Synthesis of Pyrrolidinium-Type Poly(ionic liquid) Membranes for

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Synthesis of Pyrrolidinium-type Poly(Ionic Liquid) Membranes for Antibacterial Applications Jing Qin, Jiangna Guo, Qiming Xu, Zhiqiang Zheng, Hailei Mao, and Feng Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00387 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 14, 2017

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ACS Applied Materials & Interfaces

Synthesis of Pyrrolidinium-type Poly(Ionic Liquid) Membranes for Antibacterial Applications Jing Qin,a Jiangna Guo,a Qiming Xu,b Zhiqiang Zheng,a Hailei Mao*, b and 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. bDepartment of Anesthesiology and Critical Care Medicine, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.

E-mail: [email protected];

[email protected]

1

ACS Paragon Plus Environment


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Abstract: Pyrrolidinium-type small molecule ionic liquids (ILs), poly(ionic liquid) (PIL) homopolymers, and their corresponding PIL membranes were synthesized and used for antibacterial applications. The influences of substitutions at the N position of pyrrolidinium cation on the antimicrobial activities against both Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were studied by minimum inhibitory concentration (MIC). The antibacterial efficiency of both the small molecule ILs and PIL homopolymers increased with the increase of the alkyl chain length of substitutions. Furthermore, PIL homopolymers show relatively lower MIC values, indicating better antimicrobial activities than those of corresponding small molecule ILs. However, the antibacterial properties of the PIL membranes are contrary to corresponding ILs and PIL homopolymers, which reducing with the increase of alkyl chain

length.

Furthermore, the resultant

PIL membranes

show

excellent

hemocompatibility and low cytotoxicity towards human cells, demonstrating clinical feasibility in topical applications.

Keywords: Antibacterial materials; pyrrolidinium cations; poly(ionic liquid)s; polymer membranes; cytotoxicity.

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Introduction Bacterial infection diseases have caused lots of deaths in modern healthcare due to the overuse of antibiotics and rapid increase multi-drug-resistant pathogens.1-6 Therefore, the development of efficient antibacterial materials without resistance is extremely urgent and has attracted great attention in the field of healthcare.7 Antimicrobial agents, such as antimicrobial peptides,8-10 silver ions,11, 12 carbon-based materials13 and cationic polymers (or compounds)14-16 have been intensively studied. The antibacterial mechanisms of these antibacterial agents generally involve the disturbing cell membrane, interfering the genetic material (such as DNA or RNA) synthesis or inhibiting certain enzymes.17 From the view point of safe clinical applications,

an

ideal

antimicrobial

material

should

show broad-spectrum

and efficient antibacterial properties, and be low toxic and highly biocompatible. Therefore, cationic antimicrobial polymers substituted with various cations, such as quaternary ammonium,18,

19

phosphonium,20-22 pyridinium23,

24

and imidazolium

cations25, 26 have been extensively studied. Poly(ionic liquids)s (PILs) are materials that comprised of polymeric backbone and an ionic liquid (IL) species in repeating units. The designability of ILs and the selectivity of polymer segments enrich the properties and applications of PILs, which have aroused considerable attention in polymer and material sciences.27-29 The cationic structures make it possible to develop IL and (or) PIL type antibacterial agents. More recently, quaternary ammonium (QA) and imidazolium cation based PILs have been synthesized and characterized with their antibacterial properties. For example, the antimicrobial activities of QA/poly(methyl methacrylate) hybrid films 3



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changed with QA compound content.30 Ying et al. prepared the amphiphilic imidazolium type oligomer and polymers, which can efficiently inhibit the growth of fungi and clear the fungal biofilm, and meanwhile display minimal hemolysis.31 Yan and co-workers prepared imidazolium type PILs and followed by anion-exchange with L-proline or L-tryptophan. The resulted PIL membranes exhibited high antimicrobial properties due to the synergistic attributes of imidazolium cations and anions.32 More recently, the structure-antibacterial activity relationship of bis-imidazolium cations and their corresponding polymer membranes was studied.33 The results indicated that the higher charge density (bis-cations) of imidazolium cations, the higher antimicrobial activities, although the toxicity and hemolysis rate of bis-imidazolium based polymer membranes need to be lowered. It has already been demonstrated that pyrrolidinium cation based ILs are usually less toxic if compared with QA and imidazolium analogues.34-37 However, synthesis of pyrrolidinium-based polymer materials for antibacterial applications has been rarely reported. In this work, we present the synthesis and characterization of pyrrolidinium-based ILs and PILs antibacterial materials. Pyrrolidinium-based small molecule ILs and corresponding PIL homopolymers substituted with various alkyl chains were synthesized and characterized with their antibacterial activities against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Furthermore, pyrrolidinium-based PIL membranes were prepared by photo-crosslinking of dially methyl ammonium bromide with acrylonitrile and styrene (Scheme 1). The antibacterial activity, hemolysis and cytotoxicity between pyrrolidinium-based ILs, 4


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and their corresponding PIL homopolymers and PIL membranes were investigated systematically.

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Scheme 1. (A) Chemical Structures of pyrrolidinium-based ILs and their corresponding PIL homopolymers; Synthesis of pyrrolidinium-based (B) PIL homopolymers; and (C) PIL membranes.

Experimental Section Materials. 1-Methylpyrrolidine, diallylmethylamine, 1-bromobutane, 1-bromohexane, 1-bromoctane,

1-bromodecane,

1-bromododecane,

ether,

ethanol,

styrene, 6


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acrylonitrile,

1,4-divinylbenzene

azobisisobutyronitrile

(AIBN),

(DVB),

deuterium

benzoin oxide

ethyl

(D2O),

ether,

acetone,

d6-dimethylsulfoxide

(d6-DMSO), and chloroform-d (CDCl3) were analytic grade and used as received without further purification. Phosphate buffered saline (PBS, pH=7.4) and polyethylene terephthalate (PET) was purchased from Shanghai Chemical Reagents Co.

(Shanghai,

China).

The

chain

transfer

agent

(CTA),

(S)-2-(ethyl

propionate)-(o-ethyl xanthate) was synthesized according to the literature.38 S. aureus (ATCC 6538) and E. coli (ATCC 8099) strains were kindly provided by Dr. Shengwen Shao (Huzhou University School of Medicine, China). Distilled deionized water was used throughout the whole experiments.

Synthesis of Pyrrolidinium-based ILs A series of Py-Cx (x=4, 6, 8, 10, 12, see Scheme 1) ILs were synthesized via stirring of 1-bromoalkane (2.74 g, 0.02 mol) with 1-methylpyrrolidine (1.7 g, 0.02 mol) in ethyl acetate at room temperature for 48 h (see Figure S1). The obtained product was washed three times with diethyl ether after the evaporation of solvent, and then dried under dynamic vacuum at room temperature for 24 h. 1-Butyl-1-methylpyrrolidinium bromide (Py-C4). White solid. 1H NMR (400 MHz, D2O): 3.49 (m, 4H), 3.37-3.17 (t, 2H), 2.97(s, 3H) 2.20 (s, 4H), 1.77 (m, 2H), 1.38 (m, 2H), 0.93 (t, 3H). 1-Hexyl-1-methylpyrrolidinium bromide (Py-C6). White solid. 1H NMR (400 MHz, D2O): 3.48 (d, 4H), 3.36-3.23 (m, 2H), 3.01 (s, 3H), 2.19 (s, 4H), 1.77 (m, 2H), 1.32 (t, 7



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6H), 0.86 (t, 3H). 1-Octyl-1-methylpyrrolidinium bromide (Py-C8). White viscous oil. 1H NMR (400 MHz, D2O): 3.49 (m, 4H), 3.31 (t, 2H), 2.97 (s, 3H), 2.20 (s, 4H), 1.79 (s, 2H), 1.31 (m, 10H), 0.85 (t, 3H). 1-Decyl-1-methylpyrrolidinium bromide (Py-C10). Colorless viscous oil. 1H NMR (400 MHz, D2O): 3.51 (m, 4H), 3.40-3.27 (m, 2H), 3.05 (s, 3H), 2.22 (s, 4H), 1.87-1.72 (m, 2H), 1.45-1.15 (m, 14H), 0.88 (t, 3H). 1-Dodecyl-1-methylpyrrolidinium bromide (Py-C12). Yellowish viscous oil. 1H NMR (400 MHz, D2O): 3.56 (m, 4H), 3.45-3.28 (m, 2H), 3.08 (s, 3H), 2.22 (s, 4H), 1.79 (s, 2H), 1.33 (m, 18H), 0.87 (t, 3H).

Synthesis of Pyrrolidinium-based PIL Homopolymers In brief, poly(diallyl methyl amine hydrochloride) was synthesized by a typical reversible addition–fragmentation chain transfer (RAFT) polymerization. A mixture containing diallyl methyl ammonium hydrochloride, AIBN (1 mol%, as initiator), and (S)-2-(ethyl propionate)-(o-ethyl xanthate) (1 mol%, as RAFT agent) was stirred at 60 °C for 12 h under a nitrogen atmosphere (Figure S2). The reaction was stopped by precipitating into acetone. The obtained polymer was dissolved in methanol at 0 °C, followed by slowly adding sodium methoxide solution (1.0 equiv., 30 wt % in methanol). The produced sodium chloride was filtered after stirring for 4 h. The filtrate was evaporated and dried in a vacuum oven at 50 °C for 12 h before the characterization. The relative molecular weight and molecular weight distribution of 8


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the synthesized polymers were test by GPC and summarized in Table S1. 1H NMR (400 MHz, CDCl3): δ 2.94-2.63 (s, 2H), 2.29 (d, 3H), 2.14 (d, 2H), 1.92-1.62 (s, 2H), 1.49-0.77 (m, 4H). A mixture containing synthesized poly(diallyl methyl amine) (with the same molecular weight and molecular weight distribution) and bromoalkane, was further stirred in DMF at 80 oC for 24 h. The obtained raw product was purified by precipitating into ether three times to remove the unreacted raw materials. The resultant polymers were dried in a vacuum oven at 60 oC and characterized by 1H NMR. The quaternization reaction degree of the polymer could be determined by the element ratio of Br/N based on the energy-dispersive X-ray (EDX) spectra. All the quaternization degree was found to be in the range of 88 % to 97 % (see Figure S2).

Preparation of Pyrrolidinium-based PIL Membranes A mixture containing dially methyl ammonium bromide (20 mol%), styrene (20 mol%), acrylonitrile (60 mol%), divinylbenzene (1 wt % to the formulation based on the total weight), and 1 wt % of benzoin ethyl ether (photoinitiator) was ultrasonicated to homogeneous solution. The obtained mixture solution was photo-initiated by ultraviolet irradiation (250 nm wavelength) in a glass mold for 30 min at room temperature. Subsequently, the obtained PIL membranes (∼100 μm in thickness) were immersed and ultrasoniced in ethanol at room temperature for 24 h to remove the unreacted monomer. Finally, the obtained PIL membranes were washed with deionized water. 9



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Characterization 1

H NMR spectra were recorded on a Varian 400 MHz spectrometer with CDCl3, D2O

or d6-DMSO as the solvent. A field-emission scanning electron microscope (SEM, Hitachi Model S-4700) were taken to observe the morphology of the bacteria on the polymeric membranes. The energy-dispersive X-ray (EDX) spectra were measured with

the spectrometer

attached on the

ZEISS

EVO18.

Gel

permeation

chromatography (GPC, TOSOH HLC-8320) equipped with TSK gel Muliti pore HZ-N (3) 4.6 × 150 mm column was used to measure the number-average molecular weight (Mn) and molecular weight distribution (PDI, Mw/Mn) of the synthesized homopolymers. Dimethyl formamide was served as the eluent with a flow rate of 0.6 mL min-1. The measurements were carried out at 40 oC. Fourier transform infrared (FT-IR) spectra of the prepared membranes were recorded on a Thermo scientific Nicolet 6700 FT-IR spectrometer in the range of 4000–400 cm-1. The optical density (OD) values were tested on a microplate reader (Bio Tek Instruments, Inc.).

Bacteria Culture Luria–Bertani broth medium (LB) was used to culture E. coli and S. aureus at 37 °C up to the exponential growth phase. The bacterial concentration was determined by measuring OD600 nm. The bacterial suspending solutions (OD600 nm=0.1) were used to characterize the antibacterial properties of pyrrolidinium-based ILs and corresponding PIL homopolymers.

10


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Minimum Inhibitory Concentration (MIC) Test The antibacterial activities of ILs and corresponding PIL homopolymers were investigated by the examination of MIC values using the broth microdilution format according to Clinical & Laboratory Standards Institute guidelines. ILs and corresponding PILs were diluted gradiently with broth in the 96-well plates, following by adding 100 μL microbial suspension (OD600=0.1) in each well. Plates were incubated at 37 oC and tested the optical density (OD) at set intervals with a microplate reader (Bio Tek Instruments, Inc.). MIC is expressed as the minimum IL or corresponding PIL concentration that results in 50% inhibition of bacterial growth after an incubation period of 24 h.

Antibacterial Activities of Synthesized PIL Membranes The bacterial suspension was dropped onto sterilized PET (as negative control) and PIL membranes (1.5×1.5 cm2) and incubated at 37 oC. After 4 h, 10 μL bacterial suspension was dropped onto a LB agar plate and uniformly coated. The number of viable bacterial colonies was recorded after incubated at 37 oC for 24 h. Each colony assay test was repeated three or more 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 

Anegative control  Bsample Anegative control

 100%

11



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Morphological changes of the bacteria The morphological changes of bacteria incubated on the surfaces of the PIL membranes were examined by SEM. The bacterial suspension (OD600=0.1) of E. coli or S. aureus was dropped onto the membranes surfaces and incubated at 37 °C. After 4 h, the membranes were immersed in 2.4 wt% glutaraldehyde solution for 2h, followed by dehydrating gradiently in 10 vol%, 30 vol%, 50 vol%, 70 vol%, 80 vol%, 90 vol% and 100 vol% ethanol solution for 10 min of each time, respectively.

Hemolysis assay Fresh human blood (3 mL) was centrifuged at 1500 rpm for 15 min, and washed with PBS till the supernatant was transparent. The precipitate of red blood cells was diluted to 2 vol% with PBS. The sterilized PET and PIL membranes (1.5×1.5 cm2) were dipped into the diluted blood (5 mL for each tube) solution and incubated at 37 o

C for 3 h, respectively. 100 μL aliquot of the supernatant was transferred into a

96-well plate after the treated diluted blood samples were centrifuged at 1500 rpm for 15 min. The hemoglobin release was evaluated by recording OD576 nm with the Eon microplate spectrophotometers (Bio Tek Instruments, Inc.). The red blood cells in PBS and with 2 % Triton were used as negative and positive control, respectively. The hemolysis rate was calculated according to the following formula: Hemolysis rate (%) 

OD

sample  ODnegative control

ODpositive

control  ODnegative control

100%

12


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Cytotoxicity evaluation Human dermal fibroblasts were offered by Shanghai Ninth People’s Hospital, China. The protocols for the isolation and culture of the human dermal fibroblasts were followed with the processes reported earlier.39,

40

The toxicity of PIL

homopolymers and PIL membranes against fibroblasts was evaluated via 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, human dermal fibroblasts (1×104 CFU/mL) in 10% fetal calf serum medium were cultured in a 24-well plate for 48 h. The PET and PIL membranes (1.0 × 1.0 cm2) were dipped into the fibroblast cell solutions and cultured together at 37 oC for 72 h, followed by adding 0.1 mL MTT solution (5 g L-1 in PBS) into each well and incubated at 37 oC. After 4 h, the supernatant was removed, 0.75 mL DMSO was added in each well to dissolve formazan crystals. The OD values at 490 nm were recorded to evaluate formazan release with the Eon microplate spectrophotometers (Bio Tek Instruments, Inc.). All the measurements were repeated for more than three times. The relative growth rate (RGR) of the human dermal fibroblast cells was calculated according to the following formula:41

RGR (%) 

ODsample ODcontrol

100%

Results and discussion Scheme 1A shows the chemical structures of pyrrolidinium-based small molecule ILs and PIL homopolymers synthesized in this work. 1-Butyl-1-methylpyrrolidinium bromide

(Py-C4),

1-hexyl-1-methylpyrrolidinium

bromide

(Py-C6), 13



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1-octyl-1-methylpyrrolidinium bromide (Py-C8), 1-decyl-1-methylpyrrolidinium bromide (Py-C10), and 1-dodecyl-1-methylpyrrolidinium bromide (Py-C12) were first synthesized (see Figure S1). The chemical structure and purity of the synthesized ILs were confirmed by 1HNMR spectra. Furthermore, a series of corresponding PIL homopolymers, poly(N-hexyl

poly(N-butyl

N-methyldiallylammonium

N-methyldiallylammonium

N-methyldiallylammonium N-methyldiallylammonium

bromide) bromide)

bromide)

bromide)

(PPy-C6),

(PPy-C8), (PPy-C10),

(PPy-C4),

poly(N-octyl poly(N-decyl poly(N-dodecyl

N-methyldiallylammonium bromide) (PPy-C12), were synthesized by a typical RAFT polymerization and following quaternization reaction (see Scheme 1B and Table S1). The antibacterial activities of pyrrolidinium-based small molecule ILs and PIL homopolymers were tested by minimum inhibitory concentration (MIC), the lowest concentration that can inhibit 50% visible growth of microorganisms after 24 h incubation, using Gram-negative E. coli and Gram-positive S. aureus as the model microorganisms. The MIC values were summarized and listed in Table 1. As it can be seen that the alkyl chain length of substituents at the N position of pyrrolidinium cations highly affects the MIC values of ILs and PILs against both E. coli and S. aureus in the solutions. The longer the alkyl chains, the lower MIC values are. In addition, PILs exhibited relatively lower MIC values relative to those of the small molecule ILs (except Py-C12 and PPy-C12), indicating higher antibacterial activities of PIL homopolymers. It is hypothesized that the antibacterial mechanism of the cationic compounds (or polymers) may involve two aspects. Since pyrrolidinium 14


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cations of ILs and PILs could freely move in the solution, the electrostatic interaction between the cationic pyrrolidinium groups and the electro-negative phosphate groups of the microbial cell wall may destroy the cell membrane. Then the hydrophobic segments of the compounds may insert in the hydrophobic regions of the lipid membrane of bacteria, leading to the leakage of the electrolyte out of the cell membrane (poration) and cell death.42, 43

Table 1. The antibacterial activities of pyrrolidinium-based small molecule ILs and corresponding PIL homopolymers measured as MIC.

ILs

PILs*

Samples

MIC (μmol mL−1) S. aureus

MIC (μmol mL−1) E. coli

Py-C4

64.505

83.333

Py-C6

14.920

29.88

Py-C8

1.808

1.899

Py-C10

0.559

0.622

Py-C12

0.015

0.020

PPy-C4

0.549

2.196

PPy-C6

0.236

0.548

PPy-C8

0.147

0.424

PPy-C10

0.112

0.224

PPy-C12

0.061

0.090

*

MIC of PILs represents the concentration of repeating units.

It should be noted that the MIC values to Gram-negative E. coli is higher than those to Gram-positive S. aureus. Such a difference may be due to the different structures of bacterial cell walls.44, 45 The cell wall of the Gram-negative bacteria is 15



composed of two layers, an outer membrane (about 7-8 nm) which is rich in negatively charged lipopolysaccharide, and a relatively thin peptidoglycan inner layer (about 2–7 nm). As for the Gram-positive bacterium, the cell wall is mainly made up of a thick (about 20–80 nm) but porous peptidoglycan layer, which is interconnected with negatively charged teichoic acid.46-48 Therefore, it is supposed that the hydrophobic alkyl chains are more likely to insert into the porous cell wall of the Gram-positive bacteria and disturb peptidoglycan layer to lead the cell death,49, 50 which leads to better antimicrobial properties of ILs and PILs against S. aureus, eventually. A)

Against E. coli

0.5

Control Py-C4 Py-C6 Py-C8 Py-C10 Py-C12

0.3

B)

0.2

Against S. aureus

0.7

Control Py-C4 Py-C6 Py-C8 Py-C10 Py-C12

0.6 0.5

OD600

OD600

0.4

0.4 0.3 0.2

0.1

0.1

0

10

20

30

40

50

60

70

0

10

20

Time (h)

C)

0.5 Against

30

40

50

60

70

Time (h)

E. coli

Control PPy-C4 PPy-C6 PPy-C8 PPy-C10 PPy-C12

D) 0.7 Against S. aureus

0.3 0.2

Control PPy-C4 PPy-C6 PPy-C8 PPy-C10 PPy-C12

0.6 0.5

OD600

0.4

OD600

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0.4 0.3 0.2

0.1

0.1 0

10

20

30

40

50

60

70

0

10

20

Time (h)

30

40

50

60

70

Time (h)

Figure 1. The growth curves of bacteria incubated with pyrrolidinium-based small molecule ILs and corresponding PIL homopolymer solutions under the concentration of individual MIC for 48 h against E. coli (A and C) and S. aureus (B and D). 16


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The growth curves of bacteria incubated with ILs and PILs under MIC values were further investigated by microplate reader, as shown in Figure 1. The OD600 values of both pyrrolidinium-based ILs and corresponding PILs decreased sharply within 48 h. The results indicated that the pyrrolidinium-based ILs and corresponding PILs could effectively kill the bacteria and inhibit their growth. Polymeric membranes with intrinsically antimicrobial activities and robust mechanical properties have a wide range of prospects in practical applications. Here, pyrrolidinium-based PIL membranes were synthesized via photo-cross-linking of a dially

methyl

ammonium

bromide

with

acrylonitrile

and

styrene,

using

divinylbenzene as the crosslinker (Scheme 1C). The aim of co-polymerization with styrene and acrylonitrile was to enhance their chemical resistance and robustness of the PIL membranes.51 The chemical structure and composition of the synthesized PIL membranes were characterized by FTIR spectra (Figure S3). It can be seen that all the membranes show an absorption band of cyano groups (C≡N) at about 2240 cm−1, and the absorption peak at 2945 cm−1, which belongs to −CH3 and −CH2−. The absorption peak at 1440 cm−1 is the characteristic peak of pyrrolidinium cations, while the peak at 1600 cm−1 is assigned to the benzene rings. The results confirm the successful synthesis of pyrrolidinium-based PIL membranes. Moreover, scanning electron microscopy (SEM) of the PIL membranes (Figure S4) displays that the synthesized membrane surfaces are uniform and smooth, without any obvious pores.

17



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Figure 2. Bacterial viabilities of (A) E. coli and (B) S. aureus after contacting with pyrrolidinium-based PIL membranes for 4h, using PET membranes as the control.

Figures 2 and S5 show the antibacterial activities of the synthesized PIL membranes. It can be seen that the growth of both E. coli and S. aureus bacteria are inhibited after contact with the PIL membranes for 4 h. The pyrrolidinium cations are fixed on the polymer backbone, therefore, the antibacterial activity can only be observed upon exposure to the bacteria. It should be noted that the antibacterial properties decreased with the increase of alkyl chain length of substituents of the pyrrolidinium cations. These results are contrary to the antibacterial properties of corresponding pyrrolidinium-based small molecule ILs and PIL homopolymers studied above. Such an inconsistency may be due to the effects of different carbon chain orientations in the IL and PIL bacteria suspensions, and on the PIL membrane surfaces. It is speculated that the substituted hydrophobic carbon chains of pyrrolidinium-based ILs or PILs may self-assemble (or aggregate) in the bacteria suspension.26,

52, 53

These hydrophobic carbon chains tend to insert into the 18


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hydrophobic cell membranes and thus increase the antimicrobial activities. The longer the carbon chain of pyrrolidinium-based ILs or PILs, the stronger interaction with the phospholipids bilayer of bacteria is. Therefore, the antibacterial activities of these pyrrolidinium-based IL or PIL solutions increase with the length of the carbon chains. In the case of the PIL membranes, both the chemical composition and surface molecular structure highly affect the antimicrobial properties of the material surfaces. For example, the end groups influence the PIL membrane surface, causing a different component from the bulk.54, 55 Here, the hydrophobic carbon chains tend to segregate at the air/polymer interface, while the hydrophilic pyrrolidinium cations remain in the membrane bulk. Upon the bacteria suspension contacting with the surface of PIL membrane, the pyrrolidinium cations are prone to spread to the water/polymer interface, while the carbon chains insert into the membrane bulk, resulting in dramatically covering up the effects of longer carbon chains of the materials on bacteria. Therefore, pyrrolidinium-based PIL membranes with longer carbon chains show lower antibacterial activities. SEM images were further used to characterize the micro-structure changes of S. aureus and E. coli incubated on the PIL membrane surfaces for 4 h (Figure 3). It can be seen that the bacteria on the PET membrane surfaces (as control) were smooth and complete, while aggregations of lipid vesicles, distorted and collapsed bacterial membranes were observed on the surfaces of the PIL membranes, indicating that the S. aureus and E. coli cells wholly collapsed and the bacteria died.

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Figure 3. Scanning electron microscopy (SEM) images of S. aureus (A-F) and E. coli (a-f) cultured on the PIL membranes for 4 h. PET (A, a); PPyM-C4 (B, b); PPyM-C6 (C, c); PPyM-C8 (D, d); PPyM-C10 (E, e) and PPyM-C12 (F, f). Collapses and fusion of bacterial cell membranes on the PIL membranes are observed (scar bar=1 µm).

Table 2. The hemolysis rate of the synthesized PIL membranes. Membranes

Hemolysis Rate (%)

PET

0.00±0.10

PPyM-C4

0.31±0.04

PPyM-C6

0.10±0.05

PPyM-C8

0.15±0.01

PPyM-C10

0.11±0.01

PPyM-C12

0.10±0.06

High biocompatibility is important for antibacterial materials in the practical application of medicine. Therefore, the hemocompatibility of the synthesized PIL membranes was evaluated by the hemolysis rate towards fresh human red blood cells (RBCs), and the results were listed in Table 2. As can be seen that all the PIL membranes exhibited extremely low hemolytic activities (< 0.5%) for RBCs, which is 20


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qualified as according to the criterion for non-direct contact biomedical materials (hemolysis rate

Synthesis of Pyrrolidinium-Type Poly(ionic liquid) Membranes for

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