Membranes for Antibacterial Applications

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Metal-containing Poly(ionic liquid) Membranes for Antibacterial Applications Zhiqiang Zheng, Jiangna Guo, Hailei Mao, Qiming Xu, Jing Qin, and Feng Yan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.7b00165 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on April 2, 2017

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ACS Biomaterials Science & Engineering

Metal-containing Poly(ionic liquid) Membranes for Antibacterial Applications

Zhiqiang Zheng,a Jiangna Guo,a Hailei Mao,b Qiming Xu,b Jing Qin,a and Feng Yana,* 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; b

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

University, Shanghai, China.

* E-mail address of corresponding author: [email protected];

1

ACS Paragon Plus Environment


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ABSTRACT: Imidazolium−type metal-containing ionic liquid (IL) monomers and their corresponding poly(ionic liquid) (PIL) membranes coordinated with CuCl2 (PILM-Cu), FeCl3 (PILM-Fe) or ZnCl2 (PILM-Zn) were synthesized. The effect of metal ions on the antimicrobial activities against both Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was investigated. Compared with pristine PILM-Br membrane, PILM-Cu, PILM-Fe, PILM-Zn membranes exhibit enhanced antibacterial activities due to the attributes of both imidazolium cations and metal-containing anions. Furthermore, all the metal-containing PIL membranes present low hemolysis toward human red blood cell and high long-term antibacterial stability, even after immersion in water for 90 days, demonstrating clinical feasibility in topical applications.

KEYWORDS: Ionic liquids, poly(ionic liquid), metal-containing, antibacterial, polymer membrane

2


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Introduction The innovation of materials with antimicrobial activities has attracted great attention in modern healthcare owing to the overuse of antibiotics and rapidly increasing antibiotic resistance to pathogenic bacteria.1-2 In order to obtain efficient antibacterial materials without antibiotic resistance,3-6 antibiotic peptides,7 cationic compounds and polymers,8-13 silver,14-16 copper and their salts have been intensively studied.17-19 These agents show high antibacterial activities by destroying the target bacteria cell wall (or membrane), interfering with the DNA or RNA, or delivering metal ions to inhibit some certain enzymes.20-22 Currently, most cationic antimicrobial compounds or polymers reported present antibacterial activities with high selectivity for bacteria over mammalian cells.23-26 Therefore, increasing attention has been concentrated on cationic antimicrobial compounds or polymers,27-29 such as quaternary ammonium,30 pyridinium and phosphonium based materials.31-33 Ionic liquids (ILs), which composed of an inorganic anion and an organic cation, have attracted remarkable research interest due to the distinctive physicochemical properties.34-35 Various applications of ILs or poly(ionic liquid)s (PILs) in the field of bioengineering and biomaterials have been studied recently.36-39 The influences of both cations and anions on the biological activities were extensively studied.40-41 More recently, metal-containing ILs which combine the properties of ILs and the photophysical, optical, or catalytic properties that emanate from the incorporated metal have aroused wide attention.42-43 However, applications of metal-containing PIL membranes as antibacterial agents have not been investigated as far as we know. 3



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In this work, imidazolium-type ILs coordinated with CuCl2, FeCl3 or ZnCl2 were synthesized (Figure 1). The antibacterial activities of the metal-containing IL monomers against both Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were investigated by determination of the minimum inhibitory concentration (MIC). Meanwhile, the analogous PIL membranes (PILMs) were fabricated by means of photo−crosslinking of an imidazolium-type IL monomer with acrylonitrile and styrene, and following by coordination with metal ions. The antibacterial activities of the resultant PIL membranes were investigated via colony forming units (CFU) and scanning electron microscopy (SEM), which showed efficient antibacterial activities against both E. coli and S. aureus. Furthermore, the obtained PIL membranes exhibited high biocompatibility towards human red cell.

Experimental Section Materials.

1-Vinylimidazole,

1-bromoctane,

styrene,

acrylonitrile,

1,4-divinylbenzene, benzoin ethyl ether, methanol, ethanol, ethyl acetate, diethyl ether, phosphate buffered saline (PBS, pH = 7.4), anhydrous CuCl2, FeCl3, ZnCl2, and polyethylene terephthalate (PET) membranes were purchased from Shanghai Chemical Reagents Co. (Shanghai, China). Singlet oxygen sensor Green (SOSG) was purchased from Alfa-Aesar. All the reagents were analytical grade and used as received without further purification. All of the vinyl monomers were made inhibitor-free by passing through a column filled with neutral alumina. Deionized water was used throughout the experiments. Escherichia coli (E. coli) (ATCC 8099) and Staphylococcus aureus (S. aureus) (ATCC 6538) strains were kindly provided by 4


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Dr. Shengwen Shao (Huzhou University School of Medicine, China)

Characterization 1

HNMR spectra of synthesized compounds were recorded on a Varian 400 MHz

spectrometer using D2O as the solvent. The metal ions incorporated with ionic liquids were detected via electrospray ionization mass spectrometry (ESI-MS, Aligent 1200/6220). The membranes were characterized on a Specode 75 model spectrometer to obtain fourier−transform infrared (FT-IR) spectra in the range of 4000−400 cm−1. The water contact angle of the PIL membranes was detected by static contact angle measurements (USA Kino Industry Co.,Ltd). The turbidity of bacterial suspension was recorded by microplate reader (Multiskan GO 1510). Field-emission scanning electron microscope (SEM, Hitachi Model S−4700 and ZEISS EVO18, Germany) was used to observe the bacteria on the PIL membranes. Energy dispersive X-ray microanalysis (EDX) spectroscopy measurements were performed with the spectrometer attached on the ZEISS EVO18 field−emission SEM system. Fluorescence spectra were obtained using a HITACHI F-2500 spectrometer.

Synthesis of ionic liquid monomers

1-Octyl-3-vinylimidazolium bromide (IL-Br) was synthesized via stirring a mixture containing 1-bromoctane (0.075 mol) and 1-vinylimidazole (0.075 mol) at room temperature for 72 h. The obtained product was washed with ethyl acetate and diethyl ether three times respectively, and then dried under dynamic vacuum at room 5



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temperature for 24 h. 1H NMR (400 MHz, D2O): δ 7.78 (s, 1H), 7.57 (s, 1H), 7.12 (dt, 1H), 5.99–5.72 (m, 1H), 5.48 (d, 1H), 4.31 (t, 2H), 1.93 (d, 2H), 1.53–1.12 (m, 11H), 0.88 (t, 3H).

1-Octyl-3-vinylimidazolium bromodichlorocuprate (II) (IL-Cu) was synthesized via the stirring of IL (0.025 mol) with anhydrous CuCl2 (0.025 mol) in 20 ml methanol at room temperature for 24 h. The compound was obtained after the evaporation of methanol.44

1-Octyl-3-vinylimidazolium

bromotrichloroferrate

(III)

(IL-Fe)

and

1-octyl-3-vinylimidazolium bromodichlorozincate (II) (IL-Zn) was synthesized as described for 1-octyl-3-vinylimidazolium bromodichlorocuprate (II) (IL-Cu). Equal molar of IL and anhydrous FeCl3 (or ZnCl2) was mixed and stirred in methanol at room temperature for 24 h. The compound was obtained after the evaporation of methanol.44

Preparation of PIL membranes A mixture containing IL-Br (20%, molar ratio), styrene (20%, molar ratio), acrylonitrile (60 %, molar ratio), divinylbenzene (2 wt % of the formulation based on the weight of monomer), and 1 wt % of benzoin ethyl ether (photo-initiator) was ultrasonicated to obtain a homogeneous solution, which was further photo-crosslinked in a handmade glass mold by an UV light (~250 nm wavelength) at room temperature. The membrane thickness was controlled by the standard spacer bars (~50 µm in diameter). The unreacted monomer residues were removed by immersing the polymer 6


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membranes in ethanol and ultrasonicated at room temperature. Then the depurated membranes were thoroughly washed with deionized water to obtain the PIL-Br membranes. Metal-containing PIL membranes were prepared by immersing the PIL-Br membranes in CuCl2, FeCl3 or ZnCl2 saturated ethanol solution at room temperature for 48 h. For simplicity, the obtained membranes are abbreviated as PILM-Cu, PILM-Fe, and PILM-Zn, respectively.

Bacteria culture Prior to the antibacterial experiments with the PIL membranes, S. aureus and E. coli were grown in a Luria-Bertani broth medium (LB) at 37 °C for 24 h. The concentration of bacteria used in the experiments was controlled by the optical density at a wavelength of 600 nm (OD600). Here, the OD600 nm values of bacteria suspensions were determined to be about 0.1.

Minimum inhibitory concentration (MIC) test The antibacterial activities of the ILs were evaluated by MIC test according to the Clinical & Laboratory Standards Institute guidelines by using the microdilution broth format, 5% (volume ratio) dimethyl sulfoxide was used as a co-solvent to improve the solubility of the metal-containing ionic liquids.45 The plates were incubated at 37 °C for 24 h and read visually at set intervals. The MIC values were determined as the lowest concentration of ILs where there was an obvious inhibition of the bacteria. 7



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Colony forming unit (CFU) counting method Typically, the bacteria suspension was dropped onto the surface of the polymeric membranes (1.5×1.5 cm2) and incubated at 37 °C for 4 h, using PET membranes as the control. Then, 10 μL of the bacteria suspension was spread onto the agar plate. After incubated for 24 h at 37 °C, the viable colonies of bacteria were recorded.

Morphological change of the bacteria Bacteria suspension (100 μL, OD600

nm=0.1)

was dropped onto the polymeric

membranes surfaces (1.5×1.5 cm2). After 4 h of incubation at 37 °C, the membranes were totally immersed in 3 wt% glutaraldehyde solution for 3 h. Then the membranes were stepwisely dehydrated by 10 vol%, 30 vol %, 50 vol %, 70 vol %, 80 vol %, 90 vol % and 100 vol % ethanol (10 min for each step), respectively. Then the morphological changes of bacteria were observed by scanning electron microscope (SEM, Hitachi Model S−4700).

Reactive oxygen species (ROS) generation tests The tests of singlet oxygen generation were carried out in a closed centrifuge tube under the visible light at room temperature. In the presence of singlet oxygen generation, the fluorescent probe singlet oxygen sensor green (SOSG) changes into SOSG-EP which can be measured by monitoring the absorbance at 525 nm.46 Here, 0.01 g of each membrane was immersed in 2 mL of ultrapure water containing 10 μL of SOSG. After 4 h exposure to visible light, the ultrapure water was analyzed by 8


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fluorescence spectrometer. The absorbance at 525 nm was measured on a HITACHI F-2500 spectrometer.

Hemolysis assay Fresh human blood (3 mL) was kindly provided by a healthy donor. The erythrocytes were obtained by centrifugation at 1500 rpm for 20 min, and washed with PBS until the supernatant was pellucid. The red blood cells precipitate was diluted to 2 vol% in PBS. After the process of disinfection, PIL and PET membranes (1.5×1.5 cm2) were immersed into the diluted erythrocytes solutions (5 mL for each tube), incubated at 37 oC for 3 h, respectively. Then the diluted erythrocytes samples were centrifuged at 1500 rpm for 20 min, and 100 μl aliquots of the supernatant were then transferred into a 96-well plate. The hemoglobin release was detected on the Eon microplate spectrophotometers (Bio Tek Instruments, Inc.), the OD values were recorded at 576 nm. The red blood cells with 2 % Triton served as the positive control, while the cells in PBS served as negative control. The hemolysis rate was determined by the following formula: Hemolysis rate (%) 

OD

sample  OD negative control

OD positive

control  OD negative control

 100%

9



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Results and Discussion.

Figure 1. A) Chemical structures of metal-containing imidazolium-type ILs, B) ball-and-stick model of metal-containing anions, and C) electrospray ionization mass spectrometry (ESI-MS) of the metal-containing anions, CuCl2Br−, FeCl3Br− and ZnCl2Br−, coordinated in ionic liquids.

Figure 1A and Figure S1 show the chemical structure and synthetic route of pristine

and

metal-containing

1-octyl-3-vinylimidazolium

bromide

imidazolium−type (IL-Br),

IL

monomers:

1-octyl-3-vinylimidazolium

bromodichlorocuprate (II) (IL-Cu), 1-octyl-3-vinylimidazolium bromotrichloroferrate (III) (IL-Fe), and 1-octyl-3-vinylimidazolium bromodichlorozincate (II) (IL-Zn). Before the antibacterial activity test, the chemical structures and purity of these IL monomers were confirmed by 1H NMR (see Figure S1) and electrospray ionization mass spectrometry (ESI-MS). Figure 1C shows the ESI-MS spectra of the synthesized metal-containing IL monomers. The intensive peaks corresponding to 10


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CuCl2Br−, FeCl3Br− and ZnCl2Br− can be observed at m/z 211, 239, and 212, respectively, indicating the successful formation of metal-containing anions.

Table 1. The antibacterial activities of imidazolium-type ILs coordinated with CuCl2, FeCl3 and ZnCl2 measured as MIC. MIC (μmol mL−1)

MIC (μmol mL−1)

S. aureus

E. coli

IL-Br

2.610±0.003

1.321±0.002

IL-Cu

0.056±0.002

0.222±0.001

IL-Zn

0.886±0.003

0.886±0.002

IL-Fe

1.254±0.005

1.110±0.003

Samples

The antibacterial activities of the synthesized IL monomers were tested by MIC using Gram-positive S. aureus and Gram−negative E. coli as the model bacteria. The average MIC values were calculated and listed in Table 1. It can be clearly seen that all the IL monomers synthesized in this work showed antibacterial activities against both S. aureus and E. coli. The antibacterial activities increased in the order as: IL-Br < IL-Fe < IL-Zn < IL-Cu (against E. coli and S. aureus). Therefore, it can be concluded that the presence of metal-containing anions may significantly improve the antimicrobial efficiency of the ILs against microbes. Figure S2 shows the bacteria growth in IL monomers under the concentration of MIC values for 48 h. The results indicate that the ILs could effectively inhibit the growth or even kill the bacteria.

11



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Figure 2. A) Synthesis of antibacterial imidazolium-type PIL membranes containing CuCl2Br−, FeCl3Br− and ZnCl2Br− anions, respectively; B) photographs (top) of metal-containing PILM-Cu, PILM-Fe, and PILM-Zn and corresponding metal elemental mapping of membrane surfaces (bottom).

From the view point of medical applications, flexible and tough polymer membranes with excellent antibacterial activities are highly desirable. Based on the antibacterial properties of the metal-containing IL monomers studied above, a series of analogous PIL membranes were prepared. Here, IL-Br monomer was photo-crosslinked with acrylonitrile and styrene (using divinylbenzene as the cross-linking agent), and followed by the coordination with corresponding metal halide to prepare metal-containing PIL membranes (shown in Figure 2A). Here, styrene and acrylonitrile were used as the co-monomers for the preparation of 12


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polymer membranes due to the high chemical resistance and excellent mechanical property of poly(styrene-co-acrylonitrile) membrane. It is expected that the metal-containing imidazolium units possess the antimicrobial activities, while the poly(styrene-co-acrylonitrile) region provides the high mechanical properties. The obtained polymer membranes (with the thickness of about 50 m) are flexible, and tough enough to be cut into any desired shape and size. In addition, scanning electron microscope (SEM) images show that the PIL membrane surfaces are uniform and smooth without any visible pores (see Figure S3). The prepared PIL membranes were first characterized by FTIR spectrum. The absorption bands at about 2980 cm−1 and 1490 cm−1 confirm the existence of polystyrene units, while the peak at 2220 cm−1 belongs to the cyano (C≡N) group. However, no new absorption peaks were observed after the coordination with inorganic salts. The prepared PIL membranes were further characterized by energy dispersive X-ray (EDX) microanalysis. Figure 2B and Figure S5 show the elemental mapping and composition of the PIL membrane surface, respectively. As can be seen that the metal (Cu, Fe, and Zn) atoms were uniformly distributed on the surface of the PIL membranes. The metal content (molar ratio) on the membrane surface was determined to be about 0.28%, 2.72% and 0.21% for Cu, Fe, and Zn, respectively. The different metal content is mainly due to the different coordination ability between CuCl2, FeCl3 or ZnCl2 and Br− anions. Both the charge number and radius of metal ions affect the coordination strength. The higher charge number and the smaller radius, the stronger coordination strength.47-50 The radius of Cu2+, Fe3+ and Zn2+ was determined to be 13



0.74 Å, 0.64 Å and 0.75 Å, respectively. Therefore, it is not surprising that PILM-Fe presents highest metal content. These results further confirm the successful synthesis of metal-containing PIL membranes. Figure S6 shows the water contact angle measurement of the PIL membranes. It can be seen that the hydrophobicity of PIL membranes was increased after the coordination with the metal salts due to the relatively higher hydrophobicity of metal-containing IL units. B 140 120 100

Control PILM-Br PILM-Fe

140

PILM-Cu PILM-Zn

Bacterial Viability (%)

A Bacterial Viability (%)

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80 60 40 20 0

Against S. aureus

120 100

Control PILM-Br PILM-Fe

PILM-Cu PILM-Zn

80 60 40 20 0

Against E. coli

Figure 3. Bacterial viabilities of (A) S. aureu and (B) E. coli after contacting with PIL membranes for 4 h. PET membranes were used as the control (average of five samples).

The antibacterial properties of the PIL membranes were characterized by colony forming units (CFU) counting method. The statistical results of the viabilities of E. coli and S. aureus are presented in Figure 3 and Figure S7. After the contact with the PIL membranes for 4 h, the viable colonies of E. coli and S. aureus on the surface of PILM-Cu and PILM-Zn membranes decreased sharply, if compared with PET membrane. The relative viabilities of both E. coli and S. aureus were lower than 0.1% for PILM-Cu membrane. The viabilities of S. aureus and E. coli for PILM-Zn membrane were 4.25% and 20.1%, respectively. The relatively poor antibacterial 14


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activities were found for PILM-Fe and PILM-Br membranes, with the viabilities of about 70 % and 80 % for both S. aureus and E. coli, respectively. The antibacterial activities of PIL membranes against both S. aureus and E. coli are consistent with the small molecule metal-containing IL monomers, that is, PILM-Br < PILM-Fe < PILM-Zn < PILM-Cu. Therefore, PILM-Cu shows the highest antibacterial activities, while PILM-Br membrane exhibits the lowest antibacterial activities. The antibacterial mechanism of the PIL membranes may involve two sections. Firstly, the imidazolium cations interact with the phosphate groups of the microbial cell wall via electrostatic force, then the hydrophobic segments of the polymers insert into the hydrophobic regions of the lipid membrane of bacteria and destroy the cell membrane.51 In addition, it is hypothesized that the existence of metal ions may generate reactive oxygen species (ROS), such as singlet oxygen (1O2), leading to the progressive oxidative damage of bacteria cell wall (or membrane).52-54 28000

PILM-Cu PILM-Fe PILM-Zn PILM-Br

24000

Absorbance

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


20000 16000 12000 8000 4000 0 500

550

600

650

700

Wavelength (nm)

Figure 4. Fluorescence spectra of SOSG (λem=525 nm) with the existence of PILM-Br, PILM-Cu, PILM-Fe, PILM-Zn membranes in ultrapure water. About 0.01 g of each PIL membrane was immersed in 2 mL of ultrapure water containing 10 μL of SOSG, and tested after 4 h exposure to visible light at room temperature. 15



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Figure 4 shows the fluorescence spectra of singlet oxygen sensor green (SOSG) with the existence of PILM-Br, PILM-Cu, PILM-Fe, PILM-Zn membranes in ultrapure water. The generation of 1O2 was probed by SOSG.46 As it can be seen that all the metal-containing PIL membranes generated 1O2 in water under the visible light. However, no 1O2 generation was observed for PILM-Br membrane. Therefore, it is not surprising that the PILM-Br showed the worst antibacterial activity in this work. Among the metal-containing PIL membranes investigated, PILM-Zn generated highest concentration of 1O2 in water, leading to the high antibacterial activities. As for PILM-Cu membrane, although relatively less 1O2 was generated, the excellent antibacterial activities may also be due to the strong interaction between the CuCl2Br− and bacterial outer membrane, which causing the membrane rupture and leading to the loss of vital nutrients in the cell.55

Figure 5. SEM images of S. aureus (A−E, top) and E. coli (a−e, bottom), cultured on the PIL membranes for 4 h, using PET membrane as the control. PET (A, a), PILM-Br (B, b), PILM-Cu (C, c), PILM-Fe (D, d) and PILM-Zn (E, e), respectively. Collapses and fusion of bacterial cell membranes on the PIL membranes were observed (indicated by white arrows). Scale bar: 1 μm. 16


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Figure 5 shows the morphological changes of S. aureus and E. coli on the surface of PIL membranes observed by SEM. Compared with the complete and smooth surfaces of the bacteria incubated on the PET membranes, collapsed and distorted bacterial walls were clearly observed on the PILM-Cu and PILM-Zn membranes, indicating that the bacteria cell wall or membrane structure was wholly collapsed and destroyed. However, less deformation of S. aureus and E. coli was observed on PILM-Br and PILM-Fe membranes, indicating relatively poor antibacterial activities.

Table 2. The hemolysis rate of synthesized PIL membranes. Membranes PET PILM-Br PILM-Cu PILM-Fe PILM-Zn

Hemolysis Rate (%) 0.00±0.10 0.17±0.14 0.17±0.15 0.00±0.11 0.06±0.06

The excellent biocompatibility of materials is vital and desirable for the medical application. Table 2 shows the results of the hemolysis assay of all the PIL membranes towards the fresh human red blood cells (RBCs). It can be seen that all the PIL membranes present an extreme low hemolytic rate (

Membranes for Antibacterial Applications

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