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Polyanionic Antimicrobial Membranes: An Experimental and Theoretical Study Jiangna Guo, Qiming Xu, Rongwei Shi, Zhiqiang Zheng, Hailei Mao, and Feng Yan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00185 • Publication Date (Web): 07 Apr 2017 Downloaded from http://pubs.acs.org on April 9, 2017

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Polyanionic Antimicrobial Membranes: An Experimental and Theoretical Study Jiangna Guo,a Qiming Xu,b Rongwei Shi,c Zhiqiang Zheng,a Hailei Mao,*,b Feng Yan*,a a

Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department

of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, China. E-mail: [email protected] b

Department of Anesthesiology and Critical Care Medicine, Zhongshan Hospital, Fudan

University, 180 Fenglin Road, Shanghai, 200032, China. E-mail: [email protected] c

Institute of Technical Biology & Agriculture Engineering, Hefei Institutes of Physical Science,

Chinese Academy of Sciences, 350 Shushanhu Road, Anhui, 230031, China. ABSTRACT

Polycationic polymers have been widely used as antimicrobial materials because of their broad spectrum activity and potential use as new antibiotics. Herein, we report the synthesis of polyanionic antimicrobial membranes by in situ photo-cross-linking of a sulfate based anionic monomer and followed by cation-exchange with organic (quaternary ammonium or imidazolium) or metal (Ag+, Cu2+, Fe3+, Zn2+, Na+, K+) cations. The resultant polyanionic membranes show high and broad spectrum antibacterial activities against both bacteria ( Escherichia coli (E. coli), Staphylococcus aureus ( S. aureus) ) and fungi (Candida albicans (C. albicans) ) ． In addition, the polyanionic antimicrobial membranes efficiently inhibited the

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formation of biofilms by SC5314 and its crk1 gene deleted (Δcrk1) C. albicans strains. Furthermore, the synthesized polyanionic membranes exhibit good blood compatibility, low cytotoxicity and long-term antibacterial stability, demonstrating safe antimicrobial materials in the application of healthcare.

Keywords: polyanionic membranes, antimicrobial, cation exchange, antibiofilm, antifouling

INTRODUCTION Microbial infections, including biofilm associated infections, are one of the leading causes of morbidity in hospitalized patients. With the increasing prevalence of antibiotic-resistant bacterial infections, the development of alternative and highly efficient antibacterial materials that are not easily able to develop resistance by bacteria has gained considerable attention.1-3 Therefore, antibacterial therapeutics without inducing resistance, such as antimicrobial peptides,4, bacteriophages,6,

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silver,8-10 carbon-based materials,11-13 cationic compounds (or polymers)14

have been reported as with broad-spectrum of antibacterial activities. Among the antimicrobial materials investigated, cationic polymers which substituted with quaternary ammonium,15,

16

phosphonium,17, 18 pyridinium19 or imidazolium cations20 have been extensively studied. This type of cationic polymers can disrupt (or damage) the bacteria through the electrostatic interaction of the cationic moieties with the phosphate groups of cell membrane.21 In addition, the hydrophobic segments of cationic polymers may insert into the hydrophobic regions of the cell membrane, leading to the leakage of the intracellular substances out of the cell membrane and eventually to the cell death.22 Compared with small molecular antibiotics, cationic polymers exhibit sustained inhibitory effect, broad-spectrum antibacterial features, as well as better biocompatibility.23 At present, hundreds of reports that involve cationic polymers present in


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solution,24 coating,25 hydrogel26 and membrane27 forms with selectivity for bacteria over mammalian cells have been published. These studies have demonstrated that cationic chemistry played an important role in influencing the antimicrobial activities. However, it is tedious and time-consuming for the design and synthesis of polymers with various cationic groups, low toxicity and high antibacterial efficiency. On the other hand, anionic polymers which carry negatively charged groups (such as sulfate, carboxylate or sulfamate groups) have been recently applied in biological applications. For example, anionic polymers have been applied as siRNA vectors which increased the gene silencing efficiency of the vectors in cell culture.28 Polymer membrane surfaces (or coatings) with either cationic/anionic groups or zwitterionic groups can highly reduce the nonspecific protein adsorption due to the synergistic antifouling effect of the binary components.29-33 Furthermore, the cation-exchange capability of anionic polymers enables the preparation of functional polymers with various cations without altering the main structure simply via the polymerization of one anionic monomer, and followed by cation-exchange reactions. In this work, we report a facile and effective strategy for the preparation of polyanionic (AP) membranes with inherently antimicrobial activities. The antimicrobial polymeric membranes were prepared via the photo-cross-linking of an anionic monomer, 2-acrylamido-2methylpropanesulfonic acid (AMPS), with acrylonitrile, styrene and divinylbenzene (as crosslinker) and followed by cation-exchange with organic cations (such as ammonium (NH+) or imidazolium (Im+) cations), or with metal cations (Ag+, Cu2+, Fe3+, Zn2+, Na+, K+) (see Scheme 1). The antimicrobial activities of the prepared polyanionic membranes against E. coli, S. aureus and C. albicans were investigated. For comparison, a typical polycationic membrane, CPCH3SO3 (see Scheme 1), was also synthesized and the antimicrobial property was studied under


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the same experimental conditions. Moreover, the inhibition of bioﬁlm formation of the polymeric membranes was further assessed using wide-type C. albicans (SC5314) and corresponding crk1 gene deleted (Δcrk1) strains (loss of crk1 gene could reduce the biofilm formation of the Candida strain). In order to get detailed insights into the nature of the antimicrobial activities, theoretical approaches (molecular dynamics simulations (MDS)) were carried out as well. EXPERIMENTAL SECTION Materials.

Styrene,

2-acrylamido-2-methylpropanesulfonic

acid

(AMPS),

acrylonitrile,

divinylbenzene (DVB), methylimidazole, 2-(diethylamino) ethyl methacrylate, benzoin isobutyl ether, triethylamine, sodium methanesulfonate, imidazole, acetonitrile, bromoethane, diethyl ether, and ethyl acetate, ethanol, phosphate buffer saline (PBS), sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), sodium nitrate (NaNO3), potassium nitrate (KNO3), silver nitrate (AgNO3), copper nitrate (Cu(NO3)2), ferric nitrate (Fe(NO3)3·9H2O) and zinc nitrate (Zn(NO3)2·6H2O) poly(ethylene terephthalate) (PET) membranes were used as purchased. All of the vinyl monomer oils were made inhibitor-free by passing the liquid through a column filled with basic alumina to remove the inhibitor and then stored at -5 oC. Deionized water was used throughout the experiments. Staphylococcus aureus (S. aureus) (ATCC 6538), Escherichia coli (E. coli) (ATCC 8099) and Candida albicans (C. albicans) (ATCC 76615) strains were kindly provided by Dr. Shengwen Shao (Huzhou University School of Medicine, China). Two C. albicans strains (SC5413 and Δcrk1) used for biofilm formation assay were kindly granted by Prof. Jiangye Chen (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China). Characterization. 1H NMR spectra were recorded on a Bruker Advance 400 MHz NMR spectrometer using D2O as the solvent. The Fourier transform infrared (FT-IR) spectra were


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recorded on a Varian CP-3800 spectrometer in the range of 4000–400 cm-1. Scanning electron microscopy (SEM) images were taken with a Philips Model XL 30 FEG microscope with an accelerating voltage of 10 kV. The energy-dispersive X-ray spectroscopy (EDX) measurements were performed with the spectrometer attached on the Hitachi Model S-4700 field emission scanning electron microscopy (FE-SEM) system. The optical density (OD) values were tested on an Eon microplate spectrophotometer (Bio Tek Instruments, Inc.). Synthesis of 1-triethylamine 2-acrylamido-2-methyl propanesulfonate ([TEA][AMPS]). [TEA][AMPS] was synthesized as documented in the previous literature.34 2-Acrylamido-2methylpropanesulfonic acid (AMPS) (3.58 g, 0.020 mol) with overdose molar amount of triethylamine (2.220 g, 0.022 mol) was mixed and stirred in an ice-water bath overnight. The resultant viscous oil was washed with diethyl ether and ethyl acetate three times and then rotary evaporated at room temperature. [TEA][AMPS], 1H NMR (400 MHz, D2O, δ): 8.36 (s, 1H, C=O-N-H), 7.28 (s, 1H, -NH-), 6.10 (m, 1H, C=CH-), 5.63 (d, 1H, CH2=C-), 5.40 (d, 1H, CH2=C-), 3.82 ( s, 2H, -CH2-S ), 3.38 (m, 6H, -CH2-), 1.46 (m, 15H, -CH3). Synthesis

of

1-methylimidazolium

2-acrylamido-2-methyl

propanesulfonate

([MIm][AMPS]). [MIm][AMPS] was synthesized by stirring a mixing containing AMPS (3.580 g, 0.020 mol) with methylimidazole (1.810g, 0.022 mol) in an ice-water bath overnight. Diethyl ether and ethyl acetate was used to wash the resultant viscous oil three times. The product was then rotary evaporated at room temperature. 1H NMR (400 MHz, d6-DMSO, δ): 8.64 (s, 1H, C=O-NH), 8.421 (s, 1H, N=CH-N), 7.475 (s, 1H, C=CH-N-C), 5.89 (d, 1H, N-CH=C-N), 5.76 (m, 2H, C=CH-), 5.49 (d, 1H, H-C=C), 3.81 (s, 3H, N-CH3), 2.73 (s, 2H, CH2-S), 1.43 (s, 6H, C-(CH3)2).


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Synthesis of N, N, N-triethyl-2-(methacryloyloxy)-ethanaminium [TEM][Br]. Typically, a mixture of 1.310 g (0.012 mol) bromoethane and 1.850g (0.010 mol) 2(diethylamino) ethyl methacrylate was introduced into 30 mL of CH2Cl2 and stirred in in an icewater bath for 48 h. The solvent was evaporated using a rotary evaporator and washed several times with diethyl ether. The resulting [TEM][Br] was dried under vacuum. The chemical structure of the compound was confirmed by 1H NMR (400 MHz, d6-DMSO, δ): 6.05 (d, 1H, C=CH2), 5.76 (d, 2H, -C=CH2), 4.46 (m, 2H, -CH2-), 3.59 (m, 2H, -CH2- ), 3.32 (m, 6H, -CH2-), 1.898 (m, 1H, =CH), 1.19 (m, 9H, -CH3). Preparation of polymer membranes (AP-NH and AP-M). A mixture of styrene (25 %, molar ratio), acrylonitrile (60 %, molar ratio), [TEA][AMPS]) (15 %, molar ratio), divinylbenzene (4 wt% to the formulation based on the weight of monomer), and 0.5 wt % of benzoin isobutyl ether were ultrasonicated and obtained a homogeneous solution, following by casting the solution into a handmade glass mold and photo-crosslinked by irradiation with UV light of 250 nm wavelength for about 40 min at room temperature. The resultant membranes were washed and ultrasonicated with ethanol three times to remove the unreacted monomer residues. The prepared poly [TEA] [AMPS]-based membranes were immersed in a 0.05 M AgNO3 or Cu(NO3)2, Fe(NO3)3, Zn(NO3)2, NaNO3, KNO3 aqueous solution for 1 h to convert the membrane from NH+ form to Ag+, Cu2+, Fe3+, Zn2+, Na+ and K+ form, respectively. The cation-exchanged membranes were washed and ultrasonicated with deionized water more than three times. The obtained membranes are abbreviated as AP-M (M indicates the cation of polymers) for simplicity.


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Preparation of poly([MIm][AMPS]) membranes (AP-Im). The

poly([MIm][AMPS])

poly([TEA][AMPS])

based

based

membranes

membranes

above.

were

synthesized

A homogeneous

as

described

mixture,

as

containing

styrene/acrylonitrile/[Im][AMPS] (25/60/15, molar ratio), divinylbenzene (4 wt% to the formulation based on the weight of monomer), and 0.5 wt % of benzoin isobutyl ether, was photo-crosslinked by irradiation with UV light of 250 nm wavelength for about 40 min in a handmade glass mold at room temperature. The resultant membranes were washed and ultrasonicated with ethanol three times to remove the unreacted monomer residues. Preparation of CP-CH3SO3 membranes. The poly([TEM][Br]) membranes were synthesized follow the method described for poly([MIm][AMPS]). The prepared poly([TEM][Br]) membranes were immersed in equal molecular aqueous sodium methanesulfonate solution for 24 h to convert the membrane from Br− to CH3SO3− form. Colony assay for the antibacterial activities. The S. aureus and E. coli were routinely grown on Luria–Bertani (LB) agar plates. C. albicans was grown with Yeast Peptone Dextrose (YPD) agar plates at 37 oC with shaking for 24 h at 150 rpm and diluted to 1×106 CFU/mL. Microbial suspensions (100 μL) in LB (without NaCl) or YPD were spread onto sterilized PET membranes, AP-NH, AP-Im, AP-M (M: Ag+, Cu2+, Fe3+, Zn2+, Na+, K+) and CP-CH3SO3 membranes (1.0×1.0 cm2). Microbial suspension (10 L) was streaked onto an LB or YPD agar plates after incubation with the membranes at 37 oC for 4h at a relative humidity higher than 90%. The number of the colony-forming units (CFUs) was counted


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after incubation at 37 oC for 24 h. Each colony assay test was repeated more than three times. The antibacterial rate was calculated with the number of colonies from the experimental sample (B) and negative control (A) according to the following formula:

Antibacterial rate (%) 

A negative

control

A negative

 B sample

 100%

control

Morphological changes of bacteria. The morphology of bacteria coated on the surfaces of our PET control membrane and AP-NH, AP-Im, AP-M (M: Ag+, Cu2+, Fe3+, Zn2+, Na+, K+) and CP-CH3SO3 membranes was observed by field emission scanning electron microscopy (FE-SEM). The microbial suspensions (OD =0.1) of S. aureus, E. coli or C. albicans were used to inoculate on the surfaces of these polymer membranes, the membranes were then incubated at 37 oC for 4 h (see the details in colony assay, Supporting Information). The bacteria on the membranes surfaces were fixed with 2.5% glutaraldehyde for 2 h and then dehydrated step wise with 10 vol%, 30 vol%, 50 vol%, 70 vol%, 80 vol%, 90 vol%, and 100 vol% ethanol solution (10 min for each step), respectively. Molecular Dynamics Simulations (MDS). The molecular dynamics simulations (MDS) were carried out as described in the literature.35 A mixture of dioleoylphosphatidylcholine (DOPC) and anionic dioleoylphosphatidylglycerol bilayer (DOPG) (7:3, molar ratio) were used to model the polyanionic membranes of the bacterial membranes. The DOPC-DOPG membrane was built with CHARMM-GUI Membrane Builder. The equilibrated lipid bilayer is composed of 120 lipids. The CHARMM36 force field parameters were generated by the calculation using the Gaussian03 software performed at the B3LYP/6-31G* level of theory for modeling the polyanionic and polycationic membranes, and


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then by the combined tools of Antechamber and ACPYPE. The RESP charges of main cationic/anionic functional groups and non-bonded Lennard-Jones parameters for metal ions (Ag+, Cu2+, Zn2+ and Fe3+) were listed in Table S1. Other atomic RESP charges are included in the MOL2 files and showed in Supporting Information (see Figure S1). All the MDS were performed with the GROMACS program suite version 5.1.1. The solvated bilayer-AP-cationic (or bilayer-CP-anionic) systems were subjected to neutralization by 36 sodium ions, and then stepwise energy minimizations, equilibrations followed by 30 ns of production MDS, respectively. The detailed parameter settings for the MDS were performed as following: the particle-mesh Ewald method was used to calculate the long-range electrostatic interactions. Lennard-Jones potential and forces were truncated at 1.2 nm. Bond lengths were constrained by P-LINCS with 2 fs time step. The lipid-cation (or lipid-anion) systems were equilibrated in three phases. The first phase used an isochoric-isothermal (NVT) ensemble for 1 ns, with temperature controlled using the Berendsen weak coupling algorithm. During NVT, the temperature of the system was maintained at 300 K with a coupling constant of 0.1 ps. Heavy atoms of the lipid molecules and the distances between the cation (or anion) and the sulfite for each polyanionic (or polycationic) membranes were restrained. Then the systems were equilibrated under the isobaric-isothermal (NPT) ensemble for 5 ns, using Berendsen thermostat and Berendsen barostat. Coupling constants for temperature and pressure were 0.2 and 2.0 ps, respectively. Then the restraints were removed, and the systems were further equilibrated for 2 ns under the NPT ensemble. Unconstrained production simulations were performed for 30 ns using Nose-Hoover thermostat and Parrinello-Rahman barostat. Coupling constants were set similarly to those in the NPT simulations.


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Biofilm formation assay. The biofilm formation of the CP-CH3SO3, AP-Im, AP-NH and AP-M membranes was performed against SC5314 and its crk1 gene deleted (Δcrk1) C. albicans strains by via a 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Briefly, the sterilized PET, CP-CH3SO3, AP-Im, AP-NH and AP-M (M: Ag+, Cu2+, Fe3+, Zn2+) membranes were cut into round shape (d=0.6 cm) and put into a 96-well plate, and the fungal suspensions (200 L, OD=0.1) in YPD were added and inoculated at 37 oC for 2 h. After the adhesion stage of fungi, the supernatants were discarded, and the membranes were washed with PBS for three times. Then 100 L RPMI 1640 medium was added in each well and cultured at 37 oC for 48 h, and the fresh medium was changed once per 24 h. Then 100 L MTT solution (5 g/L in PBS) was added into each well and incubated at 37 oC for 4 h. After the removal of the supernatant, 150 L DMSO was added in each well to dissolve any formazan crystals. The OD490nm values were read to assess formazan release using an Eon microplate spectrophotometer (Bio Tek Instruments, Inc.). All the assays were repeated three or more times. The wells without any membrane and with PET membrane were used as controls. Cytotoxicity evaluation. The toxicity assay of the CP-CH3SO3, AP-Im, AP-NH and AP-M (M: Ag+, Cu2+, Fe3+, Zn2+) membranes was performed against human dermal fibroblast cells and evaluated via a 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). Briefly, 3×104 human dermal fibroblasts in 1 mL 10% fetal calf serum medium were cultured in a 24-well plate for 48 h. The sterilized PET and CP-CH3SO3, AP-Im, AP-NH and AP-M membranes (1.0 × 1.0 cm2) were put into the fibroblast cell suspension and cultured together at 37 oC for 72 h. Then 0.1 mL MTT


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solution (5 g/L) was added into each well and incubated at 37 oC for 4 h. After the removal of the supernatant, 0.75 mL DMSO was added in each well to dissolve any formazan crystals. The OD490nm was read to assess the cell viability using an Eon microplate spectrophotometer (Bio Tek Instruments, Inc.). All the measurements were repeated three or more times. The relative growth rate (RGR) of the human dermal fibroblast cells was calculated according to the following formula with PET as control:

RGR(%) 

OD sample OD control

100%

Hemolysis assay. A precipitate of human red blood cells was prepared by centrifuging fresh human blood (3 mL) at 1500 rpm for 15 min and washing with PBS until the supernatant was transparent. This supernatant was then diluted to 2 vol% in PBS. The sterilized PET, CP-CH3SO3, AP-Im, AP-NH and AP-M membranes (1.5×1.5 cm2) were dipped into diluted blood (5 mL for each tube) and incubated at 37 oC for 3 h. Aliquots of 100 µL supernatant (from each tube) were transferred into a 96-well plate after the treated diluted blood samples were centrifuged at 1500 rpm for 15 min. The OD576

nm

were recorded to assess hemoglobin release by an Eon microplate

spectrophotometer (Bio Tek Instruments, Inc.). Red blood cells with 2 % Triton and in PBS were applied as positive and negative controls, respectively. The hemolysis percentage was calculated according to the following formula:

Hemolysis rate (%) 

OD sample  OD negative OD positive

control

control

 OD negative

100%

control


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Protein absorption. The adsorption of a model protein bovine serum albumin (BSA) on the surface of AP-NH and AP-M membranes was determined by a Bradford protein assay. The PBS washed PET, AP-NH and AP-M membranes (1.5×1.0 cm2) were immersed into 1 mL BSA (5 wt% in PBS) solution in a 12-well plate at 37 oC for 7 days. The samples were rinsed with PBS solution three times and transferred into an Eppendorf tube with 1 mL PBS solution containing 1 wt% sodium dodecylsulfate (SDS). Sonication at 40 kHz for 30 min was used to remove the proteins adsorbed on the membrane surface. The amount of BSA adsorbed on the membranes surfaces was measured by the absorbance at 595 nm, and determined by the Bio-Rad protein assay reagent kit (Bio-Rad, U.S.A.) based on the method of Bradford.

RESULTS ANG DISCUSSION Scheme 1A shows the chemical structures of polyanionic membranes with various cations (APNH, AP-Im and AP-M), as well as a polycationic membrane, CP-CH3SO3, investigated in this work. The polymer membranes were synthesized via in situ photo-cross-linking of a mixture containing anionic monomer pendant with sulfate (-SO3－) groups, styrene, and acrylonitrile (shown in Schemes 1B,S1). Styrene and acrylonitrile were chosen as the comonomers because poly(styrene-co-acrylonitrile) is a type of copolymer material with high chemical resistance and expected ability to form robust membranes. These polyanionic membranes were then cationexchanged with various metal cations to convert NH+ to Ag+, Cu2+, Fe3+, Zn2+, Na+ or K+ form. These forms are abbreviated as AP-M (M indicates different cations) for simplicity. Figure S2 shows the photographs of prepared polyanionic and polycationic membranes. As it can be seen


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all the membranes with the thickness of about 100 μm are flexible and strong enough to be cut into any desired shape or size. The chemical structure of the polyanionic membranes was characterized by Fourier transform infrared (FT-IR) and energy dispersive X-ray (EDX) spectra.

Scheme 1 (A) Chemical structures of polyanionic membranes with various cations (AP-NH, APIm and AP-M). A polycationic membrane, CP-CH3SO3, was synthesized for comparison. (B) The synthesis of polyanionic membranes (AP-M) and the antimicrobial strategy on the membrane surfaces.


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Figure S3 shows the FT-IR spectra of the AP-NH membranes. An absorption peak at 1604 cm-1 is assigned to the skeletal vibration of benzene rings in the polystyrene units. The stretching vibration of cyano groups (C≡N) is observed at 2235 cm-1. The absorption peaks at around 1034 and 1212 cm-1 are assigned to the symmetric and asymmetric stretching vibration of sulfonate (SO3 － ) groups.34 Absorption peaks at around 2857 and 760 cm-1 are observed based on the vibrational mode of quaternary ammonium cations. The results clearly confirm the successful preparation of polyanionic membranes. The results of EDX show that the cation exchange degree is 7.17%, 5.06%, 5.11%, 5.07%, 5.02%, and 5.39% for AP-Ag, AP-Cu, AP-Fe, AP-Zn, AP-Na and AP-K membranes, respectively (Figure S4). Figure S5 shows the scanning electron microscopy (SEM) of the surface morphology of the membranes. The surfaces of all the membranes are smooth, uniform, and without any visible pores. To investigate the antimicrobial properties of polymeric membranes, gram-positive (S. aureus), gram-negative (E. coli) bacteria and fungi (C. albicans) were chosen as model microorganisms (Scheme 1B). The antimicrobial efficiency of the AP-Im and AP-NH membranes was first investigated. It can be seen that about 76%, 75% and 78% of E. coli, S. aureus and C. albicans, respectively, were eliminated after contacting with AP-Im membrane for 4 h (see Figure S6). The corresponding antimicrobial efficiency for AP-NH was increased to 83%, 95% and 85% for E. coli, S. aureus and C. albicans, respectively, however, be highly decreased to about 60%, 38% and 30%, respectively, for CP-CH3SO3 (see Figures S6, S7).


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A10075

C 100

50 25 8 6 4 2 0

Bacterial Viability (%)

B 100 Bacterial Viability (%)

Bacterial Viability(%)

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90

8 6 4 2 0

90 80 10 8 6 4 2 0

PET AP-NH AP-Ag AP-Cu AP-Fe AP-Zn


Against E. coli

Against S. aureus


Against C.albicans

Figure 1. Bacterial viabilities of (A) E. coli, (B) S. aureus and (C) C. albicans after contacting with AP-M (M: NH+, Ag+, Cu2+, Fe3+, Zn2+) membranes for 4h, using PET membranes as the controls.

An understanding of the antimicrobial mechanism for cationic polymers is usually based on the material and bacteria interactions, especially on the electrostatic interaction of the cationic moieties with the phosphate groups of the bacterial (or fungal) cell membrane, as well as the interactions between the hydrophobic segments of the polymer and the hydrophobic regions of the lipid membrane. These interactions facilitate the membrane permeability alterations and lead to the progressive leakage of cytoplasmic material and consequential lysis of the cells.36-38 We believe that such a mechanism may also be applied to explain the antimicrobial properties of the polyanionic membranes. Compared with cationic polymers on which cationic moieties were covalently bound to the polymer backbone, the relatively free cations of anionic polymers, such as Im+ and NH+ cations, are more easily to interact with the anionic phosphate groups of the cell wall (as shown in Scheme 1B). In addition, the positive charge distribution of NH+ is more concentrated than that of Im+. The stronger electrostatic interaction between the NH+ and phosphate groups of cell wall increases the antimicrobial efficiency of AP-NH membrane. These results indicate that polyanionic membranes synthesized in this work are promising for efficient antimicrobial performance.


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The antibacterial activities of polyanionic membranes with various metal cations were further studied under the same experimental conditions. Figures 1, S6 and S8 illustrate the bactericidal activities of AP-NH, AP-Ag, AP-Cu, AP-Fe, AP-Zn, AP-Na and AP-K after 4 h incubation. It can be clearly seen that the AP-NH, AP-Ag, AP-Cu, AP-Fe, AP-Zn could kill or inhibit the growth of E. coli, S. aureus and C. albicans efficiently once these organisms contact the membrane surfaces (see Figure 1). Moreover, the antibacterial efficiencies of AP-Ag, AP-Cu, AP-Fe and AP-Zn are much higher than those of AP-NH. However, AP-Na and AP-K membranes did not show significant antimicrobial activities (see Figure S6). Since Na+ and K+ support the growth of bacteria and therefore cannot efficiently inhibit the bacteria growth. A very low antibacterial activity of AP-Na and AP-K was observed may be due to residual NH+ in the polymeric membranes. To probe the interactions between the polyanionic membrane and anionic lipid bilayer, the antimicrobial mechanism was further studied by molecular dynamics simulations (MDS). The anionic lipid bilayer was modeled with a mixture of zwittorionic dioleoylphosphatidylcholine (DOPC) and anionic dioleoylphosphatidyl-glycerol (DOPG) (7:3, molar ratio) (Figure S9). The polymer models were set about 0.7 nm away from the surface of the upper leaflet of a preequilibrated DOPC-DOPG membrane.


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Langmuir

Figure 2. Snapshots for the models of lipid bilayer combined with A) CP-CH3SO3, B) AP-Im, C) AP-NH, D) AP-Ag, E) AP-Cu, F) AP-Zn and G) AP-Fe models after 30 ns simulations. Only neighboring lipid molecules and polymeric membranes are shown in sphere for clarity. All the atoms of polymer models are colored in blue, while the carbon, phosphorus, oxygen, hydrogen and nitrogen of lipids are colored in green, orange, red, white and blue, respectively. Table 1. Interaction energies between polyanionic (or polycationic) membranes and lipid bilayers. Polymer Membranes

Electrostatic interaction (kcal/mol)

Hydrophobic interaction (kcal/mol)

Total interaction (kcal/mol)

CP-CH3SO3

-5.6

-5.5

-11.1

AP-Im

-9.2

-7.3

-16.5

AP-NH

-15.8

-10.5

-26.1

AP-Ag

-19.4

-38.8

-58.2

AP-Cu

-22.1

-27.8

-49.9

AP-Fe

-20.7

-11.6

-32.3

AP-Zn

-20.8

-25.5

-46.3


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Figure 2 presents the snapshots of polymer models and lipid bilayer after 30 ns simulations, respectively. The variations of interaction energies (including electrostatic and hydrophobic interactions) between polyanionic (or polycationic) membrane and DOPC-DOPG membrane with the simulation times are shown in Figure S10, and the calculated mean values of the interaction energy are summarized and listed in Table 1. As it can be seen from MDS trajectory (Figure S10) that AP-M (M= Ag+, Cu2+, Fe3+, Zn2+) with various metal cations show stronger interactions with the lipid bilayer than that with organic cations (AP-NH, AP-Im) and CPCH3SO3. The results indicate that strong electrostatic interactions exist between the metal cations and phosphate and (or) ester groups of lipid cell membranes. These outstanding electrostatic attractions induced by the negatively charged bacterial membrane surface and positively charged metal cations within AP-M, may potentially lead to the structural changes and the imbalance of the bacterial membrane. Moreover, it has been demonstrated that electrostatic attraction is one of the important contributions to antibacterial activity, which can disrupt the normal functions of the membrane,39-41 such as, by promoting the leakage of intracellular components or inhibiting the transport of nutrients into cells. Figure 2 and Figure S11 clearly show the interactions of the polyanionic (or polycationic) membrane with bacterial lipid membrane. For example, the metalcontaining AP-Ag, AP-Cu, AP-Zn, AP-Fe polycationic membranes are gradually integrated into the cell membrane, while AP-NH, AP-Im and CP-CH3SO3 just approach or attach to the cell membrane surface due to the relatively weak interaction. Strong electrostatic attractions are formed between the metal ions or SO3－ groups of the metal-containing polycationic membranes and the lipid phosphate head groups and (or) ester groups. Furthermore, the favorable hydrophobic interactions established between polymer backbone and alkyl chain of lipid bilayers may also facilitate these polycationic membranes penetrating the lipid bilayer, which can be


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Langmuir

easily seen from Figure 2. It is obviously, the strongest interaction is found between the AP-Ag membrane and lipids, while the polyanionic CP-CH3SO3 has the weakest interaction with the lipid membrane. In addition, our calculated interaction energies can be compared with other cationic compound-lipid membrane and protein/DNA-metal interaction energies. For example, the interaction energies (>30 kcal/mol) computed in this work between the metal-containing polycationic membranes and lipid membranes are larger than the interaction energy (27 kcal/mol) between quaternary ammonium containing compound (QAC) and DOPC–DOPG membrane.35 These interactions are also higher than that between the DNA and copper compound Casiopeínas (~10 kcal/mol),42 the Cu2+/Zn2+-human CAII binding free energies(18.8/-16.4 kcal/mol), and Ca2+-conformational free energy (13.1 kcal/mol).43,44 In a word, it seems that there is a positive relationship between the interaction energy and antimicrobial activity of polymeric membranes. Here, the simulated results agree well with the experimental results.

In addition to the electrostatic and hydrophobic interactions, other factors may also influence the antimicrobial activities. For example, the size of the metal cations may influence the antimicrobial activity. The effective ionic radius for Ag+, Cu2+, Zn2+ or Fe2+ is 115, 73, 74 and 64.5 pm, respectively.45 It seems that the total interactions (shown in Table 1) between AP-M and DOPC–DOPG membrane increase gradually with the effective ionic radius of metal ions (except Cu2+ and Zn2+) increase, which indirectly indicate that the size of the metal cations may influence the antimicrobial activity to a certain degree because the larger interaction energies manifest the higher antimicrobial activity. Furthermore, the AP-Ag exhibits the highest antimicrobial activities, may also be due to the strong binding between Ag+ and proteins of cell membrane (through the interactions with disulfide and sulfhydryl groups),46 which could damage


19

Langmuir

the structure and nature of the proteins.47 In addition, the DNA-Ag+ interactions, which caused the hydrogen bond displacement between adjacent nitrogen of purine and pyrimidine bases in DNA also enhance the antimicrobial properties.48 Potent bactericidal activities were also achieved for the AP-Cu. Since Cu2+ could produce reactive oxygen species in water, leading to the progressive oxidative damage of cell.49 In addition, the interaction between the bacterial outer membrane and Cu2+ causes the membrane to rupture (or produce holes in the outer membrane), leading to the loss of vital nutrients and water in the cell.50 The antibacterial mechanism of APFe is similar to that of AP-Cu. The existence of Fe3+ can generate a reactive oxygen species, damage DNA, lipids and proteins of cell wall, resulting in the cell death.51, 52 In the case of APZn, it is presumed that Zn2+ can bind with the teichoic acids in the microorganism membrane and prolong the lag phase of growth cycle, and thus inhibit the growth of organism generation.48 In addition, AP-Zn can inhibit cell wall synthesis, causing fungal cell lysis (bursting) and death due to the structural change of corona proteins.53 Therefore, it can be concluded that the metal cations significantly improve the antimicrobial efficiency of the polyanionic membranes against microbes as compared with organic cations.

80 60 40 20 0 0

1

2

3

Time (h)

Against E. coli

4

AP-NH AP-Ag AP-Cu AP-Fe AP-Zn

80 60 40 20 0 0

1

2

3

4

Time (h)

Against S.aureus

C100 Bacterial Viability (%)


Bacterial Viability (%)

B100

A100 Bacterial 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

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80 60 40 20 0

0

1

2 Time (h)

3

4

Against C.albicans

Figure 3. Time course of surviving E. coli (A) S. aureus (B) bacteria and C. albicans (C) fungus upon contacting with AP-M (M: NH+, Ag+, Cu2+, Fe3+, Zn2+) membranes (average of five samples).


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Langmuir

The antimicrobial activities of all the polyanionic membranes with various cations (NH+, Ag+, Cu2+, Fe3+, Zn2+) were further investigated by analyzing the survival rate of E. coli, S. aureus and C. albicans upon contact with polymeric membranes at various exposure times. Figure 3 illustrates the bactericidal activity of polyanionic membranes, using PET membranes as the control. After 1 h of incubation, all the membranes displayed the antibacterial activity. With the exposure time increased to 4 h (Figure 3A-C), more than 98% of the bacteria were killed by the polyanionic membranes. Particularly, about 100% of E. coli, S. aureus and C. albicans were eliminated by AP-Ag, AP-Cu and AP-Zn membranes, demonstrating the significant bactericidal activities.

Figure 4. Scanning electron microscopy (SEM) images of E. coli, S. aureus and C. albicans, cultured on the AP-M membranes (M: NH+, Ag+, Cu2+, Fe3+, Zn2+) for 4 h. PET (A, A’, A’’), AP-NH (B, B’, B’’), AP-Ag (C, C’, C’’), AP-Cu (D, D’, D’’), AP-Fe (E, E’, E’’) and AP-Zn (F, F’, F’’), respectively. Collapses and fusion of bacterial membrane on the AP-NH and AP-M membranes are observed.


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Scanning electron microscopy (SEM) images were further utilized to observe the morphological changes of bacteria on polyanionic membranes (Figure 4). Smooth and complete cell surfaces are observed for bacteria on the PET and CP-CH3SO3 membranes (Figure 4A, A’, A” and S6). However, a wide variety of cell disfigurations (collapses and fusion) were observed for the microbial cells on the polyanionic membranes (Figure 4B-F, 4B’-F’ and 4B”-F”), indicating the perforation of cell wall. The SEM images further confirm the antimicrobial activity of the AP-M membranes.

3.0

Biofilm Formation (%)

SC5413 △crk1

2.5 2.0 1.5 1.0 0.5

PZn A

PN H A PA g A PC u A PFe

3

A

SO 3

C

PC

H

A

PIm

0.0 B la PE nk T

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

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Figure 5. Antibiofilm activity against SC5413 and Δcrk1 C. albicans strains after contacting with AP-M (M: Im+, NH+, Ag+, Cu2+, Fe3+, Zn2+) membranes and CP-CH3SO3 for 48 h, with PET membranes as controls (left column).

The inhibition of biofilm formation is an important indicator for the assessment of antibacterial materials, especially in hospital with the real applications of medical implants. To


22

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determine the antibiofilm property of polyanionic membranes, wide-type SC5314 and its crk1 gene deleted Candida strains were employed to mimic the in vitro biofilm formation on the polymeric membrane surface. Figure 5 shows that all the AP-M (M: Im+, NH+, Ag+, Cu2+, Fe3+, Zn2+) membranes could effectively reduce more than 70% biofilm formation of C. albicans, if compared with that for CP-CH3SO3 membrane. The results may be due to the higher antimicrobial activities of polyanionic membranes which could kill the fungi within a relatively short time (Figure 1C, 3C). It is not surprising that all the polymer membranes presented better antibiofilm property for Δcrk1 strains (Δcrk1 C. albicans) since the crk1 gene is essential for the growth of C. albicans (see Figure 5). These results further validated the antibiofilm activities of the polyanionic membranes.

180

Relative Growth Rate (%)

160 140 120 100 80 60 40 20

A

P-

Zn

Fe P-

u A

P-

C

g A

P-

A

H A

N PA

3

Im

P-

SO 3

A

P-

C

H

PE

T

0

C

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

Langmuir

Figure 6. Relative growth rates (RGR) of the CP-CH3SO3 and AP-M(M: Im+, NH+, Ag+, Cu2+, Fe3+, Zn2+) and membranes to human dermal fibroblast cells detected by MTT assay.


23

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In addition to antimicrobial and antibiofilm activities, the biocompatibility of polyanionic membranes was evaluated by the MTT assay method and hemolysis.54,

55

The toxicity was

characterized by incubating with fibroblast cells with the membranes and evaluating using an MTT assay. The relative growth rate (RGR) of cells in contact with all the membranes was based on the OD values (at 490 nm) from the MTT assay. Figure 6 shows that the RGR values were higher than 80% for CP-CH3SO3 (105.3%), AP-Im (84.5%), AP-NH (86.8%), AP-Fe (157.6%) and AP-Zn (128.3%), suggesting non-toxicity to human skin fibroblast cells. While about 57.9% and 59.7% were observed for AP-Ag and AP-Cu membranes, respectively, indicating certain toxicity to cells.56 The toxicity of the AP-Ag and AP-Cu membranes may be due to the existence of Ag+ and Cu2+, which interact with the SH- in the protein, leading proteins inactivation.

Table 2. Hemolytic activity of the AP-M and CP-CH3SO3 membranes. Red blood cells treated with 2 % Triton and PBS were used as positive and negative control, respectively. Polymer membranes

Hemolysis rate (%)

PET

0.16±0.11

CP-CH3SO3

0.31±0.04

AP-Im

0.86±0.06

AP-NH

0.21±0.04

AP-Ag

4.48±0.29

AP-Cu

1.30±0.11

AP-Fe

3.63±0.06

AP-Zn

4.80±0.41

The hemolysis effect of the CP-CH3SO3 and AP-M (M: Im+, NH+, Ag+, Cu2+, Fe3+, Zn2+) membranes toward the fresh human red blood cells (RBC) was further investigated. All the


24

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membranes exhibited low hemolytic activity (5%) for RBC after 3h of contact time. Introduction of Ag+ highly increased the hemolytic activity from 0.21% for AP-NH to 4.48% for AP-Ag. In addition, AP-Cu, AP-Fe and AP-Zn are even low hemolytic, as shown in Table 2. The difference between mammalian red blood cell membranes and bacterial cell membranes induced the high selectivity. The outer membrane of mammalian cells is usually electrically neutral, while the outer membrane is normally negatively charged in a bacterium.57 The different electrostatic interactions between antibacterial agents and mammalian cells compared to microbial cells are also influential factors.25, 58 Both cytotoxicity and hemolysis results indicate that the synthesized polyanionic membranes are qualified for indirect contact biomedical materials (hemolysis rate:

Polyanionic Antimicrobial Membranes: An ... - ACS Publications

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