedded in a thin layer of vitreous ice. Frozen hydrated specimens were observed by an FEI Tecnai 20 electron microscope (LaB6) operated at 120 kV in low-dose mode. Scanning Electron Microscopy (SEM). After the treatment described in the antibacterial experiments above, S. aureus was centrifuged (7100 rmp for 5 min) at 4 °C. After removing the supernatant, S. aureus pellets were resuspended with sterile water. 5 µL S. aureus suspensions were dropped on clean silicon slices followed by naturally drying in the super clean bench, and then was fixed with 0.1% glutaraldehyde for overnight. Next, the specimens were washed with sterile water for three times, and dehydrated by adding different gradient ethanol (40%, 70%, 90%, and 100%, each for 6 min). Finally, the specimens were dried in vacuum drying oven and coated with platinum before SEM observation (Hitachi S4800, Japan). Isothermal Titration Microcalorimetry (ITC). The calorimetric measurements were conducted on a TAM III calorimeter (TA Instruments, USA) at 25.00 ± 0.01 °C. To monitor the binding processes of the guest molecules with APDB or APCB, the sample cells were initially loaded with 600 µL 500 µM APDB or APCB solution or water, and then a certain concentration of guest molecule solution was injected consecutively into the stirred sample cell in portions of 10 µL via a 500 µL Hamilton syringe until the interaction process was completed. The system was stirred at 90 rpm with a gold propeller. While studying the interaction of APCB or APCB/guest molecule mixtures with S. aureus, the sample cell was initially loaded with 750 µL S. aureus with OD or PBS. Then 200 µM APCB or APCB/guest molecule mixtures were injected consecutively into the stirred sample cell in each portion of 10 µL until the end of the interaction. Each ITC curve was repeated at least twice with a deviation of ± 4%.

RESULTS AND DISCUSSION 9



Cytotoxicity and Antibacterial Activity of APDB and APCB. The toxicity of APDB and APCB toward mammalian cells was evaluated by MTT assay. As shown in Figure 1a, APDB and APCB do not exhibit obvious cytotoxicity to HaCaT cells even when their concentrations are as high as 128 µM. Obviously, APDB and APCB have much lower toxicity to mammalian cells compared to their corresponding cationic ammonium surfactants DTAB and CTAB, which show an IC50 value of about 23 µM and 5.2 µM, respectively. Gram-positive S. aureus was selected as a representative microbe for estimating the antibacterial activity of APDB and APCB. It can be found that APDB bearing the dodecyl chain is devoid of any activity even at the high concentration of 100 µM, while APCB with the hexadecyl chain is moderately active against S. aureus, i.e. the killing efficiency is 59% at the concentration of 50 µM, and the efficiency is improved to 74% with increasing the concentration to 100 µM (Figure 1b).

b

120 100 80 APDB APCB DTAB CTAB

60 40 20 0 0

20

40

60

80

100

100

CFU reduction%

a Cell Viability (%)

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

Page 10 of 31

120

80

APDB APCB

60

40

20

0 25

50

75

100

CSurfactant (µM)

CSurfactant (µM)

Figure 1. (a) Viability of HaCaT cells after incubation with APDB and APCB, and (b) antibacterial activity of APDB and APCB to Gram-positive S. aureus at different concentrations (Csurfactant).

The self-assembling ability of cationic surfactants is responsible for their antibacterial activity.9 Thus, 10


Page 11 of 31

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


to understand the obvious discrepancy in the antibacterial activity of APDB and APCB, surface tension measurements were conducted to determine their critical aggregation concentrations (CACs). As shown in Figure 2a, APDB has a higher CAC value of 1670 µM in PBS, while APCB shows much stronger self-assembly ability with two CAC values at 180 and 1060 µM in water, and the CAC value of APCB is further decreased to 4.8 µM in PBS. Therefore, at the concentrations (50 and 100 µM) used for evaluating the antibacterial activity, APDB exists as monomers, which are not potent enough to kill S. aureus. As to APCB, the CAC of APCB is very low. The reduction of CAC value of APCB in buffer is caused by the salts in the buffer by screening the electrostatic repulsion between the quaternary ammonium head groups. DLS results and TEM images (Figure 2b-d) confirm that APCB forms spherical aggregates with diameters of 100–200 nm in water and PBS. The formation of self-assemblies increases the local APCB concentration, leading to the killing efficacy to S. aureus. Thus, the formation of aggregates is a necessary condition for the antimicrobial activity of these β-CD-attached cationic ammonium surfactants. However, although the CAC of APCB is very low, its bacterial killing efficiency is still very weak. Normally, the bacterial killing efficiency of less than 60% against Gram-positive bacteria at 50 µM is considered very weak. When the surfactant aggregates kill bacteria, two equilibria coexist, i.e., disaggregation equilibrium between surfactant aggregates and surfactant monomers, and aggregation equilibrium between surfactant molecules and bacterial phospholipid membrane. Therefore, when the stable APCB spherical aggregates are formed, the disaggregation of the stable spherical aggregates is a slower thermodynamic process, which may reverse the killing efficacy of the surfactant (APCB). So despite displaying strong self-assembly ability, APCB shows weaker antibacterial activity against S. aureus. In the following text, guest molecules will be applied to improve the antibacterial activity of APDB and APCB.

11



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

Page 12 of 31

Figure 2. (a) Variations of surface tension of APDB in PBS and APCB in H2O and PBS at 25.00 ± 0.01 °C, (b) the size distribution of APCB in H2O (CAPCB = 500 µM) and PBS (CAPCB = 50 µM), (c) and (d) cryo-TEM of APCB in H2O (CAPCB = 500 µM) and PBS (CAPCB = 50 µM), respectively. Effects of Guest Molecules on the Antibacterial Activity of APDB and APCB. Normally, cationic surfactants show antibacterial activity by electrostatic targeting to cell membrane and then inserting into the cell membrane to cause the cell lysis by hydrophobic interaction.2 Based on this understanding, three kinds of model guest molecules including AD-NH3+ carrying one positive charge, DB carrying a benzene ring group and a dodecyl chain, and single chain cationic ammonium surfactant DTAB or CTAB are chosen to improve the antibacterial activity of APDB and APCB. Firstly, ITC was employed to investigate the binding process of these guest molecules with APDB and APCB. Figure 3 shows the variation of the observed enthalpy (∆Hobs) against guest/APDB or 12


Page 13 of 31

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


guest/APCB molar ratio when the solutions of the guest molecules were separately titrated into 500 µM APDB or APCB solution. The ITC curves are approximately sigmoidal in shape, and the ∆Hobs values change gradually from exothermic value to zero, reaching the saturation of interaction of the guest molecules with APDB or APCB. These ITC curves are analyzed by the model of single set of binding sites described in Supporting Information. The obtained binding parameters of the guest molecules with APDB and APCB are listed in Table 1. As shown, the binding constants Kb of these guest molecules with APDB and APCB indicate that the binding modes are typical for the complexation with β-CD, but the complexation ability is different for the guest molecules. APDB exists as monomers at 500 µM, and thus the CD cavity of APDB is completely exposed. The Kb values suggest that AD-NH3+ exhibits stronger complexation ability with APDB than DB and DTAB. This is attributed that the size of hydrophobic group of AD-NH3+ more closely matches the β-CD cavity than DB and DTAB. However, the binding number of all the guest molecules with APDB (N) is close to 1, indicating that they all form 1 : 1 complexes with APDB. As to the APCB spherical aggregates, AD-NH3+ exhibits weaker binding ability with APCB aggregates due to the steric hindrance at the aggregate surface, while DB, DTAB and CTAB show stronger binding ability with the APCB aggregates because of the hydrophobic interaction between the alkyl chain of the guest molecules and the hydrophobic domains of the APCB aggregates. Compared to the APDB monomers, the available CD cavities on the APCB spherical aggregates are less since there is steric hindrance in the aggregates and partial APCB molecules are packed in the interior of the aggregates. Therefore, the binding ratio of the APCB aggregates with the guest molecules is about 0.5, as indicated by their binding numbers (N).

13



b

a

2

0 0

∆ Hobs (kJ/mol)

∆Hobs (kJ/mol)

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

Page 14 of 31

-3 +

AD-NH3/APDB

-6

DB/APDB DTAB/APDB

-9

-12 0.0

0.5

1.0

1.5

-2 +

AD-NH3/APCB DB/APCB DTAB/APCB CTAB/APCB

-4

-6 0.0

2.0

0.3

nguest/nAPDB

0.6

0.9

1.2

1.5

nguest/nAPCB

Figure 3. (a) The variation of observed enthalpy changes (∆Hobs) against the guest/APDB molar ratio by titrating the aqueous solutions of 1800 µM AD-NH3+, DB (1.8% DMSO) and DTAB into 500 µM APDB solution, respectively. (b) The variation of ∆Hobs against the guest/APCB molar ratio by titrating the aqueous solutions of 1800 µM AD-NH3+ and DTAB, 1000 µM DB (1.0% DMSO) and 800 µM CTAB into 500 µM APCB solution, respectively. The dilution enthalpy of the guest molecule solution has been deducted. Table 1. The Binding Constant (Kb) and Binding Number (N) of the Guest Molecules with APDB or APCB Derived from the ITC Curves in Figure 3. Guest/Host Complexes

N

Kb ( × 104 M-1)

AD-NH3+/APDB

0.94

4.71

DB/APDB

0.77

0.83

DTAB/APDB

0.80

1.89

AD-NH3 /APCB

0.43

0.84

DB/APCB

0.41

5.4

DTAB/APCB

0.59

1.56

CTAB/APCB

0.43

7.54

+

Next, the antibacterial activity of the complexes of the guest molecules with APDB or APCB was explored. To ensure the concentration and molar ratio of the guest molecules consistency in APDB and 14


Page 15 of 31

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


APCB, the mixing molar ratio of APDB or APCB with the guest molecules is kept 1 : 1. But for CTAB, due to its strong antibacterial activity, the host-guest mixing ratio is kept at 1 : 0.1 and 1: 0.01 in order to avoid the superposition effect of the antibacterial activity of CTAB itself. Figure 4a shows that the guest molecules (AD-NH3+, DB, DTAB and CTAB) themselves have no obvious influence on the growth of S. aureus, and the guest molecules do not change the antibacterial ability of APDB against S. aureus, i.e., the APDB/guest complexes still exhibit very low antibacterial activity. This is attributed that the APDB/guest complexes still cannot form self-assemblies at the concentrations used, where no reliable scatting intensity can be found in the DLS results. However, Figure 4b indicates that all the guest molecules can improve the antibacterial activity of APCB against S. aureus, but to different extents. Either AD-NH3+ or DB improves the killing efficacy of APCB at 50 µM from 59% to 75%. Particularly, DTAB or CTAB improves the killing efficacy of APCB to more than 90%. Even only adding 0.5 µM CTAB into 50 µM APCB, i.e. the APCB/0.01CTAB mixture, the killing efficacy of the APCB aggregates is improved from 59% to 80%. In addition, APCB and all the APCB/guest complexes are active against S. aureus by destroying the bacterial cell membrane, as shown in Figure 5. For S. aureus without any treatments (Figure 5a), the SEM image shows intact and smooth bacterial cells with clear edges. In contrast, the structures of S. aureus treated with APCB or the APCB/guest mixtures (Figure 5b-f) are collapsed and merged, and the membranes of some S. aureus are disintegrated. The mechanism about the effects of the guest molecules on the antibacterial activity of APCB will be further investigated as follows from the aspects of their effects on the self-assemblies of APCB and the interactions of APCB and APCB/guest mixtures with S. aureus.

15



100 100

a 80

CFU reduction%

CFU reduction%

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

Page 16 of 31

60

40

20

APDB APDB + /AD-NH3 /DB APDB + /DTAB AD-NH3 DB APDB

0

DTAB

b

APCB /DTAB APCB +

80

/AD-NH3

APCB /DB

APCB /0.1CTAB APCB /0.01CTAB

APCB

60 40 20

CTAB

0

Figure 4. (a) Antibacterial activity to S. aureus for APDB, APDB/AD-NH3+, APCB/DB and APDB/DTAB at the host-guest mixing molar ratio of 1 : 1 as well as the antibacterial activity of the guest molecules. CAPDB = 50 µM, CAD-NH3+ = 50 µM, CDB = 50 µM, CDTAB = 50 µM and CCTAB = 5 µM. (b) Antibacterial activity to S. aureus for APCB, APCB/AD-NH3+, APCB/DB and APCB/DTAB with the host-guest mixing molar ratio of 1 : 1, and the antibacterial activity of APCB/0.01CTAB and APCB/0.1CTAB with the host-guest mixing molar ratio of 1 : 0.01 and 1 : 0.1. CAPCB = 50 µM.

Figure 5. SEM images of S. aureus before (a) and after incubation with APCB (b), APCB/DTAB (c), APCB/0.1CTAB (d), APCB/AD-NH3+ (e), and APCB/DB (f) at CAPCB = 50 µM. Arrows indicate lesions and collapses of bacterial membrane. 16


Page 17 of 31

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


Roles of Guest Molecules in the Antibacterial Activity of APCB. To elucidate the role of the guest molecules in the killing efficacy of APCB to S. aureus, the size, ζ potential and morphology of the APCB self-assemblies after adding the guest molecules have been studied. Figure 6a and 6b show that all the guest molecules, AD-NH3+, DB, DTAB and CTAB, have no obvious effect on the size and morphology of the APCB spherical aggregates. For other CD-based amphiphiles, the complexation of guest molecules also did not disrupt the self-assembling structures.19,31 However, the ζ potential values of the APCB spherical aggregates in PBS before and after adding the guest molecules are different (Figure 6c). Adding AD-NH3+, DTAB or CTAB enhances the positive potential of the APCB aggregates, while DB almost does not change the ζ potential of the APCB aggregates. Furthermore, ITC was employed to investigate the binding process between S. aureus and APCB before and after adding the guest molecules. Figure 7 presents the variation of observed enthalpy (∆Hobs) while separately titrating the solutions of APCB and the APCB/guest mixtures into S. aureus solution. In the case of APCB itself, the ∆Hobs curve shows a sigmoidal shape. With the addition of APCB, the ∆Hobs value is initially exothermic and displays a platform, then the value becomes close to zero, suggesting the binding with S. aureus reaches the saturation. After incorporating the guest molecules, the ∆Hobs curves of the APCB/guest molecules with S. aureus exhibit different situations for different guest molecules. Upon adding AD-NH3+ or DB, the ∆Hobs curves are similar to that of APCB, and only a slight difference appears in the descending extent of the ∆Hobs value. However, very interestingly, DTAB and CTAB cause significant differences in the ∆Hobs curves. After incorporating DTAB into APCB, with the addition of APCB/DTAB spherical aggregates into S. aureus, the ∆Hobs value is initially larger exothermic, then gradually decreases and reaches a plateau. With further adding the APCB/DTAB mixture, ∆Hobs continuously becomes smaller and finally returns to zero. The variation situation of the 17



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

Page 18 of 31

∆Hobs curve indicates that the interaction of the APCB/DTAB complex with S. aureus involves two processes, which differs from that of APCB with S. aureus distinctly. When changing the guest molecule DTAB into CTAB, the ∆Hobs curve of the APCB/CTAB with S. aureus exhibits a different situation. With adding the APCB/0.1CTAB mixture into S. aureus, ∆Hobs increases sharply from a less exothermic value to a maximum exothermic value, and then becomes smaller and finally close to zero. But similar to the situation in the presence of DTAB, the ∆Hobs curve of APCB/0.1CTAB with S. aureus also shows two interaction processes. These ITC curves (Figure 7) were separately analyzed by the models of single binding-site set and two binding-site sets described in Supporting Information. The derived binding numbers (N) and stepwise binding numbers (N1 and N2) of APCB, APCB/guest mixtures, DTAB and CTAB with per S. aureus as well as the corresponding binding constants (K, K1 and K2) are listed in Table 2. Obviously, adding the three kinds of guest molecules causes diverse interaction modes between the APCB spherical aggregates and S. aureus. The interaction modes of the guest molecules with APCB spherical aggregates and their interaction mechanism with S. aureus are proposed in Scheme 3, which possibly accounts for the different killing efficacy of the APCB/guest molecule mixtures. As shown in Table 2, the binding constant values of both APCB/AD-NH3+ and APCB/DB with S. aureus are five times larger than that of APCB. This suggests that AD-NH3+ and DB obviously enhance the interaction of the APCB spherical aggregates with S. aureus, which is responsible for the improvement of the killing efficacy APCB at 50 µM from 59% to about 75%. However, AD-NH3+ and DB play different roles in improving the antibacterial activity of APCB. Cationic charged AD-NH3+ increases the positive zeta potential of the APCB aggregates, that is to say, the trapped AD-NH3+ by the CD cavity of APCB endows the APCB aggregates with more positive charges, which improves the electrostatic targeting of the APCB/AD-NH3+ aggregates at the negatively charged surface of S. aureus (the first line in Scheme 18


Page 19 of 31

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


3). As to DB, the benzene ring of DB complexes with the CD of the APCB aggregates, similar to the inclusion behavior of DB with CD reported previously,32 while its dodecyl chain associates with the hydrocarbon chain of APCB. However, the complexation of DB with APCB does not change the size, morphology and ζ potential of the APCB spherical aggregates. Therefore, DB enhances the ability of APCB to insert into and disintegrate the cell membrane of S. aureus by increasing the amount of the hydrophobic chains in the mixed aggregates (the second line in Scheme 3). In particular, upon incorporating DTAB or CTAB, the killing efficacy of the APCB aggregates is greatly improved to more than 90%. On one hand, when the alkyl chain of DTAB or CTAB is encapsulated into the CD cavity of APCB, the cationic ammonium headgroups exposed at the outer surface of the APCB spherical aggregates add more positive charges at the aggregate surface, as proved by the increased positive zeta potential. Thus DTAB or CTAB improves the targeting ability of the APCB aggregates to the negatively charged surface of S. aureus. On another hand, given that the binding constants of DTAB and CTAB with S. aureus are almost one order of magnitude larger than that with the APCB spherical aggregate (Table 1 and Table 2), DTAB and CTAB will be released from the CD cavities of the APCB aggregates due to the competitive interaction of the surfactants with S. aureus after the APCB/DTAB or APCB/0.1CTAB spherical aggregates targeting at the S. aureus surface. Consequently, the released DTAB or CTAB acts synergistically with APCB in interacting with bacteria, which further enhances the antibacterial activity of APCB. So the interactions of the APCB/DTAB or APCB/0.1CTAB spherical aggregates with S. aureus reflected in the enthalpy curves (Figure 7) are supposed to follow the two processes (the last line in Scheme 3): (1) the positively charged APCB/DTAB or APCB/0.1CTAB aggregates electrostatically target to the negatively charged surface of S. aureus followed by the process of the DTAB or CTAB molecules being released from the CD cavities of APCB spherical aggregates; (2) the released DTAB or CTAB molecules cooperatively kill S. aureus 19



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

Page 20 of 31

with APCB. Because the binding constant value of CTAB with S. aureus is much larger than that of DTAB (Table 2), CTAB shows much stronger interaction with S. aureus than DTAB, leading to the larger exothermic enthalpy change of APCB/0.1CTAB with S. aureus during the first step. Moreover, the binding constant K2 of APCB/DTAB or APCB/0.1CTAB with S. aureus for the second step is much larger than the binding constants of APCB, APCB/AD-NH3+ and APCB/DB with S. aureus, which accounts for much stronger killing efficacy of APCB/DTAB and APCB/0.1CTAB to S. aureus. From the aspect of cationic surfactants, during killing bacteria, they mainly undergo the electrostatic binding of cationic surfactants to the surface of bacterial cell membrane, accumulation of cationic surfactants on the surface of the cell membrane to reaching effective concentration, and the insertion of cationic surfactants into cell membrane to induce cell lysis. Without APCB, DTAB or CTAB molecules themselves need a larger concentration to meet the required accumulation on the surface of bacteria. The APCB spherical aggregates serve as a carrier to enrich DTAB or CTAB molecule on the bacteria surface through the host-guest interaction, which avoids the slow accumulation process of individual DTAB or CTAB molecules. When DTAB and CTAB are accumulated enough on the bacteria surface with the aid of APCB aggregates, DTAB or CTAB performs the antibacterial action together with APCB and in turn significantly enhance the antibacterial activity of the system..

20


Page 21 of 31

Figure 6. (a) Size distribution, (b) cryo-TEM images and (c) ζ potential of APCB and APCB/guest molecule mixtures in PBS (CAPCB = 50.0 µM).

0

∆ Hobs (kJ/mol)

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


-10 -20 APCB APCB/DTAB APCB/0.1CTAB

-30

+

APCB/AD-NH3

APCB/DB 8 DTAB (molar ratio/10 ) CTAB

-40 -50 0

3

6

9

12

7

Molar Ratio/10

Figure 7. Observed enthalpy changes (∆Hobs) by titrating 200.0 µM APCB into S. aureus PBS solution with OD600 =0.50, titrating 200.0 µM APCB/DTAB or APCB/0.1CTAB into S. aureus PBS solution with OD600 =0.75, titrating 200.0 µM APCB/AD-NH3+ and APCB/DB into S. aureus PBS solution with OD600 =0.63, titrating 1800.0 µM DTAB or 32.0 µM CTAB into S. aureus PBS solution with OD600 =0.75. The corresponding dilution enthalpy has been deducted. 21



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

Page 22 of 31

Table 2. Thermodynamic Parameters of the Binding of APCB or the APCB/guest Mixtures with S. aureus Derived from the ITC Curves in Figure 7. The Binding Numbers (N) and Stepwise Binding Numbers (N1 and N2) of APCB, APCB/Guest Molecule Mixtures, DTAB or CTAB Bound with Per S. aureus, and the Corresponding Binding Constants (K, K1 and K2). Surfactant-S.aureus APCB-S.aureus +

N

K 7

(× 10 )

(× 106 M-1)

4.34

1.06

APCB/AD-NH3 - S.aureus

4.13

5.55

APCB/DB-S.aureus

4.42

5.38

APCB/DTAB-S.aureus

1.87 (N1)

3.41 (N2)

312 (K1)

8.74 (K2)

APCB/0.1CTAB-S.aureus

3.28 (N1)

2.46 (N2)

0.47 (K1)

25.5 (K2)

DTAB-S.aureus

27.6

0.48

CTAB-S.aureus

0.72

5.65

Scheme 3. Proposed interaction modes of the guest molecules with APCB in spherical aggregates, and the antibacterial mechanism of the mixed spherical aggregates.

Cytotoxicity of APCB/Guest Molecule Mixtures. The cytotoxicity of APCB in the presence of the guest molecules was evaluated by their ability to kill mammalian cells (HaCaT) and the results are shown in Figure 8. Incorporating AD-NH3+ almost does not affect the toxicity of APCB to HaCaT cells. 22


Page 23 of 31

Incorporating DB slightly increases the toxicity of APCB to HaCaT cells. This means that either AD-NH3+ or DB maintains the low cytotoxicity of APCB while improves its antibacterial efficacy. Upon adding the cationic surfactants, the APCB/DTAB mixture exhibits much higher cytotoxicity than APCB, while the APCB/0.1CTAB mixture shows much weaker cytotoxicity than the APCB/DTAB mixture, but slightly stronger cytotoxicity than the APCB/DB mixture. The cytotoxicity is mainly caused by the strong cytotoxicity of DTAB or CTAB itself. It is worth noting that at the APCB concentration of 50 µM, the APCB/0.1CTAB mixture exhibits more than 90% antibacterial activity against S. aureus, while the viability of mammalian cell still keeps over 60%. Therefore, the APCB/0.1CTAB mixture should be a good choice with higher antibacterial activity and lower cell toxicity. 140 120

Cell Viability (%)

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


100 80 60 40

APCB +

APCB/AD-NH3

20

APCB/DB APCB/DTAB APCB/0.1CTAB

0 0

20

40

60

80

100

120

140

CAPCB (µM)

Figure 8. Cell viability of HaCaT cells after incubation with APCB, APCB/AD-NH3+ (1:1 mixing molar ratio), APCB/DB (1 : 1 mixing molar ratio), APCB/DTAB (1:1 mixing molar ratio) and APCB/0.1CTAB (1:0.1 mixing molar ratio) at different concentrations.

Among the guest molecules utilized, only DTAB and CTAB possess antibacterial potency, but the antibacterial potency is very low at the concentration used. Interestingly, DTAB and CTAB can 23



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

Page 24 of 31

effectively improve the antibacterial activity of APCB, making the antibacterial activity of APCB at 50 µM to be comparable or even higher than that at 100 µM. In particular, adding a very small amount of CTAB, i.e., 5.0 µM, can significantly increase the antibacterial activity of the APCB aggregates from 59% to more than 90%. Even only adding 0.5 µM CTAB into 50 µM APCB, the killing efficacy of the APCB/0.01CTAB aggregates increases from 59% to 80%. The enhanced effective binding at the bacteria surface, the release of CTAB at the bacteria surface, and then the cooperative action with APCB lead to the high antibacterial activity of the APCB/CTAB mixture. This method greatly decreases the dose of surfactants for effectively killing bacteria, at which the toxicity of the system can be kept at a lower level.

CONCLUSION In summary, covalent incorporation of β-CD into cationic surfactants greatly decreases their cytotoxicity while offering the opportunity to improve the antibacterial activity of cationic surfactants by adding appropriate guest molecules. The guest molecules can improve the antibacterial activity of the β-CD-attached cationic ammonium surfactant bearing a hexadecyl chain (APCB), whereas cannot improve the antibacterial activity of β-CD-attached cationic ammonium surfactant bearing a dodecyl chain (APDB). The reason is that the self-assembling ability of APDB is very weak and cannot form aggregates at the concentration used, while APCB and the APCB/guest molecules form 100–200 nm spherical aggregates, increasing the local surfactant concentration and thus leading to the high antibacterial activity. On the basis of the APCB aggregates, AD-NH3+ or DB increases the antimicrobial activity against S. aureus from 59% to 75%, while DTAB or CTAB makes the antimicrobial activity 24


Page 25 of 31

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


reach more than 90%. The different antimicrobial activities of the APCB/guest mixtures are generated by the different interaction modes of the APCB aggregates with S. aureus. The trapped AD-NH3+ supplies more positive charges to the APCB spherical aggregates, leading to stronger electrostatic interaction between the APCB aggregates and the negatively charged surface of S. aureus. DB binds with APCB through the benzene ring being encapsulated into the CD cavities of APCB while leaving the dodecyl chain to associate with the hydrophobic chains of the APCB aggregates, which enhances the ability of APCB to insert and disintegrate the cell membrane of S. aureus. Compared with AD-NH3+ and DB, incorporating DTAB and CTAB is more effective for improving the antimicrobial activity of the APCB aggregates. On one hand, similar to the situation of AD-NH3+, the cationic ammonium headgroups of DTAB or CTAB add more positive charges at the outer surface of the APCB aggregates and thus enhance the binding ability with the negatively charged surface of S. aureus. On the other hand, DTAB and CTAB can be released from the CD cavities of APCB after the aggregates of APCB/DTAB or APCB/CTAB targeting the S. aureus surface, and the released DTAB or CTAB acts synergistically with APCB in interacting with bacteria, which further enhances the antibacterial activity of APCB. Because the antibacterial efficacy of APCB is significantly improved by the guest molecules, the surfactant concentration needed to effectively kill the bacteria is reduced and low cytotoxicity is kept. Therefore, this supramolecular strategy provides an effective platform for the construction of highly efficient antibacterial agents with low cytotoxicity.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXX. 25



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

Page 26 of 31

The ITC analysis process AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (S.W.) *E-mail: [email protected] (Y.W.) 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 We greatly appreciate Prof. Hai Xu, Dr. Meiwen Cao and Dr. Dong Wang (Centre for Bioengineering and Biotechnology, China University of Petroleum) for cryo-TEM measurements. We also acknowledge National Natural Science Foundation of China (21327003, 21633002) for financial supports. REFERENCES (1) 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. (2) 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. 26


Page 27 of 31

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


(3) Buffet-Bataillon, S.; Tattevin, P.; Bonnaure-Mallet, M.; Jolivet-Gougeon, A., Emergence of Resistance to Antibacterial Agents: The Role of Quaternary Ammonium Compounds-a Critical Review. Int. J. Antimicrob. Ag. 2012, 39, 381-389. (4) Lee, W.; Li, Z.-H.; Vakulenko, S.; Mobashery, S., A Light-Inactivated Antibiotic. J. Med. Chem. 2000, 43, 128-132. (5) Velema, W. A.; van der Berg, J. P.; Hansen, M. J.; Szymanski, W.; Driessen, A. J.; Feringa, B. L., Optical Control of Antibacterial Activity. Nat. Chem. 2013, 5, 924-928. (6) Colomer, A.; Pinazo, A.; Manresa, M. A.; Vinardell, M. P.; Mitjans, M.; Infante, M. R.; Perez, L., Cationic Surfactants Derived from Lysine: Effects of Their Structure and Charge Type on Antimicrobial and Hemolytic Activities. J. Med. Chem. 2011, 54, 989-1002. (7) 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. (8) 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. (9) Zhou, C.; Wang, F.; Chen, H.; Li, M.; Qiao, F.; Liu, Z.; Hou, Y.; Wu, C.; Fan, Y.; Liu, L.; Wang, S.; Wang, Y., Selective Antimicrobial Activities and Action Mechanism of Micelles Self-Assembled by Cationic Oligomeric Surfactants. ACS Appl. Mater. Interfaces 2016, 8, 4242-4249. (10) Ghosh, C.; Manjunath, G. B.; Akkapeddi, P.; Yarlagadda, V.; Hoque, J.; Uppu, D. S. S. M.; Konai, M. M.; Haldar, J., Small Molecular Antibacterial Peptoid Mimics: The Simpler the Better! J. Med. Chem. 2014, 57, 1428-1436. (11) Haldar, J.; Kondaiah, P.; Bhattacharya, S., Synthesis and Antibacterial Properties of Novel Hydrolyzable Cationic Amphiphiles. Incorporation of Multiple Head Groups Leads to Impressive Antibacterial Activity. J. Med. Chem. 2005, 48, 3823-3831. 27



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

Page 28 of 31

(12) Sambhy, V.; Peterson, B. R.; Sen, A., Antibacterial and Hemolytic Activities of Pyridinium Polymers as a Function of the Spatial Relationship between the Positive Charge and the Pendant Alkyl Tail. Angew. Chem. Int. Ed. 2008, 47, 1250-1254. (13) Ilker, M. F.; Nusslein, K.; Tew, G. N.; Coughlin, E. B., Tuning the Hemolytic and Antibacterial Activities of Amphiphilic Polynorbornene Derivatives. J. Am. Chem. Soc. 2004, 126, 15870-15875. (14) Liu, K.; Liu, Y.; Yao, Y.; Yuan, H.; Wang, S.; Wang, Z.; Zhang, X., Supramolecular Photosensitizers with Enhanced Antibacterial Efficiency. Angew. Chem. Int. Ed. 2013, 52, 8285-8289. (15) Bai, H.; Yuan, H.; Nie, C.; Wang, B.; Lv, F.; Liu, L.; Wang, S., A Supramolecular Antibiotic Switch for Antibacterial Regulation. Angew. Chem. Int. Ed. 2015, 54, 13208-13213. (16) Zhou, C.; Wang, D.; Cao, M.; Chen, Y.; Liu, Z.; Wu, C.; Xu, H.; Wang, S.; Wang, Y., Self-Aggregation, Antibacterial Activity, and Mildness of Cyclodextrin/Cationic Trimeric Surfactant Complexes. ACS Appl. Mater. Interfaces 2016, 8, 30811-30823. (17) Bai, H.; Lv, F.; Liu, L.; Wang, S., Supramolecular Antibiotic Switches: A Potential Strategy for Combating Drug Resistance. Chem.-Eur. J. 2016, 22, 11114-11121. (18) Bai, H.; Zhang, H.; Hu, R.; Chen, H.; Lv, F.; Liu, L.; Wang, S., Supramolecular Conjugated Polymer Systems with Controlled Antibacterial Activity. Langmuir 2017, 33, 1116-1120. (19) Galstyan, A.; Kauscher, U.; Block, D.; Ravoo, B. J.; Strassert, C. A., Silicon(IV) Phthalocyanine-Decorated Cyclodextrin Vesicles as a Self-Assembled Phototherapeutic Agent against Mrsa. ACS Appl. Mater. Interfaces 2016, 8, 12631-12637. (20) Ferro, S.; Jori, G.; Sortino, S.; Stancanelli, R.; Nikolov, P.; Tognon, G.; Ricchelli, F.; Mazzaglia, A., Inclusion of 5-[4-(1-Dodecanoylpyridinium)]-10,15,20-Triphenylporphine in Supramolecular Aggregates of Cationic Amphiphilic Cyclodextrins: Physicochemical Characterization of the Complexes and Strengthening of the Antimicrobial Photosensitizing 28


Page 29 of 31

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


Activity. Biomacromolecules 2009, 10, 2592-2600. (21) Yu, G.; Jie, K.; Huang, F., Supramolecular Amphiphiles Based on Host-Guest Molecular Recognition Motifs. Chem. Rev. 2015, 115, 7240-7303. (22) Sakai, F.; Chen, G. S.; Jiang, M., A New Story of Cyclodextrin as a Bulky Pendent Group Causing Uncommon Behaviour to Random Copolymers in Solution. Polym. Chem. 2012, 3, 954-961. (23) Valente, A. J.; Söderman, O., The Formation of Host–Guest Complexes between Surfactants and Cyclodextrins. Adv. Colloid Interface Sci. 2014, 205, 156-176. (24) Messner, M.; Kurkov, S. V.; Jansook, P.; Loftsson, T., Self-Assembled Cyclodextrin Aggregates and Nanoparticles. Int. J. Pharm. 2010, 387, 199-208. (25) Tang, Y.; Zhou, L.; Li, J.; Luo, Q.; Huang, X.; Wu, P.; Wang, Y.; Xu, J.; Shen, J.; Liu, J., Giant Nanotubes Loaded with Artificial Peroxidase Centers: Self-Assembly of Supramolecular Amphiphiles as a Tool to Functionalize Nanotubes. Angew. Chem. Int. Ed. 2010, 49, 3920-3924. (26) Wang, Y.; Ma, N.; Wang, Z.; Zhang, X., Photocontrolled Reversible Supramolecular Assemblies of an Azobenzene-Containing Surfactant with α-Cyclodextrin. Angew. Chem. Int. Ed. 2007, 46, 2823-2826. (27) Zhang, B.; Yue, L.; Wang, Y.; Yang, Y.; Wu, L., A Novel Single-Side Azobenzene-Grafted Anderson-Type Polyoxometalate for Recognition-Induced Chiral Migration. Chem. Commun. 2014, 50, 10823-10826. (28) Han, Y.; Cheng, K.; Simon, K. A.; Lan, Y.; Sejwal, P.; Luk, Y.-Y., A Biocompatible Surfactant with Folded Hydrophilic Head Group: Enhancing the Stability of Self-Inclusion Complexes of Ferrocenyl in a β-Cyclodextrin Unit by Bond Rigidity. J. Am. Chem. Soc. 2006, 128, 13913-13920. (29) Michel, D.; Chitanda, J. M.; Balogh, R.; Yang, P.; Singh, J.; Das, U.; El-Aneed, A.; Dimmock, J.; Verrall, R.; Badea, I., Design and Evaluation of Cyclodextrin-Based Delivery Systems to Incorporate Poorly Soluble Curcumin Analogs for the 29



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

Page 30 of 31

Treatment of Melanoma. Eur. J. Pharm. Biopharm. 2012, 81, 548-556. (30) Funasaki, N.; Ohigashi, M.; Hada, S.; Neya, S., Surface Tensiometric Study of Multiple Complexation and Hemolysis by Mixed Surfactants and Cyclodextrins. Langmuir 2000, 16, 383-388. (31) Versluis, F.; Tomatsu, I.; Kehr, S.; Fregonese, C.; Tepper, A. W. J. W.; Stuart, M. C. A.; Ravoo, B. J.; Koning, R. I.; Kros, A., Shape and Release Control of a Peptide Decorated Vesicle through pH Sensitive Orthogonal Supramolecular Interactions. J. Am. Chem. Soc. 2009, 131, 13186-13187. (32) Li, S. Y.; Zhang, L.; Wang, B.; Ma, M. F.; Xing, P. Y.; Chu, X. X.; Zhang, Y. M.; Hao, A. Y., An Easy Approach for Constructing Vesicles by Using Aromatic Molecules with β-Cyclodextrin. Soft Matter 2015, 11, 1767-1777.

30


Page 31 of 31

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


TOC

31


Tuning Antibacterial Activity of Cyclodextrin-Attached Cationic

Recommend Documents