Preparation of an Antibacterial Poly(ionic liquid) Graft Copolymer of Hydroxyethyl Cellulose

Article abstract of DOI:10.1021/acs.biomac.5b01300

Poly(ionic liquid)s (P(IL)s) of different degrees of polymerization (10, 50, and 100) were prepared via RAFT polymerization using an alkyne-terminated xanthate as transfer agent, with a monomer conversion of up to ～80% and a D_M of 1.5 for P(IL)₁₀₀. Subsequently, P(IL) chains were coupled to ¹⁵N-labeled azido-functionalized hydroxyethyl cellulose (HEC), forming graft copolymers of HEC with different chain length and graft densities, which were characterized using (¹³C and ¹⁵N) CP-MAS NMR and FT-IR spectroscopies. The antibacterial activities of HEC-g-P(IL)s were tested against Escherichia coli and Staphylococcus aureus and were comparable to ampicillin, a well-known antibiotic, demonstrating efficient activity of the graft copolymers against bacteria. Moreover, HEC-g-P(IL)s were slightly more effective against E. coli than S. aureus. A decrease in graft density of P(IL)₁₀ on the HEC backbone decreased the activity of the graft copolymers against both bacteria. These findings suggest that HEC-g-P(IL) could find applications as an antiseptic compound, for example, in paint formulation.

Full text of DOI:10.1021/acs.biomac.5b01300

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Article
Preparation of an antibacterial poly(ionic liquid)
graft copolymer of hydroxyethyl cellulose
Fanny Joubert, Robert Paul Yeo, Gary J Sharples, Osama
M Musa, David R.W. Hodgson, and Neil R. Cameron
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01300 • Publication Date (Web): 06 Nov 2015
Downloaded from http://pubs.acs.org on November 9, 2015
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Preparation of an antibacterial poly(ionic liquid)
graft copolymer of hydroxyethyl cellulose
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a,c
b,c
b,c
d
Fanny Joubert , R. Paul Yeo , Gary J. Sharples , Osama M. Musa , David R. W. Hodgson
a,c,e
a,c,f,g
, Neil R. Cameron*
a
Department of Chemistry, Durham University, Science Laboratories, Durham DH1 3LE, UK
School of Biological and Biomedical Sciences, Durham University, Science Laboratories,
b
Durham DH1 3LE, UK
c
Biophysical Sciences Institute, Durham University, Science Laboratories, Durham DH1 3LE,
UK
d
Ashland Speciality Ingredients, 1005 Route 202/206, Bridgewater, NJ 08807, USA
e
Centre for Sustainable Chemical Processes, Durham University, Science Laboratories, Durham
DH1 3LE, UK
f
Department of Materials Science and Engineering, Monash University, Clayton, Victoria, 3800,
Australia
g
School of Engineering, University of Warwick, Coventry, CV4 7AL, UK
*
To whom correspondence should be addressed: neil.cameron@monash.edu; +61399020774.
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Keywords: Hydroxyethyl cellulose, poly(ionic liquid)s, “grafting to”, RAFT polymerization,
‘click’ chemistry, antibacterial properties.
Abstract
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Poly(ionic liquid)s (P(IL)s) of different degrees of polymerisation (10, 50 and 100) were
prepared via RAFT polymerisation using an alkyneꢀterminated xanthate as transfer agent, with a
monomer conversion of up to ~80% and a Ɖ of 1.5 for P(IL) . Subsequently, P(IL) chains
M
100
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were coupled to Nꢀlabelled azidoꢀfunctionalized HEC, forming graft copolymers of HEC with
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different chain length and graft densities which were characterised using ( C and N) CPꢀMAS
NMR and FTꢀIR spectroscopies. The antibacterial activities of HECꢀgꢀP(IL)s were tested against
E. coli and S. aureus and were comparable to ampicillin, a wellꢀknown antibiotic, demonstrating
efficient activity of the graft copolymers against bacteria. Moreover, HECꢀgꢀP(IL)s were slightly
more effective against E. coli than S. aureus. A decrease in graft density of P(IL) on the HEC
1
0
backbone decreased the activity of the graft copolymers against both bacteria. These findings
suggest that HECꢀgꢀP(IL) could find applications as an antiseptic compound, for example in
paint formulation.
Introduction
Synthetic, manꢀmade polymers are produced from petroleum, a nonꢀrenewable resource. This
will eventually result in decreased production of synthetic polymers with consequent societal
problems because of their ubiquity in everyday life (packaging, furniture, containers, electronics,
etc.). Polymers are also found in abundance in nature, in the form of biopolymers such as
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cellulose, starch, proteins, amylose and chitin. In contrast to petroleumꢀbased synthetic polymers,
these polymers come from renewable sources. Cellulose is the most abundant biopolymer on
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Earth and is principally found in the cell walls of plants . Cellulose, which is defined by the
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assembly of βꢀDꢀanhydroglucopyranose (AGU) units , presents interesting properties such as
biocompatibility and high strength, however, its principal drawback is its insolubility in both
3, 5ꢀ7
organic and aqueous solvents, limiting considerably its use in industry
. Chemical
modification of the hydroxyl groups of cellulose overcomes this problem to some extent. In fact,
hydroxyethyl cellulose (HEC) (Figure 1), where the hydroxyl groups have been modified with
ethylene oxide, shows solubility in dimethyl sulfoxide and water due to the removal of the Hꢀ
bonding networks of cellulose. Although the chemical modification of cellulose can improve
considerably its physical properties, cellulose derivatives are still less competitive than synthetic
polymers due to the latter’s better solubility and easier production.
Figure 1. Chemical structure of HEC
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Tashiro and, more recently, MuñozꢀBonilla reviewed advances in the preparation of
antibacterial polymers which contain, for instance, quaternary ammonium salts, biguanide group
and quaternary pyridinium, phosphonium or sulfonium salts. A new class of monomer, ionic
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liquid monomers (IL), which include vinylimidazolium, methacryloyl and styreneꢀbased ILs
(Figure 2), have been developed recently and present promising properties such as high thermal
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stability, high ionic conductivity, low vapour pressure and bactericidal effects
. The
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antibacterial properties arise from the presence of the positive charge which disrupts the bacterial
membrane leading to cell death.
Figure 2. Structure of ionic liquid monomers (a) methacryloylꢀ, (b) vinylꢀimidazoliumꢀ and (c)
styreneꢀ based IL
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ILs have been polymerised successfully using conventional free radical polymerisation and
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CRP techniques such as ATRP
and RAFT polymerisation
. Regarding RAFT
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polymerisation, Mori et al. used a xanthateꢀtype chain transfer agent (‘CTA’) to polymerise
vinylimidazolium saltꢀbased ionic liquid (IL) monomers leading to a monomer conversion of up
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to 78% and dispersity (Ɖ ) ranging from 1.2 to 1.4. Vijayakrishna et al. , however, used a
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dithiobenzoate CTA to polymerise methacryloylꢀbased IL monomers to a lower monomer
conversion after a longer reaction time. The Ɖ was not reported, however, the degree of
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polymerisation was estimated using H NMR spectroscopy and was in agreement with the
monomer conversion.
Although HEC is used extensively as an emulsifier, stabiliser, thickener and cosmetic filmꢀ
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former in the formulation of personal care products and paints
, it lacks antibacterial
properties. Antibacterial compounds such as antibiotics are commonly mixed with cellulose to
prevent the growth of microorganisms such as bacteria. In this work we sought to develop a
hybrid HEC material possessing antibacterial properties via the introduction of cationic
polymers, such as P(IL)s, as grafts on the HEC backbone. To prepare HECꢀgꢀP(IL), our reported
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method based on a “grafting to” approach, combining RAFT polymerisation and CuAAC, was
used. An acryloylꢀbased IL monomer was chosen for the RAFT polymerisation because of the
predicted higher reactivity of such a monomer compared to vinylꢀimidazoliumꢀbased ILs. Here,
we report the use of this method to form wellꢀdefined HEC graft copolymers containing different
graft densities and graft lengths of P(IL), as well as their antibacterial behaviours and
cytotoxicities against an immortalised lung alveolar cell line (A549).
2. EXPERIMENTAL
2
.1.
Materials
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ꢀHydroxyethyl cellulose (HEC) (M =90 kDa, MS =2.5), sodium azide (NaN ), Nꢀlabelled
w
3
1
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sodium azide (Na N), carbon tetrabromide, triphenylphosphine, propargyl bromide solution
(
80% wt. in toluene), potassium ethyl xanthogenate, 2,2′ꢀazobis(2ꢀmethylpropionitrile) (AIBN),
11ꢀbromoꢀ1ꢀundecanol, pꢀtoluene sufonic acid, monomethyl ether hydroquinone (MEHQ),
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acrylic acid, 1ꢀmethylimidazole, sodium Lꢀascorbate, copper (II) sulphate pentahydrate,
N,N,N′,N′ꢀtetramethylethylenediamine (TMEDA), dimethylformamide (DMF), pentane,
cyclohexane, sodium hydroxide, magnesium sulfate, ampicillin sodium salt and Luria broth were
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purchased from Sigma Aldrich. Isoꢀsensitest broth was obtained from Oxoid . Toluene, diethyl
ether, acetone, tetrahydrofuran (THF) and dialysis tubing of 500ꢀ1,000 Da and 3,500 Da MWCO
were supplied by Fisher Scientific. DMSOꢀd was purchased from Apollo Scientific, and dialysis
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tubing of 50 kDa MWCO was supplied by Spectrumlabs. Both bacteria, Escherichia coli Kꢀ12
wildꢀtype strain (W3110 / ATCC27325, Fꢀ, λꢀ, rpoS(Am), rphꢀ1, Inv(rrnDꢀrrnE)) and
Staphylococcus aureus (3R7089 strain Oxford / ATCC9144) were prepared in the laboratory.
The A549 immortalized lung alveolar cell line was provided from the American tissue culture
collection (ATCC). Dulbecco’s Modified Eagle Medium (DMEM), Fetal Calf Serum (FCS) and
Almar blue were purchased from Life Technologies Limited (UK).
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.2.
Characterisations
Solution state NMR spectra were recorded using a Bruker Avance 400 spectrometer at 400.13
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MHz ( H) and 100.60 MHz ( C). For solid state NMR spectroscopy, a Varian VNMRS
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spectrometer with a 9.4 T magnet was used and C (100.562 MHz) and N (40.527 MHz)
experiments were run using the cross polarisation method. IR spectra were recorded on a Perkinꢀ
Elmer 1600 Series FTꢀIR spectrometer. Molecular weight and Ɖ data were obtained using triple
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detection SEC on a Viscotek TDA 301 with refractive index, viscosity and light scattering
detectors and 2 × 300 mL PL HFIPgel 9 µm columns. 1,1,1,3,3,3ꢀHexafluoropropanꢀ2ꢀol with 25
mM of trifluoroacetic acid salt (NaTFAc) was used as the eluent at a flow rate of 0.8 mL/min
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and at a constant temperature of 40 ºC. Experiments were performed by Smithers Rapra,
Shawbury, UK.
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.3.
Synthesis and characterisation
2.3.1. Preparation of N ꢀHEC
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The procedure followed that reported in the literature (see Scheme 1). In a roundꢀbottomed
flask fitted with a condenser, 2ꢀhydroxyethyl cellulose 1 (M =90,000 g/mol, MS =2.5, 2.5 g,
w
ꢀ
3
ꢀ2
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9.19×10 mol, 1 eq), sodium azide (3.8 g, 5.85×10 mol, 6 eq.) and Nꢀlabelled sodium azide
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ꢀ3
Na N (0.1 g, 1.54×10 mol, 0.1 eq) were dissolved in DMF (100 mL). The mixture was heated
3
at 80 °C for 1 h in order to dissolve HEC. The mixture was cooled to room temperature and
ꢀ2
carbon tetrabromide (14.3 g, 4.31×10 mol, 5 eq) was added. Triphenylphosphine (11.8 g,
ꢀ2
4
.50×10 mol, 5 eq) dissolved in DMF (12.5 mL) was added carefully to the HEC mixture. The
reaction was then left for 24 h at room temperature under magnetic stirring. The product was
precipitated by addition of toluene (500 mL) and collected by filtration. The solid was dissolved
in DMF (50 mL) and reꢀprecipitated using diethyl ether (500 mL). After being filtered, the solid
was washed with acetone (100 mL) and dried under vacuum at 50 °C overnight. The product 2
was obtained as a light yellow solid in quantitative yield (3.1 g). The product was characterised
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using solid state ( C and N) CPꢀMAS NMR and FTꢀIR spectroscopies.
2
.3.2. Preparation of the alkyneꢀterminated xanthate
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The procedure followed that reported in the literature (see Scheme S1). In a oneꢀneck roundꢀ
ꢀ3
bottomed flask, propargyl bromide solution 3 (80% wt. in toluene, 1.02 g, 8.57×10 mol) and
ꢀ3
potassium ethyl xanthogenate 4 (1 g, 6.24×10 mol) were dissolved in THF (10 mL). The flask
was covered with aluminium foil and the reaction was run overnight at room temperature. THF
(100 mL) was added and the mixture was filtered to remove KOH. The excess solvent was
evaporated and distilled water (10 mL) was added to the residues. The product was extracted
with diethyl ether (3×30 mL). The diethyl ether was removed under vacuum and the final
product 5 was purified via column chromatography using pentane as eluent and dried overnight
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under vacuum. The product was obtained as a pale yellow oil in a yield of 48% (0.48 g). H
NMR (400 MHz, DMSOꢀd ): δ (ppm) 1.37 (t, J =7.0 Hz, 3H, CH ꢀCH ꢀO), 3.22 (t, J =2.6 Hz,
6
H
3
2
13
1H, CH≡Cꢀ), 3.97 (d, J =2.6 Hz, 2H, CH≡CꢀCH ꢀ), 4.65 (q, J =7.0 Hz, 2H, ꢀCH ꢀO); C NMR
2
2
(
400 MHz, DMSOꢀd ): δ (ppm) 13.5 (CH ꢀCH ꢀOꢀ), 23.5 (ꢀCH ꢀSꢀ), 70.6 (ꢀCH ꢀOꢀ), 74.1
6 C 3 2 2 2
(
CH≡Cꢀ), 78.6 (CH≡Cꢀ), 211.8 (ꢀC=S).
2
.3.3. Preparation of 1ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀmethylimidazolium bromide
1ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀmethylimidazolium bromide was prepared in a twoꢀstep
25
procedure (Scheme 2) according to Harmand et al.
First step: In a twoꢀneck roundꢀbottomed flask fitted with both a condenser and a dropping
ꢀ3
funnel, 11ꢀbromoꢀ1ꢀundecanol 6 (2.5 g, 9.95×10 mol, 1 eq.), pꢀtoluene sulfonic acid (0.2 g,
ꢀ3
ꢀ4
1
.16×10 mol, 1 eq.) and MEHQ (0.02 g, 1.6×10 mol, 0.02 eq.) were dissolved in cyclohexane
ꢀ2
(27 mL) and the mixture was refluxed for 1 h. Acrylic acid 7 (0.8 mL, 1.16×10 mol, 1 eq.) was
dissolved in cyclohexane (4 mL) and the solution was dropped into the reaction mixture using a
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dropping funnel. The final mixture was refluxed overnight. Once cooled to room temperature,
the organic solution of the product was washed with NaOH (10% w/w, 3×50 mL), dried over
MgSO and concentrated under vacuum. The product 8 was obtained in a yield of 88% (2.4 g).
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H NMR (400 MHz, DMSOꢀd ): δ (ppm) 1.30ꢀ1.80 (m, 18H, BrꢀCH ꢀ(CH ) ꢀ), 3.52 (t, J =6.7
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H
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2 9
Hz, 2H, BrꢀCH ꢀ), 4.10 (t, J =6.7 Hz, 2H, ꢀOꢀCH ꢀ), 5.94 (dd, J =10.3, 1.6 Hz, 1H, HCH=CHꢀ),
2
2
1
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.17 (dd, J =17.3, 10.3 Hz 1H, ꢀCHꢀCH ), 6.32 (dd, J =17.3, 1.6 Hz, 1H, HCH=CHꢀ); C NMR
ac 2
(100 MHz, DMSOꢀd ): δ (ppm) 25.3ꢀ35.1 (9C, BrꢀCH ꢀ(CH ) ꢀ), 40.4 (ꢀCH ꢀBr) 64.0 (ꢀCH ꢀOꢀ
6 C 2 2 9 2 2
), 128.4 (ꢀCH=CH ), 131.2 (ꢀCH=CH ), 165.4 (ꢀC=O).
2
2
ꢀ3
Second step: Bromoalkylacrylate 8 (2.4 g, 7.96×10 mol, 1 eq.), and 1ꢀmethylimidazole 9 (0.6
ꢀ3
mL, 7.96×10 mol, 1eq.) were mixed and heated at 50 °C for 24 h. Once the mixture had cooled
to room temperature, the product precipitated and was filtered. The product 10 was then washed
with ether (3×50 mL) and dried under vacuum to obtain a white powder in a yield of 85% (2.6
1
g). H NMR (400 MHz, DMSOꢀd ): δ (ppm) 1.24ꢀ1.77 (m, 18H, ꢀOꢀCH ꢀ(CH ) ꢀCH ꢀ), 3.86 (s,
6
H
2
2 9
2
3
H, ꢀNꢀCH ), 4.08 (t, J =6.6 Hz, 2H, ꢀOꢀCH ꢀ), 4.16 (t, J =7.2 Hz, 2H, CH ꢀCH ꢀNꢀ), 5.95 (dd, J
3 2 2 2
=
10.3, 1.7 Hz, 1H, HCH=CHꢀ), 6.15 (dd, J =20.3, 10.3 Hz, 1H, ꢀCHꢀCH ), 6.30 (dd, J =17.3, 1.6
2
+
13
Hz, 1H, HCH=CHꢀ), 7.77 (dd, 2H, J =27.0, 0.97 Hz, ꢀCH=CHꢀ), 9.22 (s, 1H, ꢀCH=N ꢀ); C
NMR (100 MHz, DMSOꢀd ): δ (ppm) 25.3ꢀ35.1 (9C, BrꢀCH ꢀ(CH ) ꢀ), 35.7 (NꢀCH ), 48.7
6
C
2
2 9
3
(CH ꢀCH ꢀNꢀ), 64.0 (ꢀCH ꢀOꢀ), 122.2 (NꢀCH=CHꢀN) , 123.5 (NꢀCH=CHꢀN), 128.4 (ꢀ
2 2 2
CH=CH ), 131.2 (ꢀCH=CH ), 136.5 (ꢀNꢀCH=Nꢀ), 165.4 (ꢀC=O).
2
2
2.3.4. RAFT polymerisation of 1ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀmethylimidazolium
bromide
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18
The following procedure was inspired by the work of Mori et al. and Vijayakrishna et al.
(
see Scheme 3). 1ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀmethylimidazolium bromide (IL) 10 (10, 50 or 100
eq.), xanthate 5 (1 eq.) and AIBN (0.3 eq.) were dissolved in DMF (10 mL) and the mixture was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
purged with N for 10 min. The flask was sealed and the reaction was heated at 70 °C for 17 h.
2
The mixture was dialysed against water for 2 days using a dialysis tubing of MWCO of 500ꢀ
1
,000 g/mol for P(IL)10 and 3,500 g/mol for P(IL)50 and P(IL) . Subsequently the dialysed
100
material was freezeꢀdried overnight and the polymer 11 was obtained as a sticky white solid in a
yield of 70ꢀ80% (Table S1). The polymer was characterised using solution state NMR
spectroscopy and SEC. NMR spectra were recorded in DMSOꢀd and the M and Ɖ values were
6
n
M
determined using SEC with conventional calibration (PMMA standards).
2
.3.5. CuAAC
between
N ꢀHEC
3
and
alkyneꢀterminated
poly(1ꢀ(11ꢀ
acryloyloxyundecyl)ꢀ3ꢀmethylimidazolium bromide)
General procedure: in a roundꢀbottomed flask fitted with a drying tube, N ꢀHEC 2, alkyneꢀ
3
terminated P(IL) 11, sodium Lꢀascorbate (2 eq.), copper (II) sulfate pentahydrate (1 eq.) and
N,N,N′,N′ꢀtetramethylethylenediamine (1 eq.) were dissolved in DMF (20 mL). The flask was
heated at 30 °C for 24 h. The mixture was cooled to room temperature and dialyzed for 3 days
using a 50 kDa MWCO membrane against MilliꢀQ water (18.2 Mega Ohm). HECꢀgꢀP(IL) 12
was further lyophilized and obtained in a yield ranging from 64ꢀ100% (Table S2). The graft
13
15
copolymers were characterized using solid state ( C and N) CPꢀMAS NMR and FTꢀIR
spectroscopies.
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2
.4.
Biological study
.4.1. Antiꢀbacterial testing
10
11
12
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The bacteriological effects of the HEC ꢀgꢀP(IL) s 18 containing P(IL) with different chain
m
n
lengths (n= 10, 50 and 100; m =1) and different graft densities (m =1, 0.3 and 0.17; n =10) were
2
6
investigated using an Escherichia coli Kꢀ12 wildꢀtype strain (W3110) , and Staphylococcus
2
7
aureus (3R7089 strain Oxford) selected as representative Gramꢀnegative and Gramꢀpositive
species, respectively.
Bacterial growth inhibition assay on agar plates
TM
HEC ꢀgꢀP(IL) s were dissolved in Oxoid isoꢀsensitest broth at a concentration of 10 mg/mL
m
n
and 6 sequential 2ꢀfold dilutions were prepared in the same medium. 10 µL of each dilution was
spotted onto a LuriaꢀBertani (LB) agar plate containing bacteria in a 0.6% soft agar overlay (5
mL) and the plates incubated overnight at 30 °C. Samples were analysed in triplicate and a zone
of growth inhibition was observed due to the presence of the HEC ꢀgꢀP(IL) s.
m
n
Determination of minimum inhibitory concentration (MIC)
MIC determination followed the protocol described by Andrews . 10 mg/mL dilution of
HEC ꢀgꢀP(IL) s in isoꢀsensitest broth were prepared and 11 sequential 2ꢀfold dilutions were
28
m
n
made using the medium as diluent. The inoculum was prepared from a single bacterial colony
grown on LB agar plates and inoculation of 5 mL of isoꢀsensitest broth followed by incubation
overnight at 37 °C. 20ꢀ50 µL of overnight culture was inoculated into 1 mL of isoꢀsensitest broth
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and incubated at 37 °C with aeration until an inoculum density of approximatively 10 cfu/spot
was reached as determined by an optical density A650nm of 0.07, equivalent to a 0.5 MacFarland
standard (240 µM BaCl in 0.18 M H SO4 aq.). This inoculum was diluted 10ꢀfold in isoꢀsensitest
2
2
0
1
2
3
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9
0
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0
broth for use in MIC determination.
In a 96ꢀwell microtiter plate, 50 µL of each dilution of HEC ꢀgꢀP(IL) was mixed with 50 µL
m
n
of inoculum for each dilution. Ampicillin was diluted in a similar manner as HEC ꢀgꢀP(IL) s and
m
n
was used as a positive antibacterial control. The plate was incubated at 37 °C overnight with
shaking and the optical density A650nm of each well was recorded using a Biotek Synergy HT
MultiꢀMode Microplate Reader. Samples were analysed in triplicate.
2
.4.2. Measurement of cytotoxicity
29
A549, an immortalized lung alveolar cell line , was cultured in DMEM supplemented with
3
10% FCS. Approximately 5×10 cells in a volume of 100 ꢁL per well were used to seed a 96ꢀ
well plate. Cells were allowed to grow in a humidified incubator with an atmosphere of 5% CO2
at 37 °C until 70% confluence was achieved. HEC ꢀgꢀP(IL) s to be tested were dissolved in
m
n
culture medium to a concentration of 1 mg/mL and 100 ꢁL was added to each well of the first
column of wells to give a final concentration of 500 ꢁg/mL. Subsequently 2ꢀfold serial dilutions
were performed from columns 1ꢀ11 with the final column being left as a negative control. After
further incubation the effect of the treatment was determined by a cell viability assay using
30
Almar Blue (GIBCO) that was analysed on a fluorescent plate reader (BioTek, FL500) using
the GEN5 software package. Samples were analysed four times to ensure the reliability of the
results. The fluorescence was read using a fluorescence excitation and emission wavelengths of
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5
40–570 nm (peak excitation is 570 nm) and 580–610 nm (peak emission is 585 nm)
respectively. The evolution of the fluorescence intensity as a function of the concentration of
HECꢀgꢀP(IL) permitted the graphical determination of LD .
5
0
10
11
12
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3. RESULTS AND DISCUSSION
3
3
.1.
Graft copolymers of HEC
.1.1. Azide functionalized hydroxyethyl cellulose: N ꢀHEC
3
2
3
N ꢀHEC 2 was prepared from HEC 1 using a oneꢀstep procedure (Scheme 1). In order to aid
3
15
15
the detection of nitrogen by N NMR spectroscopy, sodium azide was doped with Na N . The
3
characterisation of the partially labelled N ꢀHEC has already been described in our previous
3
2
2
work , where the efficiency of a method combining click reaction and RAFT polymerisation to
prepare graft copolymers of HEC was demonstrated, and resulted in the full functionalization of
the primary alcohols of HEC with NaN . For simplicity, Scheme 1 only shows substitution at the
3
C6 position, however, substitution at positions C2 and C3 cannot be ruled out.
1
5
Scheme 1. Synthesis of partially Nꢀlabelled N ꢀHEC 2.
3
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.1.2. Poly(1ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀmethylimidazolium bromide)
0
1
2
3
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7
8
9
0
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9
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ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀmethylimidazolium bromide 10 was synthesised in 75% overall
2
5
yield following the twoꢀstep procedure of Harmand et al. , as shown in Scheme 2. It was then
polymerised by RAFT polymerisation using xanthate 5 as chain transfer agent (Scheme 3). A
xanthate CTA was chosen because the imidazolium ring could potentially reduce the reactivity
of the acrylate functionality.
N
OH
Br
N
N
O
9
7
O
N
O
Br
HO
11 Br
11
11
O
O
6
8
10
Scheme 2. Preparation of 1ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀmethylimidazolium bromide 10.
17
The polymerisation of IL 10 (Scheme 3) was inspired by the work of Mori et al. and
1
8
Vijayakrishna et al. . RAFT polymerisation was performed at 70 °C in DMF using a ratio of
xanthate to initiator (AIBN) of 1:0.3 which led to a good comprise between monomer conversion
and dispersity. Different degrees of polymerisation (10, 50 and 100) were targeted in order to
produce graft copolymers of HEC with different chain lengths. After 17 h of polymerisation, the
1
monomer conversion determined using H NMR spectroscopy was found to be approximatively
7
0ꢀ80% (Table S1). Subsequently, the P(IL)s 11 were characterised by NMR spectroscopy and
SEC.
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Scheme 3. RAFT polymerisation of 1ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀmethylimidazolium bromide
(
IL) 10.
1
In the H NMR spectrum of P(IL) (Figure 3), signals δ ~1.2ꢀ1.8 ppm are assigned to the ꢀ
1
00
H
CH protons along the side chain and the backbone of P(IL). The signal δ ~2.2 ppm is assigned
2
H
to the methine group of the backbone and the signal δ ~3.9 ppm is assigned to the ꢀCH group
H
3
which is the substituent group of the ring. The signal δ ~4 ppm is assigned to ꢀCH ꢀ next to the
H
2
imidazolium ring and the signal δ ~4.2 ppm is assigned to the protons –CH next to the acrylate
H
2
group. The signal δ ~ 7.7ꢀ7.8 and 9.4 ppm are assigned to the protons of the imidazolium ring.
H
The signals of the end groups were not found, perhaps because they are hidden by the signals of
1
P(IL) because they are too weak. The absence of peaks in the range δ ~6.0ꢀ6.5 ppm in the H
H
NMR spectrum confirms that dialysis was successful in removing any residual monomer, and
thus the purity of the polymer can be estimated at > 99%. The spectra of the P(IL) and P(IL)
10
5
0
did not show any new signals, thus, the estimation of the chain length from end groups analysed
by NMR spectroscopy was not feasible.
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ꢀ
h,ꢀd
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9
0
ꢀ
ꢀ
l
i
h,ꢀd
e
g
jꢀ+ꢀk
m
1
Figure 3. Solution state H NMR spectrum of poly(1ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀ
methylimidazolium bromide) (DPtargeted =100)
The molecular weight and dispersity of P(IL) were determined using SEC. The SEC trace for
P(IL)100 is displayed in Figure 4. Conventional calibration with PMMA standards was used to
determine M and was found to be 26,600 g/mol. This is in good agreement with the predicted
n
M at 71% monomer conversion (27,600 g/mol). A Ɖ of 1.5 was measured, indicating some
n
M
control of the polymerisation. A value of Ɖ below 1.2 is widely accepted to indicate full control
M
of the polymerisation, while a value between 1.2 to 1.5 indicates some control of the
polymerisation. A value greater than 1.5 means the loss of control resulting in a broader
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molecular weight distribution. Typically, polymers produced via conventional free radical
polymerisation have a Ɖ value higher than 1.5. The dispersity value of 1.5 for P(IL)100 was
M
borderline for a polymer which was prepared from a reversible deactivation radical
polymerization (RDRP) process. Regarding P(IL)s with shorter chain lengths (DP = 10 and 50),
10
11
12
13
14
15
16
17
18
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Ɖ values are expected to be similar or slightly higher because the lower DP accentuates
M
differences in chain lengths.
250
2
30
10
2
190
170
150
130
110
9
7
0
0
12
14
16
18
20
22
24
Retention volume (mL)
Figure 4. SEC trace of P(IL) .
1
00
3.1.3. Graft copolymers HECꢀgꢀpoly(1ꢀ(11ꢀacryloyloxyundecyl)ꢀ3ꢀmethylimidazolium
bromide): HECꢀgꢀP(IL)s
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Ionic liquid polymers P(IL) 11 of different chain lengths (n =10, 50 or 100) were coupled to
n
each AGU unit of N ꢀHEC 2 and this resulted in the preparation of graft copolymers HECꢀgꢀ
3
P(IL) s 12 (Scheme 4) where the grafts were composed of 10, 50 or 100 repeat units of ionic
n
0
1
2
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9
0
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7
8
9
0
liquid monomer. These fully substituted HEC graft copolymers thus have a graft density of 1.
Additionally, HEC ꢀgꢀP(IL) s were prepared with different graft densities (m =0.3 and 0.17) via
m
10
the coupling of one P(IL) chain per 3 and 6 AGU units of N ꢀHEC respectively. In order to
1
0
3
remove the homopolymer P(IL) , the reaction mixture was dialysed against water using a dialysis
n
tubing of a MWCO of 50,000 g/mol ensuring the recovery of only HEC ꢀgꢀP(IL) s. The graft
m
n
13
15
copolymers were characterised using solid state ( C and N) CPꢀMAS NMR and FTꢀIR
spectroscopies. Their solubility permits characterisation by solution state NMR spectroscopy,
however the resulting spectra do not show the presence of HEC and thus evidence of the
coupling could not be demonstrated. The chain length (defined by “n”) of P(IL) did not influence
the detection of the HEC backbone by solid state NMR spectroscopy, however, decreasing the
graft density (defined by “m”) allowed the presence of HEC to be confirmed. The
characterisation of HEC ꢀgꢀP(IL) s (m =1, 0.3 and 0.17) only are reported here.
m
10
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15
Scheme 4. CuAAC between alkyneꢀended P(IL) and partially Nꢀlabelled N ꢀHEC
n
3
FTꢀIR spectra of N ꢀHEC, P(IL) and HEC ꢀgꢀP(IL) s are displayed in Figure 5. The total
3
10
m
10
ꢀ1
loss of the azide signal at 2136 cm in the spectrum of HEC ꢀgꢀP(IL) (Figure 5d) indicates the
1
10
complete consumption of the azide group of N ꢀHEC (Figure 5a) due to the formation of the
3
triazole with the alkyneꢀterminated P(IL) . This is crucial evidence of the grafting because,
1
0
otherwise, the IR spectrum of HEC ꢀgꢀP(IL) is the same as that of P(IL) (Figure 5e), and no
1
10
10
absorption bands of HEC were detected because they are probably hidden by those of P(IL) .
1
0
ꢀ1
On decreasing the graft density (Figure 5b & c), the azide signal at 2136 cm was still detected,
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however a decrease of its relative intensity was observed compared to the IR spectrum of N3ꢀ
HEC (Figure 5a) indicating partial consumption of the azide. In fact, increasing the graft density
produces a decrease of the relative intensity of the azide signal (Figure 5c vs. 5b). Except for this
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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ꢀ1
signal, the others are characteristic of the presence of P(IL) . The bands at 3400 cm are
1
0
ꢀ1
ꢀ1
assigned to ꢀOH groups, at ~3142 cm to the ꢀCH groups of the ring, at 2856ꢀ2926 cm to the
ꢀ1
ꢀ1
alkyl ꢀCH groups, at 1734 cm to the carbonyl group, at 1676 cm to the ꢀC=Cꢀ and ꢀC=Nꢀ
ꢀ1
ꢀ1
groups, at 1576 cm to the ꢀCꢀCꢀ and ꢀCꢀNꢀ groups, at 1468 cm to the ꢀCH alkyl groups and at
ꢀ1
~1170 cm to the alkyl ꢀCH groups.
(
e)
N3
(d)
c)
b)
(
(
(
a)
3
900
3400
2900
2400
1900
1400
900
400
ꢀ1
Wavenumber (cm )
Figure 5. FTꢀIR spectrum of: (a) N ꢀHEC; (b) HEC0.17ꢀgꢀP(IL) ; (c) HEC ꢀgꢀP(IL) ; (d)
3
10
0.3
10
HEC ꢀgꢀP(IL) ; and (e) P(IL) .
1
10
10
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The solid state C CPꢀMAS NMR spectrum of HEC ꢀgꢀP(IL) (Figure 6c) shows only signals
1
10
of the P(IL) grafts. The signal δ ~174.4 ppm is assigned to the carbonyl group, δ ~139.5 and
10
C
C
~
123.6 ppm are assigned to the alkyl ꢀCH groups of the ring, δ ~64.9 ppm to the ꢀCH ꢀO group,
C 2
10
11
12
13
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δ ~49.1 ppm to the ꢀCH ꢀN group, δ ~41.4 ppm to the methine group and the signals δ ~36.0ꢀ
C
2
C
C
30.6 ppm to the methylene and methyl groups. The absence of signals for the HEC backbone was
expected because of the relatively high molecular weight of the P(IL) grafts. In our previous
1
0
2
2
work , coupling of polymers with N ꢀHEC was demonstrated more easily because of the low
3
molecular weight of the grafted chains (PVP or PNIPAAM ) which did not exceeded 1,000
1
0
10
g/mol. The signals assigned to the HEC backbone, which were relatively weak compared to
1
3
22
those of the grafts, were detected in the solid state C NMR spectra , however, we suspected
that an increase in the molecular weight of the grafts would result in the disappearance of the
signals of HEC. This is what we observed with P(IL) grafts, which have a molecular weight of
1
0
approximatively 4,000 g/mol. However, a decrease of the graft density permitted the detection of
the HEC backbone. In fact, signals assigned to HEC were clearly detected in the spectrum of
HEC0.17ꢀgꢀP(IL) (Figure 6a) which possesses the lowest graft density of P(IL) . The signal δ
C
10
10
~103.6 ppm is assigned to the carbon at the C1 position, ~82.6 ppm to carbon at the C4 position,
74.9 ppm to the set of carbons at C2, C3 and C5 positions, ~71.2 ppm to the set of carbons at
~
C6, C7, C8 and C9 positions and ~51.1 ppm to the carbon at C10 position. These signals
assigned to the HEC backbone were still detected in the spectrum of HEC ꢀgꢀP(IL) (Figure
0
.3
10
6b) but their relative intensities are lower due to the increase of the graft density of P(IL)10
compared to those in the spectrum of HEC0.17ꢀgꢀP(IL)10.
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l,ꢀh
C6ꢀtoꢀC9
(
a)
b)
C2,ꢀC3,ꢀC5
C4
ꢀ
C10
C1
0
1
2
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3
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5
6
7
8
9
0
(
(c)
e
g
f
j,ꢀk
i
m
1
3
Figure 6. Solid state C CPꢀMAS NMR spectra of HEC ꢀgꢀP(IL) : (a) HEC ꢀgꢀP(IL) ; (b)
m
10
0.17
10
HEC ꢀgꢀP(IL) ; (c) HEC ꢀgꢀP(IL) .
0
.3
10
1
10
1
5
The solid state N spectra of HEC ꢀgꢀP(IL) with n =10, 50 and 100 were all identical, thus
1
n
only the spectrum of HEC ꢀgꢀP(IL) is reported here (Figure 7b) with the spectrum of N ꢀHEC
1
10
3
(
Figure 7a) used as a reference. The spectrum of N ꢀHEC showed two signals δ ~ –310 ppm
3
N
3
1, 32
and ~ –170 ppm which are assigned to N and N respectively (Scheme 4)
The differences in
.
α
γ
intensity can be explained by the differences in their proton environment. N is next to a
α
methylene group resulting in a higher intensity of its signal compared to that of N because
γ
2
2
nitrogen is detected through the excitation of the protons. From our previous investigation , only
1
5
the N signal remained in the N CPꢀMAS NMR spectrum after cycloaddition of N ꢀHEC with
α
3
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alkyneꢀterminated synthetic polymers, however, the signal was shifted from ~ –310 ppm to ~ –
1
34 ppm, providing evidence of the success of the “click” reaction. Here, the spectrum of HEC1ꢀ
gꢀP(IL)10 showed two signals δ ~ –208.0 and ~ –194.3 ppm which are assigned to the
N
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33
imidazolium ring, based on the literature . More precisely, the former is assigned to the ꢀNꢀCH
3
group and the latter to the NꢀCH ꢀ group. The absence of the signal δ ~ –134 ppm, assigned to
2
N
N after the cycloaddition, is most likely caused by the high molecular weight of the P(IL)10
α
grafts which decreases the relative intensity of the N signal. Decreasing the graft density of
α
P(IL) resulted in the loss of signals assigned to the nitrogen atoms nitrogen present in the
1
0
grafts, however, the signals assigned to the triazole and azide were also absent. In fact, a
decrease of the graft density did not change the ratio of N (after cycloaddition) to P(IL) , and
α
10
this is why N remains undetectable. N and N (before cycloaddition) were also not detected
α
α
γ
upon decreasing the graft density because their concentration is too low compared to the rest of
the sample.
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(
a)
0
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0
(b)
15
Figure 7. Solid state N CPꢀMAS spectra: (a) N ꢀHEC; (b) HEC ꢀgꢀP(IL)
10
3
1
To summarise, the characterisation of the graft copolymers demonstrated the presence of P(IL)
grafts by NMR and FTꢀIR spectroscopies, however, detection of the HEC backbone was more
challenging due to the high molecular weight of the IL monomer. When the graft density of
1
3
P(IL) was decreased, the HEC backbone was detected by C CPꢀMAS NMR spectroscopy
1
0
15
indicating the presence of HEC in the product. The use of N CPꢀMAS NMR spectroscopy did
not demonstrate the formation of the triazole, most probably because of the relatively low level
of enrichment over the 0.4% background. However, evidence of coupling between P(IL)s and
ꢀ1
N ꢀHEC came from the disappearance of the azide band at 2100 cm in the FTꢀIR spectrum of
3
the graft copolymers. Furthermore, a quantitative yield for each coupling reaction (Table S2)
strongly implies coupling of the azide in N ꢀHEC, resulting in the preparation of HEC based
3
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P(IL) graft copolymers. The final graft copolymers were purified by extensive dialysis using a
high molecular weight cut off membrane (50,000 g/mol) so it can reasonably be expected that
any uncoupled P(IL) chains were removed.
10
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3.2.
Biological activities of HECꢀgꢀP(IL)s
The successful preparation of HECꢀgꢀP(IL)s 12 permitted the study of their effects on bacteria
and on an immortalized lung alveolar cell line (A549) to investigate potential applications.
Furthermore, the influence of the chain length and the graft density of P(IL) on biological
activity were investigated.
3
.2.1. Antibacterial effects
Two tests were performed to evaluate the bacteriological effects of the HECꢀgꢀP(IL)s. The first
was a qualitative test to visualise any effects on growth inhibition, whereas the second was a
quantitative test permitting a determination of the minimum inhibition concentration (MIC)
which is the lowest concentration of a compound which inhibits bacterial growth. E. coli (Gramꢀ
negative) and S. aureus (Gramꢀpositive) were chosen as representative bacteria.
Solutions of each HEC ꢀgꢀP(IL) were prepared using isoꢀsensitest broth and series of 11 x 2ꢀ
m
n
fold dilutions were prepared resulting in concentrations ranging from 10 mg/mL to 2 ꢁg/mL. In
the first test, which shall be referred to as the zone inhibition assay, 10 ꢁL of each solution of the
first 6 dilutions were spotted onto an agar plate containing an overlay of E. coli or S. aureus
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bacteria. The plates were incubated overnight to allow the lawn of bacteria to grow (Figure 8).
nd
To determine the MIC (2 test), 50 ꢁL of each graft copolymer solution was pipetted into a 96ꢀ
well plate containing 50 ꢁL of bacteria solution (both in isoꢀsensitest broth). The 96ꢀwell plate
was incubated overnight and the optical density (OD) at A650nm was determined in each well to
monitor bacterial growth (Figure 9). A 10 mg/mL dilution series of ampicillin was used as a
positive control.
0
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9
0
For E. coli, growth inhibition (Figure 8a) was obtained from solutions of P(IL) ꢀgꢀHEC s with
n
1
concentrations ranging from 10 to 1.75 mg/mL, independent of the graft chain length (n = 10, 50
and 100). At 0.875 mg/mL HEC ꢀgꢀP(IL) (with n = 100, 50 and 10) concentration, inhibition
1
n
was not observed indicating the loss of antibacterial effect at this concentration. At 1.75 mg/mL,
the size of the zones were similar independent of the length of P(IL) grafts, indicating there was
no major influence of the chain length on antibacterial properties. However, the graft density of
P(IL) on HEC affected to a slight extent the inhibitory effect. The inhibitory effect of the graft
10
copolymers with lower graft densities (m =0.3 and 0.17) was shown at higher concentration than
HEC ꢀgꢀP(IL) . At a 1.75 mg/mL HEC ꢀgꢀP(IL) (with m =0.3 and 0.17) concentration,
1
10
m
10
inhibition zones were not observed indicating the lack of an inhibitory effect. Furthermore, the
size of the inhibition zones for HEC ꢀgꢀP(IL) and HEC0.17ꢀgꢀP(IL)10 were similar at a
0
.3
10
concentration of 2.5 mg/mL, demonstrating a limited influence of the graft density on inhibition
at these densities. However, HEC ꢀgꢀP(IL) (n =10, 50 and 100) also have a higher inhibitory
1
n
effect compared to those of lower graft density.
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(a)
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(b)
Figure 8. Results of the zone inhibition assays for the graft copolymers HECꢀgꢀP(IL)s against
a) E. coli (b) S. aureus.
(
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For S. aureus, zones of growth inhibition were observed at 10 and 5 mg/mL concentrations for
each graft copolymers (Figure 8b), however, at 2.5 mg/mL concentration, HEC ꢀgꢀP(IL) and
1
10
HEC ꢀgꢀP(IL) still showed a slight growth inhibition, whereas HEC ꢀgꢀP(IL) , HEC ꢀgꢀ
0
.3
10
1
50
1
0
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0
P(IL)100 and HEC0.17ꢀgꢀP(IL)10 did not. Samples with longer grafts of IL (50 and 100) or a
decreased graft density (HEC0.17ꢀgꢀP(IL) ) had a similar effect on the growth of S. aureus
10
bacteria.
In order to corroborate these observations, MICs for each bacterial strain with the different
graft copolymers were determined. Regarding E. coli, the graft copolymers HEC ꢀgꢀP(IL) s (n
1
n
=
10, 50 and 100) and HEC ꢀgꢀP(IL) s (m =0.3 and 0.17) gave MIC values of 10 and 19 ꢁg/mL
m 10
respectively (Figure 9a). Furthermore, a MIC value of 39 ꢁg/mL was measured for ampicillin,
higher than that determined for HECꢀgꢀP(IL)s demonstrating a stronger inhibitory effect of the
graft copolymers against Gramꢀnegative bacteria. For S. aureus, MIC values for HEC ꢀgꢀP(IL)10
1
and HEC ꢀgꢀP(IL) were estimated to be 19 ꢁg/mL whereas a MIC value of 39 ꢁg/mL was
0.3
10
determined for HEC ꢀgꢀP(IL) (n =50 and 100) and HEC ꢀgꢀP(IL) (Figure 9b). The MICs of
1
n
0.17
10
HEC ꢀgꢀP(IL) (n =100 and 50) and HEC0.17ꢀgꢀP(IL)10 were similar to that of ampicillin,
1
n
however, the MICs of HEC ꢀgꢀP(IL) s (m =1 and 0.3) were lower, suggesting a potentially
m
10
stronger efficacy of these graft copolymers against Gramꢀpositive bacteria compared to a wellꢀ
known antibiotic.
To summarise, growth inhibition effects of the graft copolymers on both Gramꢀpositive and
Gramꢀnegative bacteria were observed. The magnitude of the effects were comparable to those
produced by ampicillin, a commonly used antibiotic. The cationic charge of the imidazolium ring
8
is likely to be responsible for this effect . Surprisingly, the chain length of the grafts, which
determines the number of charges, did not significantly influence the antibacterial properties of
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HECꢀgꢀP(IL)s against E. coli.. However, a decreased graft density of P(IL)10 on the HEC
backbone increased the MIC, suggesting a reduction of the antibacterial effect. This suggests that
the antibacterial activity results from the presence of the cationic charge at the outer surface and
does not rely on the number of charges in the graft copolymer. A decreased chain length
therefore improved the antibacterial effect whereas a decreased graft density reduced the
antibacterial action. The antibacterial activity was slightly stronger using graft copolymers with
shorter chains and one plausible explanation could be the accessibility of the cationic charges
which disrupt the cell wall envelope of the bacteria resulting in its death. Graft copolymers with
long grafts could adopt different conformations limiting the accessibility of the cationic charge
for the bacteria and thus the antibacterial power. The MIC values against S. aureus were higher
than against E. coli suggesting that the antibacterial activity of the graft copolymers is slightly
stronger against E. coli than against S. aureus; the improved resistance of S. aureus over E. coli
was also evident in zone inhibition assay (Figure 8). The mechanism of the inhibitory effect most
likely resides in the interaction between the cationic charge and the bacterial membrane. Gramꢀ
positive and Gramꢀnegative bacteria cell envelopes differ significantly in terms of structure,
permeability and density of charge. The S. aureus phospholipid bilayer is protected by a thick
10
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3
4
outer layer of peptidoglycan further stabilised by teichoic acids which may partially restrict
direct contact of graft copolymers with the bacterial membrane. In order to destabilise the S.
aureus membrane the concentration of HECꢀgꢀP(IL) had to be higher compared to that for E.
n
coli where the outer membrane lipopolysaccharide is directly available for attack. However,
HEC ꢀgꢀP(IL) with lower graft densities (m =0.3 and 0.17) have similar antibacterial effects
m
10
against both bacteria (E. coli and S. aureus) and this could be due to relatively good accessibility
of the cationic charge to destabilise readily Gramꢀpositive bacteria. The graft copolymers
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