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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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Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

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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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View Article OnlineDOI: 10.1039/C8PY01101J

http://dx.doi.org/10.1039/c8py01101j

ARTICLE Polymer Chemistry

2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx



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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Polymer Chemistry ARTICLE




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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c consult author(s) regarding copyright matterspolynitroxide copolymers to reduce biofilm fouling on...

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