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Boase, Nathan, Torres, Marcelo, Fletcher, Nicholas, Fuente-Nunez, Cesar,& Fairfull-Smith, Kathryn(2018)Polynitroxide copolymers to reduce biofilm fouling on surfaces.Polymer Chemistry, 9, pp. 5308-5318.
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https://doi.org/10.1039/C8PY01101J
This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1
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Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Polynitroxide copolymers to reduce biofilm fouling on surfaces
Nathan R.B. Boase,a Marcelo D.T. Torres,
b,c,d Nicholas L. Fletcher,
e,f Cesar de la Fuente-Nunez,
b,c
and Kathryn E. Fairfull-Smith*a
Biofilms are highly organised colonies of microorganisms, at a surface or an interface, which are extremely resistant to
treatment with biocides or antimicrobials. Consequently, biofilms are the primary cause of fouling and persistent
infections in both industrial and clinical settings. Thus, there is a clear need to develop coatings that are able to prevent
the growth of biofilms on surfaces. Herein, we report on the development of polynitroxides for use as anti-biofilm
coatings. We demonstrate that we can tune the composition of the nitroxide 2,2,6,6-tetramethyl-4-piperidyl methacrylate
in a copolymer with methyl methacrylate in order to control the surface concentration of nitroxides in the resulting thin
films. The prepared films are able to reduce biofilm fouling by Pseudomonas aeruginosa (PAO1) at nitroxide monomer
concentrations as low as 30 wt%. The nitroxide containing materials show no difference in toxicity to mammalian cells,
compared to poly(methyl methacrylate), in a proliferation assay with 3T3 fibroblast cells. This is the first demonstration of
surface tethered nitroxides with activity against biofilms and provides new opportunities for developing antifouling surface
coatings.
Introduction
Nosocomial (hospital acquired) infections are a huge burden
on healthcare systems worldwide. It has been estimated that
there are 1.7 million infections and 99,000 deaths reported in
the USA annually, and 3 million infections and 50,000 deaths in
the European Union.1, 2 Of these infections, it has been
estimated that at least half are associated with an in-dwelling
or implanted device.3 It is now widely accepted that device
related infections are caused by microbes living as sessile
colonies, called biofilms.4, 5 Beyond device related infections, it
has also been shown that biofilms are strongly associated with
chronic infections.5
Biofilms are multicellular colonies of microbes that are
associated with surface-liquid or air-liquid interfaces. A biofilm
develops over a number of key steps, starting with irreversible
attachment and coordination of micro-colonies, before
maturation of the biofilm by bacterial expansion and
production of a polysaccharide extracellular matrix, which
allows for construction of complex architectures.4, 6 Biofilms
are the predominant life stage for most microorganisms, as
they provide the organisms with protection from
environmental stresses. It is this evolved ability to exist in a
coordinated fashion that protects biofilms from treatment by
common antibiotics or disinfectants.7
Biofilms are not just a problem in healthcare. Persistent
bacterial infections on the surface of industrial equipment are
a huge economic burden in a range of industries, including
marine shipping,8 fuel storage9 and food processing.10, 11 In
these cases, biofilm fouling reduces the quality of products
and reduces equipment efficiency, leading to increased
operational costs and reduced profitability. In the case of food
manufacturing there is the additional problem of bacterial
contamination of the food product, leading to food poisoning
of consumers.10 Given the wide range of clinical and industrial
problems in which biofilms are problematic, it is clear there is
a distinct need to develop new approaches to prevent biofilm
fouling of surfaces.
There are a wide range of approaches being developed to
overcome biofilm fouling of surfaces, from the modification of
the energy and structure of the surface to prevent initial
attachment, to chemical or biological therapies that can
disrupt the extracellular matrix.12-14 There have also been
significant advances into understanding how microorganisms
communicate through quorum sensing and to find chemical
cues that can trigger biofilm dispersal.15 Nitric oxide (NO) has
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been reported as one of the integral signalling molecules
involved in biofilm regulation.16-20
When NO is used at low, sub-toxic doses, it is able to
trigger planktonic microbes present in the biofilm to enter a
motile life phase, which causes dispersal of the biofilm. It has
also been shown to have an inhibitory effect on biofilm
formation, preventing microbes from entering the biofilm
forming phase.16, 21 NO has been shown to be able to bind to
H-NOX domains (heme-nitric oxide/oxygen-binding). Binding of
NO to H-NOX can regulate the expression of cyclic di-GMP, a
secondary messenger molecule that is known to play a critical
role in biofilm development.22 More recently a new family of
NO binding proteins have been discovered, NosP (NO-sensing
proteins). Binding of NO to this family of proteins has been
shown to lead to regulation of activity of a hybrid histidine
kinase in Pseudomonas aeruginosa.19, 23
Due to the gaseous nature of nitric oxide, NO donors are
typically used to release the molecule of interest and induce
biofilm dispersion.24 These donors can be used independently
as small molecule agents,21, 25 attached to a surface,26 or
incorporated into a polymeric delivery agent to modify their
physicochemical and pharmacokinetic properties.27-29 Despite
their success, there are still difficulties associated with the use
of such donors, including their stability and accurate
determination of NO dose.
As an alternative approach, we have investigated the use
of nitroxides as potential regulators of biofilm formation and
growth. Nitroxides are small organic molecules, which possess
a stabilised free radical.30, 31 They share similar electronic
properties to nitric oxide with the added advantages of being
solid or liquid at room temperature, and having a chemical
structure that can be modified to improve drug
pharmacokinetics. Nitroxides have been used to both inhibit
biofilm formation on a clean surface, or to trigger dispersal of
a mature biofilm.32-36 A link between nitroxides and the
metabolism of nitric oxide has been established through the
use of P. aeruginosa mutants with knockouts of key nitric
oxide metabolic enzymes.35 Nitroxides disrupt biofilm growth
and development but do not kill bacteria directly. When used
in combination with antibiotics, they have been shown to
greatly improve the efficacy of antibiotics as biofilm
treatments.36-38
All reported examples of nitroxides as anti-biofilm agents
have involved their use as small molecules or drug-hybrids.
With the high association of biofilms with surfaces, it would be
particularly beneficial to create active surfaces that prevent
biofilm formation. There has been significant work to achieve
this using surfaces that release disinfectants, antibiotics and
nitric oxide.12, 39 So far there has been only one reported
attempt at using surface tethered nitroxides as antibacterial
coatings.40 In this work, the authors only explored the ability of
nitroxides to modulate the bactericidal effects of nanosilver.
They hypothesised that the enhanced effect was due to
oxidation of the nitroxide to the N-oxoammonium cation and
subsequent interaction with the bacterial cell wall. Specific
anti-biofilm effects, which have been reported for nitroxides,
were not investigated. Nitroxide functionalised surfaces have
been developed for other applications, including catalysis and
redox active surfaces.41-43 These materials utilize one of three
approaches: encapsulating the nitroxide, conjugating the
nitroxide to a preformed material, or making a polynitroxide
that can be used to fabricate a material.
Polynitroxides, polymers bearing pendant nitroxide
sidechains, are an interesting class of molecules that have
been investigated as organic radical batteries, oxidation
catalysts and for use in exchange reactions for constructing
complex architectures.41, 43-47 Many of these reports focus on
using homopolymers of nitroxide monomers, with only a
limited number of examples of copolymers.45, 46, 48, 49
Copolymerisation is an interesting approach as it allows
control over both the concentration of radicals in the material
and its physical properties, by incorporation of a second
monomer (e.g. PEG for water solubility and biocompatibility).50
In this work, we demonstrate the use of polynitroxides to
fabricate nitroxide bearing surface coatings that are able to
reduce the formation of biofilms on a surface. A series of
copolymers of controlled composition of the nitroxide
monomer (2,2,6,6-tetramethyl-1-piperidinyl)oxyl methacrylate
(TMA) and methyl methacrylate (MMA) were synthesized
(Scheme 1). These polymers were then used to form thin films
via spin coating, allowing fine control over the concentration
of nitroxide radicals at the surface, whilst keeping all other
surface properties constant. Finally, the anti-biofilm activity of
the prepared materials was examined using P. aeruginosa in
an in vitro flow cell assay.
Methods
Materials
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Unless otherwise stated, all solvents and reagents were
purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) or
Thermofisher Scientific (Scoresby, Australia) at analytical grade
(or higher) and used without further purification. Methyl
methacrylate (MMA) was purchased from Sigma Aldrich
(Castle Hill, Australia) and the stabiliser was removed by
passing over a plug of aluminium oxide immediately prior to
use. 2,2,6,6-Tetramethyl-4-piperidinyl methacrylate (TMPM)
was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo,
Japan). 2,2′-Azobis(2-methylpropionitrile) (AIBN) was
recrystallised from methanol prior to use. 2-Cyanopropan-2-yl
pentyl trithiocarbonate (CPPTTC) was synthesised using
adapted literature procedures and purified by medium-
pressure liquid chromatography (MPLC).51-53 P-type, boron
doped silicon wafers, 525 ± 25 µm thickness (Si-Mat,
Kaufering, Germany), were purchased as 100 mm discs and cut
into 10 x 10 mm wafers using a carbide tipped pencil.
Convertible flow cells (7.7 cm3 – 24 mm x 40 mm x 8 mm) were
purchased from Fischer Scientific (Hampton, NH, USA). Silicone
tubing, 96-wells plates and petri dishes were purchased from
VWR (Radnor, PA, USA). Pseudomonas aeruginosa (PAO1)
bacteria were purchased from ATCC and Pseudomonas
isolation agar was purchased from Sigma-Aldrich (Waltham,
MA, USA).
Characterisation
1H and 13C NMR spectra were recorded on a Bruker System
600 Ascend LH (Bruker, Billerica, USA), equipped with an BBO-
Probe (5 mm) with z-gradient (1H: 600.13 MHz, 13C 150.90
MHz). The δ-scale was normalized relative to the solvent signal
of CDCl3: 1H NMR spectra (7.26 ppm) and for 13C NMR spectra
(77.16 ppm).
Size exclusion chromatography (SEC) experiments were
recorded on a system consisting of a 1515 Isocratic HPLC
Pump, 2414 RI detector and a 717 Plus autosampler (Waters,
Milford, USA), and a column set (PSS, Mainz, Germany)
consisting of a guard column (50 x 8 mm, 10 µm) and two 1000
Å GRAM columns (300 x 8 mm, 10 µm). The eluent was HPLC
grade dimethylacetamide with 0.1 wt% LiBr, and was degassed
by an inline Lab Hut solvent degasser. All molar mass data is
reported relative to polystyrene standards (EasyCal, Agilent,
Santa Clara, USA).
Electron paramagnetic resonance (EPR) spectra were
recorded on a MiniScope MS400 spectrometer (Magnettech
GMBH, Berlin, Germany) using dichloromethane as the solvent
and capillary tubes for liquid handling.
UV-Vis spectroscopy was recorded on a UV-1800
spectrophotometer (Shimadzu, Kyoto, Japan). All samples
were measured in quartz cuvettes at atmospheric conditions in
a dual beam experiment, using the corresponding solvent as
the reference.
Contact angle measurements were performed on a FTÅ200
(First Ten Angstroms Inc., Portsmouth, USA). Equilibrium
contact angle values were recorded using the following
protocol. A droplet of deionised water was dispensed at 1
µL/s, until the droplet fell off onto the surface under its own
weight. 20 images of the droplet were recorded for the first 60
s, until the droplet had reached equilibrium. The contact angle
was measured on both sides of the droplet and the average
equilibrium value was recorded.
AFM measurements were performed on a Flex-Bio atomic
force microscope (Nanosurf – Nanosurf AG, Liestal,
Switzerland). For the contact mode, a ContGD-G cantilever
(BudgetSensors, Sofia, Bulgaria) was used with a typical
resonant frequency of 13 kHz and a force constant of 0.2 N/m.
A setpoint of 10 nN was applied. A scan frequency of 1 Hz was
set. The images were evaluated via the software Gwyddion
2.49 (David Nečas and Peter Klapetek).
XPS measurements were performed on a Kratos Axis Supra
photoelectron spectrometer (Kratos Analytical, Manchester,
United Kingdom) incorporating a 165 nm hemi-spherical
electron energy analyser. The incident radiation was
monochromatic Al X-rays (1486.6 eV) at 225W (15 kV, 15 mA).
Survey (wide) scans were taken at analysing pass energy of 160
eV and multiplex (narrow) higher resolution scans at 20 eV.
Survey scans were carried out over 1360 - 0 eV binding energy
range with 1.0 eV steps and a dwell time of 100 ms. Narrow
higher resolution scans were run with 0.2 eV steps and 250 ms
dwell time. During analysis, the charge compensation system
was employed to prevent any localised charge build-up. The
spectra were evaluated using CasaXPS software. All spectra
were calibrated to the 284.80 eV for the C 1s peak.
Polymer film thickness on silicon substrates was
determined by spectroscopic ellipsometry, using a M-2000UI
Ellipsometer (J. A. Woollam, Lincoln, USA). Measurements
were made in the wavelength range of 245-1690 nm at three
angles of incidence (60°, 65°, 70°). The data was fitted and
evaluated using the CompleteEASE software package. A
Cauchy model was applied for the polymer film layer (A=1.45,
B=0.01) on a Si/SiO sublayer. All thickness values are the
average of at least four independently coated replicates, with
the mean and standard deviation for the layer thickness
reported.
Synthesis of PTMPM-PMMA copolymers
All copolymers were synthesised by RAFT polymerisation in a
50 wt/v% solution of dioxane at 85 °C for 8 h. The molar ratio
for each copolymer is reported in Table 1. A demonstrative
example of the polymerisation procedure is given below for
the 100 wt% TMPM polymer.
TMPM (1 g, 222 eq, 4.4 x 10-3 mol), CPPTTC (5 mg, 1 eq, 2 x
10-3 mol) and AIBN (0.6 mg, 0.2 eq, 4 x 10-6 mol) were added to
a glass reaction vial and dissolved in dioxane (2 mL). The vial
was sealed with a rubber septum and degassed by bubbling
with argon gas for 15 minutes. The vial was transferred to a
solid heating block and allowed to react, with stirring, at 85 °C
for 8 h. The reaction was quenched by placing the vial in an ice
bath and the polymer recovered and purified by three
repeated precipitations into ice cold hexane, yielding a pale
yellow powder. The powder was dried in a vacuum oven at 40
°C for 24 h, prior to analysis by 1H NMR spectroscopy and SEC.
Removal of trithiocarbonate end groups
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AIBN (60 mg) was dissolved in THF (40 mL) in a round bottom
flask and reacted at 65 °C for 1 h under atmospheric conditions
to generate pre-treated THF solution.
Each polymer (400 mg) was added to a long test tube with
the pre-treated THF solution (6 mL). These solutions were
bubbled with compressed air for 2 minutes, before being
immersed in an oil bath at 65 °C to the solvent level, open to
the atmosphere. The long test tubes allowed the reactions to
be open to air, but the solvent to recondense inside the tubes
and prevent total evaporation.
After 6 h, all reactions appeared colourless, which was
taken as the end point of the reactions. The temperature of
the reactions was reduced to 40 °C and triphenylphosphine
(PPh3) solution in THF (1 mL, 6 mg/mL) was added to each
reaction. The reaction was then heated at 40 °C for 45
minutes. All polymers were recovered by precipitation from
hexane and collected by vacuum filtration, yielding white
powders. After drying in a vacuum oven at 50 °C for 90
minutes, all reactions yielded greater than 80 % recovered
copolymer. Removal of the trithiocarbonate end groups was
confirmed by UV-vis and 1H NMR spectroscopic
characterisation.
Oxidation of PTMPM to PTMA
Oxidation of the tetramethylpiperidine starting material to the
desired (2,2,6,6-tetramethylpiperidin-1-yl)oxyl methacrylate
(TMA) nitroxide was achieved by oxidation with
3-chloroperbenzoic acid (mCPBA). Each polymer (100 mg, ~3 x
10-6 mol) was dissolved in dichloromethane (5 mL). A saturated
DCM solution of mCPBA (2 mL, 0.5 M) was added to each
reaction. For all reactions with TMPM present, the solution
quickly formed a precipitate that redissolved over a few
minutes to give a clear, orange solution. The reaction was
allowed to proceed at room temperature (23 °C) for 2 h. Each
reaction was worked up by washing the organic layer with
saturated Na2CO3 (× 2) and deionised water (× 1). The organic
layer was dried with MgSO4 and concentrated by rotary
evaporation. The concentrated solution was precipitated once,
allowed to stand for 16 h and collected by vacuum filtration.
The final polymers were dried in a vacuum oven at 50 °C for 3
h, yielding a white powder for 0 wt% PTMA, and pale orange
polymers for 30-100 wt% PTMA. Oxidation of the nitroxide was
quantified by UV-Vis and EPR spectroscopies.
Thin film fabrication by spin coating
All films were prepared by spin coating using a POLOS 200 spin
coater (SPS-Europe B.V., Putten, Netherlands). Optimised spin
coating conditions are reported here. Polymers were prepared
as 10 wt% solutions in HPLC grade toluene and allowed to
stand for a minimum of 4 h to ensure complete dissolution. All
solutions were filtered prior to spin coating. 10 µL of polymer
solution was dispensed dynamically at 8,000 RPM and allowed
to dry at the same speed for 1 min. Film thickness was
measured by ellipsometry and surface energy measured by
static contact angle.
In vitro testing of anti-biofilm activity of polynitroxide films
Biofilms of P. aeruginosa strain PAO1 were grown for 24 h in
the presence of the polynitroxide copolymer films at 37 °C in
convertible flow chambers with channel dimensions of 1 x 4 x
40 mm. The medium used was BM2 minimal medium (62
mmol L-1 potassium phosphate buffer, pH 7.0, 7 mmol L-1
(NH4)2SO4, 2 mmol L-1 MgSO4, 10 mol L-1 FeSO4) containing 0.4
% (wt/vol) glucose as a carbon source. Silicone tubing (inner
diameter, 1.5 mm; outer diameter, 3.0 mm; wall thickness, 0.8
mm) was used, and the system was assembled and sterilized
by pumping a 10% hypochlorite solution through the system
for 5 min using a multichannel peristaltic pump. The system
was then rinsed with sterile water and medium for 5 min each.
Flow chambers were inoculated by injecting 1 mL of an
overnight culture diluted to approximately 104 bacterial
cells/mL. After inoculation, the chambers were left without
flow for 4 h, after which medium was pumped through the
system at a constant rate of 2.6 mL/h). The silicon wafers
coated with polynitroxide films were then collected from the
convertible flow cells, washed to remove non-adherent
bacteria and homogenized using a bead beater for 20 min at
25 Hz, and serially diluted for CFU quantification. Two
independent experiments were performed with 2 silicon
wafers per group in each condition (N = 4).
In vitro testing of mammalian cell viability on polynitroxide films
The 3T3 Murine Fibroblast cell line was maintained in DMEM
medium (Gibco, Thermo Fisher Scientific, Australia)
supplemented with 10% (v/v) Foetal Bovine Serum (FBS;
Bovogen, Australia), 100 U/mL penicillin, 100 μg/mL
streptomycin and 2 mM L-glutamine (Gibco, Thermo Fisher
Scientific). Cells were grown in a 37 °C incubator with 5%
CO2/air and passaged every 3-4 days.
Spin coated wafers (N=3 per polymer sample) were
sterilized by spraying with 70% ethanol and then incubating for
30 minutes in 1 mL 70% ethanol in sterilised 12-well plates.
(Ellipsometry did not show any change in the films upon
exposure to 70% ethanol under these conditions.) Wells were
then washed twice with 2 mL phosphate buffered saline.
Control wafers of bare silicon, that had been spin coated under
the same conditions but with toluene only, were used as
controls for the assay. Control wells containing no wafers were
treated following the same methods to account for any change
in viability following incubation and washing. 3T3 cells were
then seeded onto the polymer wafers or empty control wells
at 100,000 cells per well in 2 mL complete media and allowed
to proliferate for 48 h.
To assess viability on polymer coated wafers, sterile
forceps were used to transfer wafers to a new sterilised 12-
well plate. Cell viability on the wafers was then assessed by 3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium (MTS) assay (CellTiter 96 Aqueous
One Solution Cell Proliferation Assay; Promega, USA) following
manufacturers methods. 1 mL of growth media containing 20%
(v/v) MTS assay reagent was added to each well containing a
wafer. Plates were incubated for 40 minutes at 37 °C, before
0.5 mL of supernatant was transferred to a 48-well plate and
absorbance at 490 nm of each well was measured. Absorbance
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readings of blank wells (no cells) were subtracted from all
readings and then values were normalized to cells grown on
silicon wafers with no polymer coating.
Results and discussion
Synthesis of polynitroxide copolymers
The aim of this work was to produce nitroxide polymer
films which could be coated onto a variety of substrates.
Poly(methyl methacrylate) was chosen as the base material as
it does not possess inherent anti-biofilm properties, so would
enable assessment of the ability of the nitroxides to prevent
biofilm formation. By using copolymer blends of PTMA with
PMMA, the surface concentration of nitroxide can be
controlled, whilst ensuring good film properties and low
aqueous solubility. The synthesis of the copolymers was
achieved using a multi-step sequence commonly employed in
the literature, involving polymerising of a
tetramethylpiperidine methacylate monomer (TMPM) and
then oxidising this species to the desired PTMA (Scheme 1).44
To synthesise the initial PTMPM-PMMA copolymers, reversible
addition−fragmentaVon chain-transfer (RAFT) polymerization
was used, to allow control of the molecular weight, but also to
assess its suitability for potential future use to develop
structurally more advanced systems, such as block copolymers
or brushes.
Some complications for using RAFT polymerisation in the
synthesis of PTMPM have been reported in the literature, with
one potential complication being aminolysis of the
thiocarbonyl chain transfer agent.44 It has been suggested that
the steric hindrance around the secondary amine, and the
extra stability of the trithiocarbonate RAFT agents can
overcome this potential limitation, so this was investigated
first using UV-Vis spectroscopy.54 A solution of TMPM and the
RAFT agent (CPPTTC) in dioxane was made up at
polymerisation concentrations, without radical initiator, and
allowed to react at 85 °C for up to 24 h. The solution was
periodically sampled and analysed by UV-Vis spectroscopy.
Over the time course of the experiment, there was no
reduction in the characteristic trithiocarbonate absorbance at
308 nm (ESI, Fig 1). The small increase in absorbance at 24 h
was due to evaporation of the solvent, which was evident by a
visible reduction in the sample volume and swelling of the
rubber septum. This result was confirmed with 1H NMR
spectroscopy, where the α-methylene of the Z-group (3.3
ppm) showed no detectable evidence for aminolysis of the
RAFT agent (ESI, Fig. 2).
With the stability of the RAFT agent confirmed, a series of
statistical copolymers of MMA and TMPM were prepared,
from 0 – 100 wt% TMPM. It was found that the
polymerisations would proceed to reasonably high conversion
when using 0.2 equivalents of initiator to RAFT agent. The one
exception is the 50 wt% polymerisation, which only ran to 46%
conversion. This is an anomaly in this series, but was kept in
the sample set, as physical characterisation of films by
ellipsometry, AFM and XPS, showed no discernible difference
in properties caused by this lower conversion. The molar mass
dispersities of the obtained polymers ranged from 1.3-1.5,
indicating that perfect control over polymerization was
difficult. These values are an overestimation of the dispersity,
as some interaction between the polymers and the column
material was apparent in the SEC traces (ESI. Fig 3). While
these SEC experiments do not demonstrate ideal conditions,
they were the most suitable conditions found. When SEC was
run on a THF system, the polymers did not elute from the
column. Despite this, the measured incorporation of the
nitroxide monomer in the polymers showed a good correlation
with the feed ratio, and the molar mass was close to the
targeted 50 kDa (Table 1). This reduced control over
polymerization may affect the use of the resulting polymers in
more advanced systems, where high end group fidelity is
required, and this is currently being investigated further.
The next step of the synthetic route was to remove the
trithiocarbonate end groups prior to oxidation of the
secondary amine to the target nitroxide. This was done to
ensure there was no oxidation of these groups, which could
lead to chain coupling or other unwanted side reactions.41 This
was achieved using a previously reported facile oxidation of
the polymers by AIBN in air. This method converts the RAFT
groups to peroxide groups, followed by reduction to hydroxyl
groups by triphenylphosphine.55, 56 The removal of the
trithiocarbonate groups was evidenced by the loss of the
characteristic trithiocarbonate UV-Vis absorbance at 308 nm
and of the α-methylene resonance of the pentyl Z-group in 1H
NMR spectrum (ESI, Fig. 4 and 5).
With the synthesis of the PTMPM-PMMA copolymers
Feed wt % TMPM Feed molar ratio
MMA:TMPM:RAFT:AIBN
% conversion
(1H NMR)
a
wt % TMPM in
copolymerb
Mn (kDa)
SECc
Mw (kDa)
SECc
ÐM
SECc
0 500 : 0 : 1 : 0.2 80 0 28.1 37.7 1.34
25 375 : 55 : 1 : 0.2 64 29.8 29.6 40.8 1.38
50 250 : 111 : 1 : 0.2 46 59.8 20.9 30.9 1.48
75 166 : 125 : 1 : 0.2 74 81.6 36.2 49.3 1.36
100 222 : 0 : 1 : 0.2 74 100 30.9 44.5 1.44 a % conversion reported as the average for both monomers, measured by 1H NMR, b wt% incorporation of 2,2,6,6-tetramethyl-4-piperidinyl methacrylate in copolymer measured by 1H NMR, relative to methyl methacrylate. c All SEC measurements are reported as relative to poly(methyl methacrylate) standards.
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completed, and the RAFT end groups removed, the final step
in the synthesis was oxidation of the tetramethyl piperidine
group to the desired TEMPO nitroxide. This was achieved using
the oxidising agent mCPBA. This is a rapid method, with the
oxidation of the polymers achieved in less than four hours. It
was found that aqueous work up was required immediately
after reaction, to ensure complete removal of the mCPBA,
before isolation of the polymers by precipitation and drying. If
this was not undertaken, then insoluble polymer gels would be
recovered after vacuum drying.
Oxidation to the TEMPO nitroxide was characterised using
two complementary techniques. Firstly, electron paramagnetic
resonance (EPR) spectroscopy was used to detect and quantify
the presence of the free radical species in each of the
copolymers (Figure 1.a). At low incorporation of the nitroxide
monomer, the characteristic three-line EPR spectrum for
TEMPO at 336 mT can be seen. The signal is broadened
compared to the reference spectrum of the small molecule
TEMPO (ESI, Fig 6.a), due to the different chemical
environments of the monomer units in the polymer and spin-
spin coupling between multiple nitroxide units. This
broadening increases with further incorporation of the
nitroxide monomer, as the localised concentration of nitroxide
rises, and therefore the spin-spin coupling effect increases.
The concentration of nitroxide in each sample was quantified
by comparing the double integral of EPR signal to a standard
curve of the reference TEMPO (ESI, Fig. 6). This comparison
suggests that only 50-70% of the secondary amine groups
within the polymer have been oxidised to the corresponding
nitroxide. This could be an underestimation due to the
broadened signal of the TEMPO in the polymer, compared to
the small molecule TEMPO, or may result from over-oxidation
of the secondary amine groups to the corresponding N-
oxoammonium cations (which are EPR silent). This hypothesis
is supported when looking at the normalised ratio of integrals,
compared to the 100 wt% PTMA sample, which correlate well
with the measured incorporation of TMPM from 1H NMR
spectroscopy.
To confirm the successful oxidation of the nitroxide
polymers, UV-Vis spectroscopy was used as a complementary
technique to measure the absorbance of the nitroxyl group at
460 nm. With increasing incorporation of the piperidine
monomer in the precursor polymer, a corresponding increase
in the concentration of nitroxide after oxidation was observed
in the UV-Vis spectra (Figure 1.b). Quantification of the
recorded absorbance measurements against a TEMPO
standard curve (ESI, Fig. 7) revealed approximately 80%
conversion to the nitroxide. This value is consistent with
reports in the literature, where it can be difficult to achieve
quantitative conversion to the nitroxide species.57 Once again,
as observed with the EPR spectroscopy data, there is
agreement between the normalised nitroxide concentration
values compared to the wt% incorporation of the TMPM
monomer, across the range of copolymers, indicating that
control of the nitroxide concentration in the material was
achieved.
Thin-film fabrication and characterisation
To investigate the potential relationship between nitroxide
concentration at the surface and the resulting anti-biofilm
Figure 1. Characterisation of nitroxide concentration in PTMA-PMMA copolymers after oxidation by mCPBA. (a) EPR spectra of unpaired nitroxide radical, (b) UV-Vis spectra of
polymers showing nitroxide absorbance at 460 nm.
wt %
TMPM
(1H NMR)
UV-Vis EPR
%
conversiona
Normalised
ratio
absorbanceb
%
conversiona
Normalised
ratio
integralb
100 80 100 68 100
82 82 84 69 82
60 79 59 58 51
30 69 26 51 22
0 0 0 0 0 a % conversion was calculated by comparison to standard curves prepared of TEMPO and are based on wt% incorporation of precursor monomer calculated from 1H NMR b ratio of absorbance maximum at 308 nm (UV-Vis) or the double integral (EPR) with 100 wt% TMPM normalised to 100 %.
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activity, we used spin coating to fabricate homogeneous and
smooth polymer thin films with no surface architecture. Firstly,
a pure PMMA polymer was used to optimise the spin coating
conditions, varying the solvent and examining the effect of
angular speed and polymer concentration on the resulting film
thickness. All films were characterised using multi-wavelength
ellipsometry for thickness and roughness (as a qualitative
measure of film quality). When comparing the use of
chloroform and toluene as spin coating solvents, it was found
that toluene was easier to handle and gave more consistent,
and slightly thinner and smoother films than chloroform. Using
toluene, we then investigated the effect of polymer
concentration and angular speed on the resulting film
thicknesses (ESI. Fig 8). From this study, it was established that
a polymer concentration of 10 wt% and a spin speed of 8000
RPM for 60 s would provide consistent films with thicknesses
of around 350 nm for our spin coater.
Using the optimised conditions described above, a series of
10 x 10 mm silicon wafers were coated with the PTMA-PMMA
copolymers. The spin coating provided films of consistent
thickness, on average 355 ± 16 nm, that were not greatly
influenced by the chemical composition of the polymers
(Figure 2.a). Atomic force microscopy confirmed the smooth
surface of the polymer films, with an RMS roughness of ± 3.0
nm over an area of 255 µm2 (ESI, Fig 9).
As we wanted to establish the influence of the nitroxide at
the surface, we wanted to ensure there was no change in the
surface energy for the different copolymers. It has been well
established that surface energy (or wettability), measured by
contact angle, has a strong influence on biofouling and biofilm
attachment.14, 58-60 From static drop contact angle
measurements, the concentration of nitroxide used in the
copolymers was shown to have no significant influence on the
contact angle of the resulting spin coated films, when
compared to the pure PMMA films (Figure 2.c). The contact
angle measured for these films ranged from 78° (100% PMMA)
to a maximum of 91° (80 wt% PTMA-PMMA). Previous work
has shown that polystyrene surfaces of moderate
hydrophobicity showed the highest levels of bacterial
adhesion, with either significant increases (28°) or decreases
(115°) in hydrophilicity leading to a decrease in adhesion.58
Therefore these materials offer an ideal model for testing the
influence of the nitroxide on the anti-biofilm properties of
these films, without the influence of the physical properties of
the surface.
The aim of this work was to use polynitroxide copolymers,
as a means to control the concentration of nitroxides at a
surface. To confirm the spin coated films prepared using
different ratios of PTMA and PMMA followed this behaviour,
XPS was used to measure the atomic composition of the
surfaces of our polymer films. From the XPS survey spectra of
the two homopolymer films, the presence of carbon (290 eV)
and oxygen (538 eV) from the methacrylate components can
be detected, plus some peaks for silicon from the underlying
silicon wafer (160 and 107 eV, ESI, Fig. 10). At a thickness of
more than 300 nm, the silicon will not be detected through the
film, so it is believed that this peak arises from small defects in
the film, such as scratches from handling. The survey spectrum
Figure 2. Characterisation of PTMA-PMMA polymer thin films, spin coated onto silicon wafers (10 x 10 mm). (a) Average thickness of spin coated films as measured by
ellipsometry. All measurements are an average of six individually coated wafers. (b) Images of water droplet on surface of coated wafer during contact angle measurements. Top –
0 wt% PTMA, bottom 100 wt% PTMA. (c) Average static drop contact angle measured for each surface as a function of composition.
Figure 3. Average atomic composition of nitrogen for each PTMA-PMMA polymer
film used in the biofilm tests. One film sample was measured, with each data point
representing at least three locations on the film. For 0 % and 100 % PTMA, two
independent film samples were measured.
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of PTMA shows an additional nitrogen 1s peak (407 eV),
confirming the presence of the nitroxide monomer. Using the
high resolution spectra for carbon, oxygen and nitrogen, it is
possible to quantify the concentration of nitroxide at the
surface, and they showed good correlation with predicted
values from the composition measured by 1H NMR
spectroscopy (Figure 3). These results indicate that the surface
composition of the spin coated material matches closely with
the concentration of the bulk material.
Examination of the high resolution nitrogen 1s region of
the XPS spectra provides information about the chemical state
of the nitrogen atoms present, including detailed information
about nitroxide species on the surfaces (Figure 4). In each of
the samples, the N 1s peaks can be deconvoluted to give 3
components, which can be assigned as nitrogen bound only to
carbon and hydrogen (400 eV), and nitrogen with an oxidation
state of one (402 eV) and two (406 eV). The unoxidised species
at 400 eV can be assigned to the starting piperidine TMPM
monomer, which was not oxidised to the nitroxide. The peak
at 402 eV corresponds to the desired nitroxide species or the
related hydroxylamine. The more oxidised species at 406 eV
can be assigned to the N-oxoammonium cation.41, 42 This peak
is not consistent in intensity across samples or across different
positions of the same sample (ESI. Fig 13). It is also not
apparent when the XPS spectrum is recorded for the polymer
powders (ESI. Fig 14). We hypothesise that during the thin film
formation, the high localised concentration of nitroxides can
lead to bimolecular redox reactions, leading to the formation
of the anionic N-oxide and the N-oxoammonium cation.61
While this species appears to be semi-stable in the dry,
condensed state, it is anticipated that after exposure to
biological media it will be quickly reduced back to the
nitroxide.62 When the relative concentration of the
components representing the nitroxide (402 eV) and N-
oxoammonium cation (406 eV) are compared to the
component for the piperidine precursor (400 eV), we see a
similar concentration of oxidised species, as measured by UV-
Vis and EPR spectroscopy (Figure 1).
In-vitro evaluation of anti-biofilm coatings
To evaluate whether the polynitroxide coatings could function
as anti-biofilm materials, we tested their ability to inhibit the
formation of Pseudomonas aeruginosa (PAO1) biofilms in a
flow cell assay. The assay used in this work, while limited in
scope, in our hands is a robust assay that has been validated to
be reliable in providing preliminary results of biofilm activity.
The flow cell is a common in vitro model for biofilms, as it
better mimics environmental conditions and the flow helps to
encourage mature biofilm development.63 The assay
developed uses a convertible flow cell, allowing for a coated
silicon wafer to be placed inside. They were inoculated with
104 bacterial cells/mL and incubated for 24 hours. After this
time the silicon wafer was removed from the flow cell,
extensively washed to remove non-adherent bacteria, before
the biofilm was disrupted using a bead beater. The amount of
bacteria attached to the wafer in the biofilm was then
evaluated by performing a colony forming assay on the
supernatant (expressed as CFU/mL). This data was log
transformed and is shown in Figure 5, where it is clear that all
of the surfaces containing nitroxide copolymers had a lower
amount of adherent bacteria, when compared to the 100%
PMMA control film (one-way ANOVA, p < 0.005, summary of
statistics in ESI, Table 1). In all cases this is greater than a
99.6% reduction in the quantity of bacteria attached to the
surface (99.96% for 100 wt% PTMA, ESI, Table 1).
From previous studies on small molecule nitroxides, a
bactericidal effect is not expected for nitroxides, which is
supported by the presence of viable cells adhered to the
nitroxide surfaces.13 From this assay, we cannot confirm if the
reduction in biofilm is due to a decrease in adherent bacteria
during the colonisation phase, or a change in the life cycle of
the attached cells, preventing them from forming biofilms.
Additional testing is currently being performed to understand
the molecular mechanism of protection.
Another interesting observation from the data is that the
relative concentration of nitroxide within the copolymers, and
therefore at the surface, does not appear to have a strong
effect on the amount of attached bacteria, in this model. This
is a somewhat surprising but interesting result, as it suggests
that only relatively small amounts of nitroxide need to be
incorporated into the coatings to maintain the anti-biofilm
activity. This is favourable for producing large scale quantities
of these materials for both medical and industrial applications.
Figure 4. High resolution XPS of the N1s region showing the different chemical
states of the nitrogen atom in either the PTMPM monomer (N-H), PTMA
monomer (N-O), or the N-oxoammonium cation (N=O).
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Figure 6. Evaluation of 3T3 mouse fibroblast cell viability by an MTS assay,
demonstrating no effect on cell proliferation after 48 h incubation on silicon
wafers spin coated by polynitroxide films, All data has been normalised to blank
For these materials to be used as a biomaterial coating,
they must be non-toxic to mammalian tissues. To evaluate
this, the cell proliferation of 3T3 fibroblasts on the
polynitroxide films was investigated and compared to bare
silicon and the PMMA control. Spin coated wafers, as were
used in the bacterial testing, were incubated with the
fibroblasts for 48 h of growth, and an MTS cell proliferation
assay was used to quantify cell viability (Figure 6). The assay
showed no statistical difference in cell viability between the
silicon only control wafers, the PMMA control, the 50 wt %
copolymer and the PTMA homopolymer (one-way ANOVA, p >
0.05). This assay indicates that the nitroxide component of the
methacrylate copolymers do not increase toxicity in
comparison to PMMA, and that these materials are very
similar to PMMA, a widely used biomaterial.64
Conclusions
In this report it has been demonstrated that polynitroxides can
be used as anti-biofilm surface coatings. Polynitroxide
copolymers were prepared by RAFT polymerisation of MMA
and TMPM, a piperidine precursor, to prevent inhibition of the
radical polymerisation by the nitroxide. After removal of
trithiocarbonate end groups, the piperidine monomer was
directly oxidised to the desired TMA nitroxide by treatment
with mCPBA. Characterisation by EPR and UV-Vis spectroscopy
showed that the nitroxide composition of the polymers was
controlled by the feed ratio of the precursor TMPM monomer.
These materials were then spin coated onto surfaces to
provide an active nitroxide surface, where the surface
concentration, as measured by XPS, was dictated by the
composition of the copolymers.
In vitro testing has demonstrated that these polynitroxide
films are able to reduce the amount of bacteria that form a
biofilm on a surface by up to 99.96 %. This effect was not
strongly concentration dependant after 30 wt% incorporation
of the nitroxide monomer. This is the first demonstration of a
surface attached nitroxide maintaining the anti-biofilm activity
seen for the small molecule and drug analogues. This opens up
opportunities to develop new surface coatings which are able
to prevent biofilm fouling, which could be used to reduce the
prevalence of nosocomial infections and industrial fouling. In
particular, anti-biofilm nitroxides could be used in combination
with other approaches that prevent bacterial attachment or
are bactericidal, to develop multidimensional materials,
capable of fighting the biofilm through multiple mechanisms.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was funded by a Future Fellowship from the
Australian Research Council (KFS, FT140100746) and the Asian
Office of Aerospace Research and Development (Grant No.
FA2386-16-1-4094, R&D 16IOA094) and supported by the Ian
Potter Foundation (NRBB), Ramon Areces Foundation (CFN)
and Fundação de Amparo à Pesquisa do Estado de São Paulo -
2016/24413-0 (MDTT). The characterisation data reported in
this paper was obtained at the Central Analytical Research
Facility operated by the Institute for Future Environments
(QUT). Access to CARF is supported by generous funding from
the Science and Engineering Faculty (QUT). The authors wish
to acknowledge the assistance of Dr Aaron Micallef for
technical NMR spectroscopy support.
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Polynitroxide copolymers to reduce biofilm fouling on surfaces
Polynitroxide films—the first example of surface tethered nitroxides reducing biofilm fouling.
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