Dual functional ionic liquids as plasticisers and antimicrobial agents for medical polymers

Article abstract of DOI:10.1039/c1gc15132k

Contamination of medical devices with bacteria such as Meticillin resistant Staphylococcus aureus (MRSA) is of great clinical concern. Poly(vinyl chloride) is widely used in the production of medical devices, such as catheters. The flexibility of catheter tubing is derived from the addition of plasticisers. Here, we report the design of two dual functional ionic liquids, 1-ethylpyridinium docusate and tributyl(2-hydroxyethyl)phosphonium docusate, which uniquely provide a plasticising effect, and exhibit antimicrobial and antibiofilm-forming activity to a range of antibiotic resistant bacteria. The plasticisation of poly(vinyl chloride) was tailored as a function of ionic liquid concentration. The effective antimicrobial behaviour of both ionic liquids originates from the chemical structure of the anion or cation and is not limited to the length of the alkyl chain on the anion/cation. The design approach adopted will be useful in developing ionic liquids as multi-functional additives for polymers.

Full text of DOI:10.1039/c1gc15132k

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PAPER
Dual functional ionic liquids as plasticisers and antimicrobial agents for
medical polymers†
Seong Ying Choi,a He´ctor Rodr´ıguez,b,c Arsalan Mirjafari,‡c Deirdre F. Gilpin,d Stephanie McGrath,d
Karl R. Malcolm,e Michael M. Tunney,d Robin D. Rogersc, f and Tony McNally*a
Received 2nd February 2011, Accepted 9th March 2011
DOI: 10.1039/c1gc15132k
Contamination of medical devices with bacteria such as Meticillin resistant Staphylococcus aureus
(MRSA) is of great clinical concern. Poly(vinyl chloride) is widely used in the production of
medical devices, such as catheters. The flexibility of catheter tubing is derived from the addition of
plasticisers. Here, we report the design of two dual functional ionic liquids, 1-ethylpyridinium
docusate and tributyl(2-hydroxyethyl)phosphonium docusate, which uniquely provide a
plasticising effect, and exhibit antimicrobial and antibiofilm-forming activity to a range of
antibiotic resistant bacteria. The plasticisation of poly(vinyl chloride) was tailored as a function of
ionic liquid concentration. The effective antimicrobial behaviour of both ionic liquids originates
from the chemical structure of the anion or cation and is not limited to the length of the alkyl
chain on the anion/cation. The design approach adopted will be useful in developing ionic liquids
as multi-functional additives for polymers.
Poly(vinyl chloride) (PVC) is widely used in the manufacture
Introduction
of a range of medical devices, including in endotracheal tubes
Nosocomial infections caused by antibiotic resistant pathogens
and catheters. The unique flexibility of PVC for use in such
such as Meticillin resistant Staphylococcus aureus (MRSA) are a
applications is derived from the use of phthalate esters as
plasticisers. However, the possible carcinogenic and reprotoxic
effects of phthalates has been a concern for some decades
major problem in hospitals worldwide.1 Furthermore, S. aureus
and coagulase-negative Staphylococci such as Staphylococcus
epidermidis can colonise and form a biofilm on the surface of
a range of biomaterials, thereby causing infection of indwelling
medical devices. As device-related infections are recalcitrant
to conventional antimicrobial therapy and host defences,
alternative agents for their treatment are required. Ionic liquids
(ILs) are molten salts which typically have a melting point
below 100 ◦C, negligible vapour pressure, very low volatility and
have been shown in separate experiments to have a plasticising
effect on polymeric materials,2,3 and antimicrobial activity.4–6
since their identification as an environmental contaminant,7
and more recently some have been prohibited for use in toys.8
Here, we report the design and use of novel dual functional
ionic liquids, which uniquely act as both a plasticiser for PVC
and exhibit antimicrobial activity, with particular efficacy
against Staphylococci, including MRSA. The glass transition
temperature (Tg) of even a rigid PVC can be tailored as a
function of IL loading, such that a decrease in Tg of up to 45 ◦C
is achievable and thus device flexibility may be engineered.
PVC comprises 25% of all plastics used in medical appli-
cations, with major applications in blood, plasma and intra-
venous fluid bags and tubing, enteral feeding and dialysis
equipment, catheters, oxygen masks and tents, goggles, gloves
and aprons.9,10 This widespread use is due to specific properties
that make it attractive in such applications, which include
flexibility, suitability for sterilization, resistance to kinking,
optical clarity, processibility and low cost. However, without
the presence of additives, PVC itself is a relatively rigid and
brittle polymer with limited function. Therefore, plasticisers,
which are normally a group of low molecular weight substances
that act as a molecular lubricant, are added to facilitate
melt processing and increase flexibility and toughness in the
final product. Phthalate esters have been the most common
group of plasticisers employed with PVC, including di-isononyl
aSchool of Mechanical and Aerospace Engineering, Queen’s University
Belfast, UK, BT9 5AH. E-mail: t.mcnally@qub.ac.uk
bDepartment of Chemical Engineering, University of Santiago de
Compostela, Santiago de Compostela, E-15782, Spain
cQUILL, School of Chemistry and Chemical Engineering, Queen’s
University Belfast, UK, BT9 5AG
dClinical and Practice Research Group, School of Pharmacy, Queen’s
University Belfast, UK, BT9 7BL
eDrug Delivery and Biomaterials Group, School of Pharmacy, Queen’s
University Belfast, UK, BT9 7BL
f
Department of Chemistry and Center for Green Manufacturing, The
University of Alabama, Tuscaloosa, AL, 35487, USA
† Electronic supplementary information (ESI) available: Figures S1, S2,
S3 and S4, and Table S1. See DOI: 10.1039/c1gc15132k
‡ Present address: Department of Chemistry, University of South
Alabama, Mobile, AL, 36688, USA
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phthalate (DINP), di-isodecyl phthalate (DIDP) and di-
ethylhexyl phthalate (DEHP). Although there is a lack of
definitive proof that phthalates directly affect human health,
research has shown that phthalates may induce reproductive
birth defects, infertility in animal models and worsen respiratory
function of mechanically ventilated preterm infants.11,12 It has
also been shown that phthalates contaminate their immediate
environment by leaching into surrounding fluids (such as when
in contact with body fluid or by volatilization in air, from medical
devices). This results in phthalates being indirectly introduced
into the human body through body fluid transfer or inhalation.
The effect of phthalates on renal function,13,14 particularly on
exposure to newborn infants15 and patients on hemodialysis16 is
of particular concern. Various attempts have been made to pre-
vent phthalate leaching, such as through the use of coatings.17,18
However, coatings can only slow down the rate of phthalate
release but are unable to prevent leaching entirely.19 Thus there
is a compelling need to develop replacements for phthalates.
Ionic liquids (ILs) are organic salts in liquid form typically
at temperatures below 100 ◦C and find application, among
others, as heat-transfer fluids, electrolytes, separation media,
and as anticorrosion agents.20,21 ILs generally have very low
flammability and volatility, and are increasingly investigated as
replacements for volatile organic solvents22 and employed in the
synthesis and modification of polymers.23,24 As ILs are in the
liquid state at room temperature and have negligible vapour
pressure, it has been proposed that ILs are less likely to leach
into the environment.25 Some ILs have been found to have a
plasticising effect on poly(methyl methacrylate) (PMMA),2,26
poly(L-lactide) (PLLA)27 and PVC,3 with some shown to have
less leaching compared to that of phthalates.3 Furthermore,
some existing ILs have displayed antimicrobial activity,28 and
in particular have been shown to prevent biofilm formation.29
Previous attempts to tailor bioactive ILs revealed the potential
of a single IL to serve more than one function.30
Consequently, and with the specific application for use in PVC
based medical devices (e.g. catheters), ILs were designed which
could provide both a plasticising effect, and exhibit antimicro-
bial activity including prevention of bacterial adherence and
biofilm formation. To achieve this, the cation/anion pair was
designed specificallyto meetthe criteriaofbeing an efficient plas-
ticizer, namely (1) being liquid at room temperature (required
to assist polymer chain mobility), (2) having molecular weight
greater than 300 g mol-1 (a critical minimum molar volume is
required to fill the available free volume), (3) high thermal stabil-
ity, >200 ◦C, (e.g. to facilitate melt extrusion of catheter tubing)
and, (4) containing polar functionality, in either the cation (e.g.,
hydroxyl group) and/or the anion (e.g., ester carbonyl group),31
to interact with the polar carbon-chlorine bonds in PVC.
Furthermore, the cation/anion pair was selected to provide an-
tibacterial activity with minimum toxicity (achieved via reduced
alkyl chain length and incorporation of an –OH group).32
and then, a metathesis reaction with sodium docusate. The
chemical structures of the constituent ions of the ILs selected
are shown in Scheme 1.
Scheme 1 Chemical structures of the IL precursors used in this study:
(a) 1-ethylpyridinium, (b) tributyl(2-hydroxyethyl)phosphonium and (c)
bis(2-ethylhexyl)sulfosuccinate (docusate).
1-Ethylpyridinium docusate (IL1)
An excess of bromoethane (Aldrich, 98%) was added drop-wise
to pyridine (BDH, >99.0%) in a round-bottomed flask at 0 ◦C
under nitrogen, with a reflux condenser attached to avoid evap-
oration of the reactants. The mixture was stirred and allowed to
warm to room temperature overnight, to ensure complete for-
mation of 1-ethylpyridinium bromide. The un-reacted materials
were removed in a rotary evaporator, and the solid obtained was
washed three times with anhydrous tetrahydrofuran (THF). The
white product was finally dried under high vacuum (<0.05 mbar)
at 70 ◦C overnight. Identity and purity of the 1-ethylpyridinium
1
bromide were confirmed by H and 13C NMR spectroscopy.
Sodium docusate (kindly supplied by Cytec, 99.0%) was dis-
solved in a 3 : 1 acetonitrile/acetone mixture and added drop-
wise to a solution of 1-ethylpyridinium bromide in acetonitrile
at room temperature, in stoichiometric amounts. After stirring
overnight, the precipitated NaBr was filtered off and the solvent
removed in a rotary evaporator. The residue was re-dissolved
in dichloromethane and this solution was washed several times
with water until no bromide ions could be detected in the wash-
ings by the silver nitrate test. The organic layer was dried over
Na2SO4, filtered and subsequently connected to the rotary evap-
orator for removal of the solvent. The remaining volatile materi-
als were removed under reduced pressure (<0.05 mbar) at 70 ◦C
overnight, giving IL1 as a viscous liquid in 82% yield. 1H and 13
C
NMR spectroscopy confirmed the structure and purity of the
desired product, and its water content was found to be <100 ppm
◦
by Karl–Fischer titration. IL1 had a melting point of 6 C, by
differential scanning calorimetry (Mettler-Toledo, model DSC
1), and a glass transition at -40 ◦C. Thermo-gravimetric analysis
(Mettler-Toledo, model TGA/DSC 1, ramp rate 5 K min-1) gave
a decomposition temperature (5% onset) of 248 ◦C.
Tributyl(2-hydroxyethyl)phosphonium docusate (IL2)
2-Chloroethanol (Fluka, >98.0%) was mixed with tributylphos-
phine (Aldrich, 97%) in a round-bottomed flask under nitrogen.
The liquid mixture was stirred at 100 ◦C for 2 days until
complete conversion to tributyl(2-hydroxyethyl)phosphonium
chloride, as monitored by 1H NMR spectroscopy. The remaining
volatiles were removed under reduced pressure (<0.05 mbar) at
Experimental
Synthesis of ILs
The synthesis of both ILs used consisted of two steps: first, an
alkylation reaction to obtain a halide salt of the desired cation;
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◦
90 C overnight, to give tributyl(2-hydroxyethyl)phosphonium
samples. The concentration of the docusate anion was then
chloride in 98% yield as a colourless viscous liquid. Equimolar
amounts of tributyl(2-hydroxyethyl)phosphonium chloride and
sodium docusate (Cytec, 99.0%) were dissolved in a 1 : 1 ace-
tone/water mixture and stirred overnight at room temperature.
The resulting suspension was diluted with H2O and extracted
with dichloromethane. The organic layer was washed several
times with H2O until no chloride ions were detected in the
washings by a silver nitrate test. The organic layer was dried over
MgSO4, filtered and the solvent removed by rotary evaporation.
The remaining volatile materials were eliminated under reduced
pressure (<0.05 mbar) to give IL2 in 92% yield as a colourless
liquid. Its structure and purity were verified by 1H and 13C
NMR spectroscopy, and its water content was <500 ppm, as
measured by◦Karl–Fischer titration. A glass transition temper-
ature of -45 C and decomposition temperature (5% onset) of
280 ◦C were determined by differential scanning calorimetry and
thermogravimetric analysis, respectively.
calculated from absorbance values obtained at 225 nm after
deducting the estimated absorbance values of the cations at this
wavelength.
Calibration curves were also established by FT-IR spec-
troscopy, using sample discs not immersed in PBS, in a spec-
trometer equipped with a single-bounce universal attenuated
total reflection (FTIR-UATR, Perkin–Elmer) attachment and
128 accumulated scans within the wavelength range 4000–
650 cm-1. The internal reflection element was a diamond-coated
zinc selenide crystal. Peak areas indicating the presence of
sulfonate (docusate) within the wavelength range 1070–990 cm-1
were calculated using Spectrum software, version 6.3.4. The
weight (%) of docusate anion leached from the sample discs was
estimated from a calibration curve and plotted as a function of
time.
Contact angle measurements
Contact angle measurements were carried out using an Easy
Drop Standard drop shape analysis system (Kru¨ss) equipped
with DSA 1 v1.9 Drop Shape Analysis software. Millipore
water droplets were applied onto the surface of PVC and
PVC-IL samples through a syringe needle and videoed using
a monochrome interline CCD (25/30 fps) camera. Images were
then captured for contact angle measurement using DSA1 v1.9
software.
Fabrication of PVC-IL blends
A PVC compound, K-Value = 60, (kindly supplied by Colorite
Europe Ltd.) was melt mixed with 0, 10, 20 and 30 wt% of
IL1 and IL2 using a co-rotating twin screw micro-extruder
(MiniLab, Thermo Haake). The composites of PVC-IL1 were
proc◦essed using a screw speed of 50 rpm at a temperature of
150 C, while those of PVC-IL2 were processed using a screw
◦
speed of 40 rpm at 140 C. A slit die with dimensions 5 mm ¥
Assessment of antimicrobial activity
Zone of inhibition assay
0.5 mm was used in producing tape-shape extrudate.
Dynamic mechanical thermal analysis (DMTA)
Sample discs of diameter 4 to 4.2 mm were stamped out
from tape-shape extrudate and used to assess the degree of
inhibition of planktonic growth of type strains and clinical
isolates of Gram-negative and Gram-positive bacteria. Fol-
The glass transition temperature (Tg) of these composite ma-
terials was determined from DMTA. A Triton thermoanalyzer
DMA Tritec 2000 was employed, using test specimens having a
width of 4.425 0.26 mm and thickness of 0.557 0.065 mm, in
tensile mode at 1 Hz, with a clamp distance of 5.32 mm, heating
rate of 2 K min-1 and displacement of 0.01 mm.
◦
lowing overnight incubation at 37 C on Mu¨ller–Hinton agar,
bacterial suspensions were prepared in accordance with BSAC
guidelines.33 Bacterial suspensions were adjusted to an optical
density equivalent to a 0.5 MacFarland standard (approximately
1 ¥ 108 colony forming units/mL). A sterile cotton tipped swab
was immersed in the inoculum and spread in three directions
onto a Mu¨ller–Hinton agar plate. The sample discs were placed
onto the inoculated plates (in triplicate) and incubated overnight
at 37 ◦C in air. Following incubation the diameter of any zone
of inhibition was measured (mm) and an image of each plate
captured digitally using a GelDocII imaging system.
Thermogravimetric analysis (TGA)
Samples (<8 mg) were tested using a thermogravimetric analyzer
TGA/SDTA 851e/LF/1600/1382 (Mettler-Toledo) over the
temperature range 25 to 600 ◦C at heating rate of 10 K min-1 in
a nitrogen atmosphere.
Leaching experiments
Approximately 10 mg of sample discs (∅ = 4 mm) were immersed
in phosphate buffered saline (PBS, prepared from Dulbecco
A (Oxoid)) for 1 h, 8 h, 1 day, 2 days, 4 days and 7 days to
observe the leaching of IL1 and IL2. Calibration curves for the
halide or sodium salt precursors of both ILs (1-ethylpyridinium
bromide, tributyl(2-hydroxyethyl)phosphonium chloride and
sodium docusate) in PBS solution were established using UV
spectrophotometry (Lamda 950, Perkin–Elmer) and using the
original PBS solution as a blank. Since the absorbance of the
docusate anion at wavelengths higher than 250 nm is negligible,
the concentration of the cations of IL1 and IL2 could be
calculated from absorbance values measured at 259 and 260 nm
respectively, after deduction of the absorbance of the control
Bacterial adherence and biofilm formation on IL-loaded PVC
The test sample that generated the optimal antimicrobial
effect against planktonically grown bacteria was then assessed
for antimicrobial efficacy against bacterial isolates grown in
bio-film. The isolates, which were all CoNS, were cultured
from continuous ambulatory peritoneal dialysis catheters, and
were provided by the Microbiology Department, Belfast City
Hospital, Belfast Health and Social Care Trust, UK. For each
isolate, bacterial suspensions were prepared as described above
and 2 mL placed into each well of a 12-well cell culture plate,
containing either the test sample (in three separate wells) or the
control. The sample size of the selected material and control
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was approximately 10 mm ¥ 4 mm ¥ 0.5 mm. The bacterial
suspensions and samples were incubated overnight at 37 ◦C.
The samples were then removed from the bacterial suspension,
rinsed gently in PBS and transferred to a fresh 12-well plate
containing 1 mL PBS. Adherent bacteria were removed from the
samples by ultrasonication for 5 min and the number of colony
forming units on each sample were counted and expressed
relative to the surface area of the section (cfu cm-2). Percentage
of inhibition was calculated based on the ratio of the number of
colony forming units of PVC-IL samples to that of PVC control
samples.
Results and discussion
Among a number of candidates, two ILs, 1-ethylpyridinium
docusate (IL1) and tributyl(2-hydroxyethyl)phosphonium do-
cusate (IL2) were synthesised and studied further (Scheme
1). These ILs were melt mixed into a rigid PVC compound
at loadings of 10, 20 and 30 wt% using a micro-twin screw
extruder. From dynamic mechanical thermal analysis (DMTA),
a plasticising effect was apparent by a significant reduction in
storage modulus (E¢), a broadening of the tand peak and shifting
of the tan d peak maximum to lower temperatures, indicating
a gradual decrease in glass transition temperature (Tg) of the
PVC-IL composites.
Time-kill assay
Both E¢ and the tan d peak maxima decreased monotonically
with increasing IL concentration (Fig. 1). Interestingly, IL2 had
a much more pronounced plasticising effect in that the Tg of
the PVC compound decreased by almost 50 ◦C on addition
of 30 wt% IL2. The decrease in Tg was only 10 ◦C when
a similar concentration of IL1 was incorporated in the PVC
compound. Moreover, a dramatic decrease in the stiffness of the
PVC compound at 37.5 ◦C (body temperature) was recorded.
The addition of IL1 or IL2 at the 30 wt% level resulted in a
significant diminution of modulus, greater than 90% in both
instances. The behaviour observed confirms that ILs can be used
to tailor the flexibility of PVC compounds.
The enhanced plasticising effect obtained for IL2 may
be derived from the more polar tributyl(2-hydroxylethyl)-
phosphonium cation relative to the 1-ethylpyridinium cation,
although the presence of ester groups in the docusate anion
will contribute to any plasticisation of the PVC compound
also. Thermal stability, an important consideration for the
melt processing of PVC into medical devices, of the PVC-IL
composite materials was also studied using TGA in nitrogen
gas (ESI, Fig. S1†). An interesting phenomenon was observed,
in that the onset of degradation of the PVC-IL materials via
a dehydrochlorination process of PVC was shifted to slightly
lower temperatures. We propose that this is due to the ability of
different ILs to promote or delay dehydrochlorination of labile
chlorine atoms in PVC.35–37
Time-kill assays were performed in sterile McCartney bottles
containing a final volume of 10 mL. Each assay included a
growth control McCartney bottle with no IL or precursor.
Furthermore, an ethanol in water mixture was also included
for comparison (6.62% v/v and 2.04% v/v for IL1- and
IL2-related solutions, respectively), as ethanol was used as
the solvent to dissolve anion components of both ILs. The
concentration of IL1 and IL2, and their respective cationic
and anionic precursors tested, are shown in Table S1 (note
that the concentration of cationic and anionic precursor was
prepared based on the relative concentration of cation and anion
in each IL; for example, 2.02 mg mL-1 of IL1 corresponds
to 0.41 and 1.61 mg mL-1 of cationic and anionic precursor,
respectively).† The inoculum to be tested was prepared by
adjusting the turbidity of an actively growing broth culture in
Mu¨ller–Hinton to give the desired in◦itial inoculum of 105 cfu
mL-1. All bottles were incubated at 37 C with samples (100 mL)
removed at 0, 2, 4, 6, 8 and 24 h. Viable counts were performed
by preparing 10-fold serial dilutions of the initial 100 mL sample
in quarter strength Ringers solution and plating by the Miles
and Misra technique (Miles and Misra, 1938) onto Mu¨ller–
Hinton agar plates. Plates were incubated for 24 h and colony
counts performed with the problem of drug carryover addressed
by dilution, as described previously by Pankuch et al.34 Killing
curves were then constructed by plotting log10 cfu mL-1 against
time over 24 h.
In determining the antimicrobial properties of the PVC-IL
composites, the samples were initially screened for antimicrobial
activity against a range of Gram-positive and Gram-negative
laboratory and clinical isolates using a zone of inhibition assay.
Neither PVC-IL combination exhibited measurable antimicro-
bial activity against Gram-negative bacteria (Table 1). However,
both ILs displayed excellent antimicrobial activity against
Gram-positive bacteria, with PVC-IL1 active at all IL concen-
trations tested (Table 1). The Gram-positive bacteria inhibited
included 6 clinical MRSA strains. This finding, that Gram-
positive bacteria were more sensitive to ILs than Gram-negative
bacteria, is consistent with that reported for other ILs.29,38
We next investigated the efficacy of PVC loaded with the most
potent antimicrobial IL (IL1) at preventing bacterial adherence
and biofilm formation by clinical coagulase-negative Staphylo-
cocci (CoNS) isolates (Fig. 2a) and PVC with the equivalent
amount of IL2 for comparison (Fig. 2b). These isolates were
selected as S. epidermidis is the leading cause of infection
related to implanted medical devices. Furthermore, they had
previously been shown to be strong biofilm formers based
Scanning electron microscopy (SEM) of biofilms
Disc samples of PVC and PVC-IL composites (Ø = 4 mm) were
immersed in a bacterial suspension of a clinical CoNS isolate
◦
in a 12-well cell culture plate overnight at 37 C. The samples
were then removed from the bacterial suspension and rinsed
gently in PBS. The samples were immersed in 3% glutaraldehyde
in 0.1 M sodium cacodylate buffer (C2H6AsNaO2) at 4 ◦C
overnight, followed by washing using 0.1 M cacodylate buffer at
4 ◦C. Dehydration was then carried out at room temperature by
transferring the samples to a 96-well plate containing 50, 70 and
90% ethanol for 30 min, respectively, and 100% ethanol for three
consecutive 30 min periods. Samples were then transferred to a
96-well plate containing hexamethyldisilazane (HMDS) and left
to dry overnight in a fume cupboard. After drying, samples were
mounted on discs and gold-coated. SEM images were captured
using a JEOL JSM-840A scanning microscope at magnification
of 3000¥ and an operating voltage of 15 kV.
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Fig. 1 DMTA curves (storage modulus, E¢ and tan d) for blends of PVC with (a) 1-ethylpyridinium docusate (IL1) and (b) tributyl(2-
hydroxyethyl)phosphonium docusate (IL2), and the variation in (c) Tg and (d) E¢ at 37.5 ◦C as a function of IL content.
on the classification of biofilm adherence reported previously
by Stepanovic et al.39,40 Three of the five isolates had also
previously been shown to be resistant to 3 or more conventional
antibiotics when grown planktonically. The IL1-loaded PVC
was challenged for different time periods between 0 and 25 h
with a high initial inoculum of each isolate to represent the
number of bacteria that could be present as a result of an overt
medical device related infection. With the exception of isolate
B11R, incorporation of IL1 into PVC resulted in a significant
reduction, ranging from 1 to a 3 log10 in the number of bacteria
adhering to the surface compared with the unloaded control.
In contrast, there was no reduction in bacterial adherence for
PVC loaded with 10 wt% IL2. Moreover, for some isolates,
bacterial adherence was greater for IL2-loaded PVC (at 10 wt%)
compared to control samples.
The reduced antimicrobial activity observed for PVC-IL2
in comparison with PVC-IL1 is due to incorporation of an
–OH group on one of the alkyl chains of the tributyl(2-
hydroxyethyl)phosphonium cation which is known to reduce the
antimicrobial effect of ILs.41 There is some evidence to suggest
that the antibacterial effect in phosphonium-based ILs could be
less related to the presence of alkyl chains on the cation and
more likely to be affected by the type of anion used.32
PVC was confirmed by scanning electron microscopy (SEM)
examination of the surfaces of the materials. For unloaded PVC,
the CoNS isolate can be seen to readily colonise the surface (Fig.
2c). In contrast, no bacteria have adhered to IL-loaded PVC
(Fig. 2d). This antibiofilm activity of 1-ethylpyridinium docusate
is in keeping with the findings of Carson et al., who reported that
1-alkyl-3-methylimidazolium chloride ILs displayed antibiofilm
activity against biofilms grown by Gram-positive bacteria on
polystyrene pegs.29
In addition to determining the antimicrobial efficacy of IL
loaded PVC, we also investigated the effectiveness of the ILs
and their cationic and anionic precursors in killing 3 clinical
strains (CoNS, S. aureus (MRSA) and S. aureus (MSSA) using
a time-kill assay (Fig. 3). The cationic precursor of IL1 (the
bromide salt of the 1-ethylpyridinium cation) did not exhibit
a significant antimicrobial effect, despite having a structure
commonly employed in a wide range of antimicrobials and disin-
fectants. Nevertheless, a similar rate of kill was observed for both
IL1 and its anionic precursor (the sodium salt of the docusate
anion) for all 3 strains, confirming that the antimicrobial efficacy
of IL1 originates from the anion. In contrast, strain specific
differences were apparent for IL2 and its precursors; for MSSA,
IL2 and its anionic precursor had a similar rate of kill whereas
IL2 and its anionic precursor exhibited higher rates of kill
against CoNS and MRSA, respectively. The cationic precursor
The prevention of bacterial adherence and biofilm formation
on the surface of the PVC-IL1 compound relative to unloaded
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Table 1 Results of zone of inhibition assay for PVC-IL1 and PVC-IL2a
Zones of inhibition/mm
Weight fraction of IL1 in PVC (%)
Weight fraction of IL2 in PVC (%)
Tested organism
10
20
30
10
20
30
Staphylococcus aureus ATCC 29213b
Staphylococcus epidermidis RP62Ac
Staphylococcus E-MRSA 16c
Staphylococcus aureus AH-7c
CoNS B5c
10.17 0.29
9.33 0.58
10.00 0.00
7.67 0.58
6.67 0.29
7.00 0.50
8.00 0.50
10.00 0.00
7.33 0.29
9.67 0.58
9.16 0.29
12.17 0.76
14.00 0.00
10.33 0.29
13.00 0.00
—
11.17 0.29
8.33 0.58
11.00 0.00
8.67 0.58
6.83 0.29
7.00 0.00
7.83 0.29
10.67 0.29
9.00 0.50
10.17 0.29
10.00 0.00
15.33 0.58
15.67 0.58
12.33 1.26
13.33 0.58
—
12.00 0.00
8.67 0.58
12.00 0.00
9.00 0.00
7.83 0.29
8.00 0.00
8.67 0.29
11.50 0.00
9.67 0.29
11.67 0.29
11.67 0.29
16.50 0.50
16.00 0.00
13.67 0.58
13.33 0.76
—
—
5.50 0.50
Insig.
Insig.
Insig.
—
—
—
—
Insig.
—
—
—
8.83 0.29
5.67 0.58
5.33 0.58
7.33 0.58
Insig.
Insig.
Insig.
8.00 0.00
8.50 0.00
Insig.
7.33 0.29
9.83 0.29
9.33 0.29
9.17 0.29
11.33 0.29
—
N/A
N/A
N/A
—
—
—
—
—
—
—
—
—
—
—
—
—
CoNS B7Rc
CoNS B8Rc
CoNS B10c
CoNS B11Rc
CoNS B12c
MRSA CFP16c
MRSA CFP17c
MRSA CFP18c
—
—
—
—
MRSA CFP19c
MRSA 4Dc
Enterococcus faecalis NCTC 12697b
Pseudomonas aeruginosa NCTC
12934b
—
—
—
—
—
Pseudomonas aeruginosa B1c
Pseudomonas aeruginosa C1c
Burkholderia cepacia complex BB1c
Burkholderia cepacia complex BB2c
Proteus mirabilis NCTC 11938b
Klebsiella aerogenes NCTC 10896b
Escherichia coli NCTC 10418b
—
—
Insig.
—
Insig.
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Insig.
Insig.
Insig.
Insig.
—
Insig.
Insig.
Insig.
Insig.
Insig.
—
—
—
a ‘—’ no inhibition; ‘Insig.’ insignificant inhibition; ‘N/A’ not available. b Type strains. c Clinical isolates.
Fig. 2 Percentage of inhibition of PVC loaded with (a) 10 wt% IL1 and (b) 10 wt% IL2 innoculated with clinical coagulase-negative Staphyloccoci
isolates for 24 h, and SEM images showing adherence of a CoNS isolate to (c) unloaded PVC sample and (d) PVC with 10 wt% IL1.
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Fig. 3 Rate of kill determined for (a) IL1 and its precursors, and (b) IL2 and its precursors against a clinical CoNS isolate, (c) IL1 and its precursors,
and (d) IL2 and its precursors against a clinical MRSA isolate, (e) IL1 and its precursors, and (f) IL2 and its precursors against a clinical MSSA
isolate.
for IL2, tributyl(2-hydroxyethyl)phosphonium chloride, also
showed different levels of antimicrobial activity against CoNS,
prevention of further growth with MSSA and a killing effect
with MRSA.
leaching of the cation did not induce high leaching of the anion
and as such low concentrations of anion were detected. It is also
possible that the apparent low amount of anion is due to strong
absorbance of the cation at 225 nm and very weak absorbance
of the anion at 260 nm. Thus, the amount of docusate anion was
also determined from FTIR-ATR of the sample discs at the end
of the leaching experiment. In the resulting spectra, a reduction
of the peak intensity at 1034 cm-1 corresponding to sulfonate
(functional group present in the docusate anion) was observed,
compared to the original discs, providing further evidence that
the anion had leached from the solid disc samples to the aqueous
medium (ESI, Fig. S4†).
Additional evidence for the leaching of IL1 and IL2 from the
PVC compound was obtained from contact angle measurements
of the neat PVC compound and with varying levels of IL1 and
IL2 added (Fig. 4). The contact angle for unloaded PVC was 89◦,
which on addition of IL1, irrespective of the percentage loading,
decreased to 16◦, confirming that the surface of the PVC-
IL1 composite was much more hydrophilic than the neat PVC
compound. In contrast, for the PVC-IL2 composites, the contact
angle increased monotonically from 89 to 118◦ on addition
of up to 30 wt% IL2, indicating that the surface of the PVC-
IL2 composites are more hydrophobic than the unloaded PVC
compound. It is obvious from both the antimicrobial testing
and leaching experiments that the excellent antimicrobial and
anti-adherence properties of PVC-IL1 originate from leaching
of the cation (to the PVC surface and then to the PBS aqueous
media (in leaching tests) and agar media (in zone of inhibition
assays)).
The antimicrobial and anti-adherence behaviour are directly
related to the extent of IL leaching from the PVC compound.
The degree of leaching was studied using a combination of UV-
vis spectrophotometry and FTIR-ATR spectroscopy on solid
samples (ESI, Fig. S2 and Fig. S3†), with the weight loss, and the
cation and anion concentration determined. The absorption of
both ILs at different wavelengths was identified by first preparing
calibration curves established from the spectra of the IL
precursors alone, in which 1-ethylpyridinium bromide (cationic
precursor for IL1), tributyl(2-hydroxyethyl)phosphonium chlo-
ride (cationic precursor for IL2) and sodium docusate (anionic
precursor for both ILs) had absorption maxima at 259, 260 and
225 nm, respectively. Although the absorbance at 260 nm for the
cationic precursor of IL2 was not the strongest, it was selected
for calculation to exclude the absorbance values contributed by
the presence of the anionic precursor, which occurred in the
range 210–250 nm.
The data obtained showed that both cation and anion leached
from PVC-IL1 composites at a faster rate than from PVC-IL2,
the highest absorbance values being obtained within the first
24 h after immersion of the composites in phosphate buffered
solution (PBS). However, it is not clear whether the leaching
of the anion is induced by leaching of the cation or vice versa,
although it is more likely to be the former as the cation is smaller
and more mobile. In contrast, for PVC-IL2 composites, the
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S. Y. C. thanks QUB for funding a studentship. H. R. is grateful
to the Ministry for Science and Innovation of Spain for support
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