Antibacterial effects of poly(2-(dimethylamino ethyl)methacrylate) against selected gram-positive and gram-negative bacteria

Article abstract of DOI:10.1021/bm901166y

Antimicrobial coatings can reduce the occurrence of medical device-related bacterial infections. Poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) is one such polymer that is being researched in this regard. The aims of this study were to (1) elucidate pDMAEMA's antimicrobial activity against a range of Gram-positive and Gram-negative bacteria and (2) to investigate its antimicrobial mode of action. The methods used include determination of minimum inhibitory concentration (MIC) values against various bacteria and the effect of pH and temperature on antimicrobial activity. The ability of pDMAEMA to permeabilise bacterial membranes was determined using the dyes 1-N-phenyl-naphthylamine and calcein-AM. Flow cytometry was used to investigate pDMAEMA's capacity to be internalized by bacteria and to determine effects on bacterial cell cycling. pDMAEMA was bacteriostatic against Gram-negative bacteria with MIC values between 0.1-1 mg/mL. MIC values against Gram-positive bacteria were variable. pDMAEMA was active against Gram-positive bacteria around its pK_a and at lower pH values, while it was active against Gram-negative bacteria around its pK_a and at higher pH values. pDMAEMA inhibited bacterial growth by binding to the outside of the bacteria, permeabilizing the outer membrane and disrupting the cytoplasmic membrane. By incorporating pDMAEMA with erythromycin, it was found that the efficacy of the latter was increased against Gram-negative bacteria. Together, the results illustrate that pDMAEMA acts in a similar fashion to other cationic biocides.

Full text of DOI:10.1021/bm901166y

Biomacromolecules 2010, 11, 443–453
443
Antibacterial Effects of Poly(2-(dimethylamino
ethyl)methacrylate) against Selected Gram-Positive and
Gram-Negative Bacteria
Lee-Anne B. Rawlinson,† Sine´ad M. Ryan,† Giuseppe Mantovani,‡ Jay A. Syrett,§
David M. Haddleton,§ and David J. Brayden*,†
School of Agriculture, Food Science, and Veterinary Medicine, and UCD Conway Institute, University
College Dublin, Dublin 4, Ireland, School of Pharmacy, University of Nottingham, NG7 2RD, United
Kingdom, and Department of Chemistry, University of Warwick, CV4 7AL, United Kingdom
Received October 12, 2009; Revised Manuscript Received November 27, 2009
Antimicrobial coatings can reduce the occurrence of medical device-related bacterial infections. Poly(2-
(dimethylamino ethyl)methacrylate) (pDMAEMA) is one such polymer that is being researched in this regard.
The aims of this study were to (1) elucidate pDMAEMA’s antimicrobial activity against a range of Gram-positive
and Gram-negative bacteria and (2) to investigate its antimicrobial mode of action. The methods used include
determination of minimum inhibitory concentration (MIC) values against various bacteria and the effect of pH
and temperature on antimicrobial activity. The ability of pDMAEMA to permeabilise bacterial membranes was
determined using the dyes 1-N-phenyl-naphthylamine and calcein-AM. Flow cytometry was used to investigate
pDMAEMA’s capacity to be internalized by bacteria and to determine effects on bacterial cell cycling. pDMAEMA
was bacteriostatic against Gram-negative bacteria with MIC values between 0.1-1 mg/mL. MIC values against
Gram-positive bacteria were variable. pDMAEMA was active against Gram-positive bacteria around its pKa and
at lower pH values, while it was active against Gram-negative bacteria around its pKa and at higher pH values.
pDMAEMA inhibited bacterial growth by binding to the outside of the bacteria, permeabilizing the outer membrane
and disrupting the cytoplasmic membrane. By incorporating pDMAEMA with erythromycin, it was found that
the efficacy of the latter was increased against Gram-negative bacteria. Together, the results illustrate that
pDMAEMA acts in a similar fashion to other cationic biocides.
Poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) is
a mucoadhesive polymer, that is cationic if dissolved into
acidified media or if quaternized by using an alkylating
agent.13,14 Recent advances in living radical polymerization
(LRP)15,16 and reversible addition-fragmentation transfer (RAFT)
polymerization17 allowed for the synthesis of pDMAEMA with
tunable polymer chain length and macromolecular architecture,
as well as a narrow molecular weight distribution. It is a
thermoresponsive polymer and, at increased temperatures, the
polymer phase separates from solution due to a breakdown in
hydrogen bonding interactions.18 The solubility of pDMAEMA
in aqueous solution is also pH-dependent. At pH 7, pDMAEMA
is partially charged (hydrophilic) and partially uncharged
(hydrophobic), resulting in an amphiphilic molecule.19 At lower
pH the polymer becomes more positively charged, and at
pH values greater than 7, it becomes mostly uncharged.19
pDMAEMA has numerous potential uses that include use as a
nonviral gene delivery vector20,21 in water purification22,23 and
in drug delivery.24 It is also used as a coating for soil-resistant
surfaces,25 as an ion exchange media for protein separation,26
and to adapt the wettability of surfaces, including microfluidic
devices.27 In addition to this, pDMAEMA has been attached to
glass,28,29 filter paper,28,30 polystyrene,31 and polypropylene32
and also as an antimicrobial surface coating to inhibit the growth
of Escherichia coli (E. coli) and Bacillus subtilis. It has also
been incorporated in antimicrobial copolymers to inhibit growth
of E. coli33 and S. aureus.34 The monomer DMAEMA has been
shown to decrease the binding of various coagulase negative
and positive Staphylococcus, Streptococcus pyogenes, E. coli,
and P. aeruginosa strains when attached to the surface of PVC
Introduction
Microbial contamination is a major concern in areas such as
food packaging and storage and water treatment. Growth of
bacteria on implanted medical devices is a particular problem1,2
and contamination can lead to development of severe infections.
A potential method to prevent microbial contamination is to
coat susceptible surfaces with antimicrobial agents including
polymers, that inhibit growth of microorganisms.1 Numerous
polymers have been found to have antimicrobial activities
against Gram-negative and Gram-positive bacteria, yeast, and
viruses.3,4 For this reason they have been investigated as
potential antimicrobial coatings and inhibit bacterial growth
when attached to glass, paper, plastic, and metal.5-8 For
example, methacryloyloxydodecylpyridinium was incorporated
into dental resin composite and reduced microbial activity
without leaching.9 Polymers have also been used to deliver
encapsulated antimicrobial agents allowing localized, controlled
release of the drug into the target site.10,11 In addition, in vivo
experiments have recently demonstrated antimicrobial potential
of selected polymers. For example, oral administration of a
chitosan-oligosaccharide formulation over 7 days to mice
subsequently challenged with Staphylococcus aureus by the
intraperitoneal route resulted in a higher survival rate compared
with that of controls.12
* To whom correspondence should be addressed. Tel.: +353 1 716 6013.
Fax: +353 1 716 6219. E-mail: david.brayden@ucd.ie.
† University College Dublin.
‡ University of Nottingham.
§ University of Warwick.
10.1021/bm901166y
 2010 American Chemical Society
Published on Web 12/21/2009

444 Biomacromolecules, Vol. 11, No. 2, 2010
Rawlinson et al.
Scheme 1. Synthesis of Disulfide-Based Bifunctional Polymerisation Initiator, Bis[2-(2-bromoisobutyryloxy)ethyl] Disulfide (BiBOE)2S2
Scheme 2. Cu(I)Br Catalyzed Polymerization of Dimethyl Amino Ethyl Methacrylate (DMAEMA) Initiated by (BiBOE)2S2
catheters.35 pDMAEMA has also been incorporated in an
zation step. In addition to these molecules the following pDMAEMA
polymers were also made for testing.
antiadherent coating on poly(methyl methacrylate) disks to
inhibit binding of E. coli, macrophages, and fibroblasts.25
Furthermore, pretreatment of human intestinal epithelial cell
cultures with pDMAEMA led to reduced adhesion and invasion
of Salmonella ser. Typhimurium.36
Synthesis of Reducible pDMAEMA. Reducible pDMAEMA has
been reported to be less toxic than unconjugated pDMAEMA so it was
also tested for its antimicrobial activity.39
Bis(2-hydroxyethyl) disulfide (4.00 mL, 32.4 mmol) and an excess
of triethylamine (9.9 mL, 71 mmol) was added to a 500 mL round-
bottom flask, along with a magnetic stir bar, and was purged with
nitrogen for 15 min on an ice bath. Anhydrous THF (150 mL) was
then added, and the resulting solution was allowed to cool to 0 °C.
Under a nitrogen atmosphere, 2-bromoisobutyryl bromide (8.4 mL, 68
mmol) was added dropwise via a degassed syringe (dropwise addition
is essential to minimize the exotherm). The solution was allowed to
reach ambient temperature and left to stir for 6 h. The resulting
triethylammonium bromide salt was removed by filtration, and the
solvent was removed under reduced pressure. The resulting pale yellow
solution was stirred with 0.1 M aqueous Na2CO3 to hydrolyze any
residual 2-bromoisobutyryl bromide. The crude product was then
extracted three times with dichloromethane, and the organic layers were
combined and dried over anhydrous magnesium sulfate, filtered, and
the volatiles removed under reduced pressure, yielding the (BiBOE)2S2
initiator as a clear yellow oil (Scheme 1; 10.1 g, 22.0 mmol, 74.1%),
The mode of action of cationic biocides has been suggested
to progress as follows: (1) adsorption onto the bacterial cell
surface, (2) diffusion through the cell wall, (3) binding to the
cytoplasmic membrane, (4) disruption of the cytoplasmic
membrane, (5) release of cell cytoplasmic constituents, and (6)
cell death.37 pDMAEMA may work in a similar manner by
adsorbing to the cell surface through electrostatic interactions
and disrupting the cytoplasmic membrane through hydrophobic
interactions.28,30 The aims of the study were therefore to (1)
investigate the range of pDMAEMA’s antimicrobial activity
against a wide group of pathogenic and commensal organisms
using a range of pDMAEMA derivatives and (2) to investigate
the antimicrobial mode of action against selected bacteria.
Materials and Methods
Materials. All chemical syntheses were carried out using standard
Schlenk techniques under an inert atmosphere of oxygen-free nitrogen,
unless otherwise stated. Copper(I) bromide (98%) was purified accord-
ing to the method of Keller and Wycoff.38 N-(n-Propyl)-2-pyridyl-
methanimine was prepared as described earlier.15 Triethylamine (99%,
Thermo Fischer Scientific, Waltham, MA) was stored over sodium
hydroxide pellets. Anhydrous tetrahydrofuran (THF; “Hi-Dry”, 99.99%,
Romil, Cambridge, U.K.) was stored over activated 4 Å molecular
sieves under dry nitrogen. All the other general chemicals and reagents
used were of analytical grade and were obtained from Sigma-Aldrich
Company Ltd. (Dorset, U.K.), unless otherwise stated.
Synthesis of Unconjugated and Hostasol-Conjugated pDMAEMA
Polymers. Unconjugated pDMAEMA was tested to show antimicrobial
effects of the polymer without any modifications and hostasol-
conjugated pDMAEMA was also tested as it was used in some
experiments to aid in visualization. Hostasol (thioxantheno[2,1,9-
def]isochromene-1,3-dione) (389.5 Da, CAS Registry Number: 14121-
49-4) was supplied by Clariant (Muttenz, Switzerland). Fluorescent
hostasol-conjugated pDMAEMA was prepared, as described previ-
ously,14 using ethyl-2-bromo isobutyrate as the polymerization initiator
and lowering the pH of the final isolated polymer aqueous solution to
pH 5.5 with 0.5 M aqueous HCl before freeze-drying. Hostasol was
used as a fluorescent marker to track the polymer in imaging and flow
cytometry studies. Nonfluorescent pDMAEMA (unconjugated pD-
MAEMA) was prepared in the same way, except that no hostasol
methacrylate fluorescent comonomer was employed in the polymeri-
1
which was stored at 4 °C. H, 13C[1H], and CHN elemental analysis
were in line with previously published data.40
Cu(I)Br (0.33 g, 2.3 mmol) was added to a clean, oven-dried Schlenk
tube, along with a magnetic follower. The Schlenk tube was sealed
with a suba-seal, evacuated, and filled with nitrogen. Toluene (25.0
mL), DMAEMA (25.0 mL, 148 mmol), and bis[2-(2-bromoisobutyryl-
oxy)ethyl] disulfide initiator (0.25 mL, 1.2 mmol) were sequentially
added via a degassed syringe and the resulting solution was degassed
by five freeze-pump-thaw cycles (Scheme 2). The solution was heated
to 70 °C in a thermostatically controlled oil bath, and then N-(n-propyl)-
2-pyridylmethanimine (0.72 mL, 4.7 mmol) was added via a degassed
syringe (t ) 0). Samples were taken every 15 min via a degassed syringe
for conversion and molecular weight analysis. Once the reaction had
reached a satisfactory conversion, the suba-seal was removed, and air
was bubbled through for 1 h. The polymer solution was passed through
a basic alumina column and then precipitated in petroleum ether, 40-60
°C (1 L), at ambient temperature.
Synthesis of 50% Quaternized pDMAEMA. As pDMAEMA is a
charged molecule, a 50% quaternized pDMAEMA polymer was used
to investigate the impact of charge on antimicrobial activity.
The disulfide-containing polymer (3.0 g, 0.17 mmol) was dissolved
in THF (20 mL) and placed in a round-bottom flask equipped with a
stirring bar. Once heated to 40 °C, Bu3P (210 µL, 0.87 mmol) was
added to the system. The reduction was complete after 10 min, the

Poly(2-(dimethylamino ethyl)methacrylate)
Biomacromolecules, Vol. 11, No. 2, 2010 445
Scheme 3. Reduction of the Disulfide Bridge and Subsequent Quaternization with MeI
solution was cooled down to ambient temperature, passed through a
short neutral alumina pad and the volatiles were removed under reduced
pressure.
An aliquot of this reduced polymer (1.0 g, 6.4 mmol of quaternizable
amine repeating units) was dissolved in THF (25 mL) and MeI (0.20
mL, 3.2 mmol) via syringe and stirred at ambient temperature for 48 h
(Scheme 3). The solvent was then removed under reduced pressure
and the solid residue was dissolved in 20 mL of deionized water and
freeze-dried, affording the desired quaternized polymer.
Standards Institute (formerly NCCLS) protocol.43 For anaerobic bacteria
the method was adapted from the microdilution broth dilution procedure
from the NCCLS protocol,44 and for yeast the method was adapted
from the microdilution broth dilution procedure from the NCCLS
protocol.45 Aerobically- and anaerobically-grown bacterial cells were
seeded in microtiter plates at 5 × 105 CFU/mL per well and 1 × 105
CFU/mL per well, respectively. Yeast cells were seeded at 0.5-2.5 ×
103 CFU/mL per well. Cells were incubated with varying concentrations
of pDMAEMA in fresh media. Aerobic bacteria plates were incubated
at 37 °C for 18 h and microbial growth was determined using a
microplate spectrophotometer (UVM340, Asys Hitech GmbH, Eugen-
dorf, Austria) at 600 nm. Anaerobic bacteria and yeast plates were
incubated at 37 °C for 48 h and quantitative cell growth was determined
by eye. The results are presented as the mean of a minimum of three
independent replicates.
Time-Kill Curves. Time-kill studies were adapted from the time-
kill method for determining bactericidal activity as outlined in the
NCCLS protocol.46 The bacteria were inoculated into flasks at 5 ×
105 CFU/mL and incubated for 90 min at 37 °C, 170 rpm. An initial
sample was taken for serial dilution and colony counting and then
polymer (unconjugated pDMAEMA) or media alone (as a control),
were added to the flasks. Incubation was continued at 37 °C, 170 rpm,
and samples taken at timed intervals up to 48 h for colony counts.
Viable counts were calculated to give CFU/mL and time-kill curves
were plotted with log10CFU/mL against time. A bactericidal effect was
defined as a g3 log10 decrease in CFU/mL after 24 h. The results are
presented as a mean ( standard error of the mean (SEM) of a minimum
of three independent replicates.
Analysis of Polymers. Molar mass distributions were measured
using size exclusion chromatography (SEC), on a system equipped with
two PL gel 5 µm mixed D-columns (300 × 7.5 mm) and one PL gel
5 mm guard column (50 × 7.5 mm; Polymer Laboratories, suitable
for molecular weights between 200 and 400000 g/mol) with differential
refractive index detection, using THF/triethylamine 95:5 (v/v), at 1.0
mL/min, as the eluent. Poly(MMA) standards (200-3 × 105 g/mol)
were used to calibrate the SEC. Analyte samples contained (0.2% vol)
toluene as the flow marker. For purity determination, pDMAEMA
polymers were subjected to SEC-HPLC with a Varian 920 HPLC using
a BioSep-SEC-S-2000 column 300 × 7.8 mm (Phenomenex, U.K.).
Samples were eluted with 50 mM phosphate buffer (pH 6.8) at a flow
rate of 1 mL/min and monitored at a UV absorbance of 280 nm.
Bacterial Strains, Media, and Culture Conditions. Salmonella
enterica subspecies enterica serovar Typhimurium (Salmonella ser.
Typhimurium) IMD 57441 was obtained from Dr. Rebecca O’Mahony,
Centre for Food Safety, School of Agriculture, Food Science and
Veterinary Medicine, University College, Dublin. Escherichia coli (E.
coli) ATCC 10536 and Micrococcus luteus (M. luteus) ATCC 9341
were obtained from Dr. Siobhan McClean, Institute of Technology,
Tallaght, Dublin. E. coli equine isolate, Salmonella enterica subspecies
enterica serovar Enteritidis (Salmonella ser. Enteritidis) ATCC 13076,
Salmonella ser. Typhimurium bovine isolate, Lactobacillus saliVarius
(L. saliVarius) UCC118, Listeria monocytogenes (L. monocytogenes)
NCTC 11994, Listeria spp. Wild type #28, and Pseudomonas aerugi-
nosa (P. aeruginosa) QC strain were obtained from Dr. Denise Drudy,
Centre for Food Safety, School of Agriculture, Food Science and
Veterinary Medicine, University College Dublin. Bifidobacterium breVe
(B. breVe) DSMZ 20213, Bifidobacterium bifidum (B. bifidum) DSMZ
20456 and Candida albicans (C. albicans) C were obtained from Mr.
Michael Folan, Westgate Biologicals Ltd., Donegal Town, Ireland.
Staphylococcus epidermidis (S. epidermidis) 145742 was obtained from
Dr. James O’Gara, School of Biomolecular and Biomedical Science,
University College, Dublin. Aerobic bacterial strains, including faculta-
tive anaerobes, except L. saliVarius, were cultured aerobically in tryptic
soy broth (TSB) or agar (TSA) at 37 °C, unless otherwise stated.
Anaerobic bacterial strains, including L. saliVarius, were cultured in
de Man Rogosa and Sharpe (MRS) media, supplemented with 0.05%
L-cysteine-HCl, at 37 °C. L. saliVarius was cultured aerobically and B.
bifidum and B. breVe were cultured under anaerobic conditions
maintained using an AnaeroGen oxygen depleting system (Oxoid,
Cambridge, U.K.) in an anaerobic chamber. C. albicans C was cultured
aerobically in TSB supplemented with 0.5% yeast extract (TSBYE) at
37 °C. All organisms were grown from frozen stocks and subcultured
at least twice before use in experiments to ensure normal growth
patterns.
Determination of Optimum pH and Temperature for Antibacte-
rial Activity. pH and temperature optimums were determined by
calculating MIC values at varying pH values and temperatures with
increasing concentrations of polymer (unconjugated pDMAEMA and
50% quaternized pDMAEMA). Growth is represented as a percentage
of cultures containing no polymer (100% growth). The results are
presented as a mean ( SEM of a minimum of three independent
replicates.
Estimation of pKa Values for Unconjugated and 50% Quater-
nized pDMAEMAs. The pKas of unconjugated pDMAEMA and 50%
quaternized pDMAEMA were determined by pH titration. Briefly, 10
mL of 1 mg/mL polymer, in distilled water, was titrated against 0.01
or 1 M NaOH to the equivalence point (unconjugated pDMAEMA,
pH ) 9.3-9.8; 50% quaternized pDMAEMA, pH ) 11.3-11.6) to
obtain the basic form of the polymers. At this point, the titration was
continued with 0.01 M HCl and the pH of the solution was monitored.
The pH at equivalence of this curve is equal to the pKa of the polymer.
The results are presented as the average of three independent replicates.
Visualization of Hostasol-Conjugated pDMAEMA Binding to
Bacteria. To visualize the interaction of the polymer with the bacteria,
hostasol-conjugated pDMAEMA was incubated with 1 × 109 CFU/
mL Salmonella ser. Typhimurium IMD 574 and S. epidermidis 1457
at concentrations of 1 and 0.1 mg/mL, respectively. At 30 and 120
min, samples were centrifuged and the pellet was washed 3 times in
PBS to remove unbound polymer. Samples were resuspended in PBS,
mounted on slides, and viewed on a Nikon Eclipse E400 fluorescent
microscope (Nikon, Japan) at 60× magnification, using a FITC filter,
excitation wavelength (Ex) ) 465-495 nm, and emission wavelength
(Em) ) 515-555 nm. Samples containing no polymer were also viewed
as controls and brightfield pictures were taken for comparisons. Pictures
MIC. The MIC is defined as the lowest concentration of polymer
to completely inhibit growth of the bacterial cultures examined. The
method of MIC calculation for aerobic bacteria was adapted from the
microdilution broth dilution procedure from the Clinical and Laboratory

446 Biomacromolecules, Vol. 11, No. 2, 2010
Rawlinson et al.
were captured using QCapture Pro software, version 5.0 (QImaging
Corporation, BC, Canada).
in PBS, was added to bacteria in the 96-well tissue culture plate and
the fluorescence was continued to be monitored every 5 min for 2 h.
Final concentrations added were 0.1 and 1 mg/mL for S. epidermidis
and 2 mg/mL for Salmonella ser. Typhimurium. Controls included
bacteria incubated without pDMAEMA and bacteria incubated with
0.1 and 1% Triton X-100. Bacteria incubated without pDMAEMA were
considered 100% controls. The results are presented as a mean ( SEM
of a minimum of three independent replicates.
Internalization of pDMAEMA by Bacteria. To investigate the
ability of pDMAEMA to enter bacteria overnight cultures of Salmonella
ser. Typhimurium IMD 574 and S. epidermidis 1457 were grown. The
cultures were incubated at approximately 5 × 108 CFU/mL with and
without hostasol-conjugated pDMAEMA (10.7 kDa) at 1 and 0.1 mg/
mL, respectively, for 0, 0.5, 2, and 4 h at 37 °C, 170 rpm. Cells were
centrifuged at 3000 × g for 10 min at 4 °C, washed 3 times in PBS
then resuspended in 1 mL PBS. A total of 50 µL from each time point
was resuspended in 10 mL PBS at 2.5 × 106 CFU/mL and another 50
µL was resuspended in 10 mL of 1 mg/mL trypan blue (Beckman
Coulter, CA) in PBS at 2.5 × 106 CFU/mL.37 Samples were incubated
at ambient temperature for at least 30 min but not more than 6 h to
ensure bacterial survival. Samples were analyzed on a Cyan ADP flow
cytometer (Beckman Coulter, CA) using Summit version 4.3 software
(Beckman Coulter, CA). At least 10000 bacteria were analyzed per
sample. The excitation laser was set at 488 nm. For analysis of hostasol-
conjugated pDMAEMA fluorescence a 530/40 nm bandpass filter (FL
1) was used. After initial analysis, 100 µg/mL propidium iodide (PI)
was added to samples, which were incubated at 4 °C for 5 min in the
dark,47 and then reanalyzed under the same conditions. PI stains DNA
of the bacteria but is only able to enter dead cells so was used as an
indicator of cell survival. PI fluorescence was determined using a 613/
20 nm bandpass filter (PE, Texas Red, FL 3). Experiments were
repeated on at least three different occasions and representative results
are shown.
Effect of pDMAEMA on Bacterial Cell Growth. One mL aliquots
from overnight cultures of Salmonella ser. Typhimurium IMD 574 and
S. epidermidis 1457 were incubated at approximately 5 × 108 CFU/
mL with and without unconjugated pDMAEMA at concentrations of
1 and 0.1 mg/mL, respectively. They were incubated for 0, 0.5, and
4 h at 37 °C, 170 rpm. Cells were centrifuged at 3000 × g for 10 min
at 4 °C, washed 3 times in PBS, then resuspended in 1 mL PBS. 50
µL resuspended sample was added to 1 mL of 70% ethanol and
incubated at ambient temperature for 24 h. Fixed cells were centrifuged
at 3000 × g for 10 min at 4 °C, washed 3 times in PBS and then
resuspended in 1 mL PBS. 100 µL from each time point was
resuspended in 1 mL PBS at 2 × 106 CFU/mL. Twenty µL RNase (10
mg/mL stock) and 10 µL PI (10 mg/mL stock) were added then samples
were incubated at 37 °C for 30 min. Samples were analyzed on a Cyan
ADP flow cytometer (Beckman Coulter, CA, USA) using Summit
version 4.3 software (Beckman Coulter, CA, USA). At least 10,000
bacteria were analyzed per sample. The excitation laser was set at 488
nm. PI fluorescence was determined using a 613/20 nm bandpass filter
(PE- Texas Red - FL 3). Experiments were repeated on at least 3
different occasions and representative results are shown.
Outer Membrane Permeabilization. Fluorescence of the probe 1-N-
phenyl-naphthylamine (NPN) increases when incorporated into the
hydrophobic core of a bacterial cell membrane (after permeation)
compared with the fluorescence of a nonpermeated bacterial cell.48,49
Salmonella ser. Typhimurium IMD 574, Salmonella ser. Enteritidis
ATCC 13076 and E. coli ATCC 10536 were grown to an optical density
at 600 nm (OD600) of 0.5. Cells were harvested and washed three times
in PBS then resuspended in 10 mM sodium phosphate buffer with 100
mM NaCl, at a pH of 7.5. Unconjugated pDMAEMA was added to
Salmonella ser. Typhimurium, Salmonella ser. Enteritidis and E. coli
suspensions at concentrations of 2, 2, or 0.5 mg/mL, respectively.
Experiments were also carried out at pH 6 and 7 for Salmonella ser.
Typhimurium IMD 574. A total of 200 µL of bacterial suspension was
added in five replicates to 96-well plates. A 20 µL aliquot of 0.2 mM
NPN was added, and the increase in fluorescence measured on a
fluorescent microplate spectrophotometer (Spectra Max Gemini, Mo-
lecular Devices, CA) every minute for 20 min with Ex ) 350 nm and
Em ) 429 nm. The results are presented as a mean ( SEM of a
minimum of three independent replicates.
Cytoplasmic Membrane Permeabilization. The method for deter-
mination of cytoplasmic membrane permeability was adapted from the
methods of Essodaigui et al. and Edgerton et al.50,51 Calcein-AM is a
nonfluorescent derivative of the dye calcein.50,52 It is lipid soluble and
therefore able to transport across cell membranes into the cytoplasm
where it is cleaved by cytoplasmic esterases to form the hydrophilic,
fluorescent molecule calcein.50,52 Once inside, fluorescent calcein is
unable to transport back across the membrane, unless the membrane
of the cell becomes permeabilized.52 Stock solutions of calcein-
acetoxymethylester (calcein-AM) (1 mM) were prepared in DMSO and
stored at -20 °C. Overnight cultures of Salmonella ser. Typhimurium
IMD 574 and S. epidermidis 1457 were centrifuged at 3000 × g for
10 min at 4 °C and washed 3 times in PBS. The cells were then
resuspended in PBS containing 5 µM calcein-AM (Invitrogen Corpora-
tion, CA, USA) supplemented with 10% vol/vol Brain Heart Infusion
broth at a final concentration of 5 × 107 CFU/mL. 180 µL of
resuspended culture was incubated for 90 min at 37 °C in a 96-well
tissue culture plate. Fluorescence, at Ex ) 496 nm and Em ) 517 nm,
was monitored every 10 min in a fluorescent microplate spectropho-
tometer (Spectra Max Gemini, Molecular Devices, CA, USA). Twenty
µL of 10 × final concentration of unconjugated pDMAEMA, diluted
Enhancement of the Efficacy of Erythromycin by
pDMAEMA. To test whether pDMAEMA may be used to enhance
the efficacy of other antimicrobial agents, it was added at concentrations
of 4 and 10 times less than its MIC concentration to erythromycin MIC
experiments against Salmonella ser. Typhimurium IMD 574, S.
epidermidis 1457, Salmonella ser. Enteritidis ATCC 13076 and E. coli
ATCC 10536. Growth of bacteria was determined visually. The results
are presented as the mean of a minimum of three independent replicates.
Cytotoxicity Analysis: Sheep Red Blood Cell Hemolysis. The
method for determination of hemolytic potential of pDMAEMA was
adapted from the protocol outlined by Shin et al.53 Briefly, 1 mL
mechanically defibrinated sheep blood (TCS Biosciences, Buckingham,
UK) was centrifuged (2000 × g, 5 min, 4 °C) and the pellet of
erythrocytes was washed 3 times in PBS. The final pellet was
resuspended in PBS (4% v/v) and 100 µL aliquots of the suspension
were plated in 96-well microtiter plates. Cells were exposed to polymers
at varying concentrations, incubated for 60 min at 37 °C, and then
plates were centrifuged at 3000 rpm for 5 min. Aliquots (100 µL) of
the supernatant were transferred to a fresh 96 well microtiter plate,
where hemoglobin release was monitored spectrophotometrically at 414
nm using a microplate spectrophotometer (UVM340, Asys Hitech
GmbH, Eugendorf, Austria). Percent hemolysis was calculated relative
to that detected with 1% Triton X-100. The results are presented as
the mean of a minimum of three independent replicates.
Cytotoxicity Analysis: MTT. This method is based on the reduction
of the tetrazolium salt, methylthiazolyldiphenyl-tetrazolium bromide
(MTT) into a crystalline formazan product by the cellular oxidoreduc-
tases of viable cells.54,55 All cell culture reagents were from Invitrogen
Corporation (CA, USA). Cell lines were obtained from the American
Tissue Type Culture Collection (ATCC, MA, USA). Caco-2 human
intestinal epithelial cells (ATCC: HTB-37, passage numbers 56-64)
were cultured in Dulbecco’s Modified Eagles Medium (DMEM)
containing GlutaMAX, supplemented with 10% fetal bovine serum
(FBS), 1% nonessential amino acids (NEAA) and 1% penicillin/
streptomycin (Pen-Strep). U937 human monocyte-like cells (ATCC:
CRL-1593.2, passage numbers 11-17) were cultured using RPMI
medium supplemented with FBS, NEAA, Pen-Strep and 1% L-
glutamine. All cells were grown in a humidified 37 °C incubator, with
5% CO2, in air. At 70-80% confluence, cells were seeded, at a density

Poly(2-(dimethylamino ethyl)methacrylate)
Biomacromolecules, Vol. 11, No. 2, 2010 447
of 2 × 104 and 2 × 105 cells/well, for Caco-2 and U937 cells
respectively, in 96-well tissue culture plates and allowed to grow for
20 - 24 h. Caco-2 monolayers were rinsed with fresh growth medium
and allowed to equilibrate at 37 °C, 5% CO2 for 60 min. Media was
removed after equilibration and 200 µL fresh supplemented DMEM
media with or without pDMAEMA was added to the wells. For U937
cells, plates were centrifuged at 3000 rpm for 5 min then media was
removed. 200 µL fresh supplemented RPMI media with or without
pDMAEMA was added to the wells. Monolayers were incubated at 37
°C, 5% CO2 for 1 and 24 h. Following incubation, 20 µL of MTT (5
mg/mL in PBS, pH 7.4) was added and cells were incubated for a
further 3-4 h at 37 °C, 5% CO2. The U937 plates were centrifuged at
3000 rpm, no centrifuge step was necessary for the Caco-2 cells, then
the media was gently removed and 100 µL dimethyl sulphoxide
(DMSO) was added to all wells. The plates were shaken for 2 - 5
min to dissolve the formazan crystals and the absorbance was read at
550 nm. 0.1% Triton X-100 was used as a positive control for
cytotoxicity. The results are presented as the mean of a minimum of
three independent replicates.
Table 1. MIC Values (mg/mL) of pDMAEMA Polymers against
Salmonella ser. Typhimurium IMD 574 and S. epidermidis 1457
Salmonella
ser. Typhimurium
Polymer
S. epidermidis
Unconjugated
Reducible
50% Quaternized
Hostasol alone
1
0.1
0.05
0.1
1
>8a
>0.016
>0.016
a P
< 0.001 compared with unconjugated pDMAEMA against
Salmonella ser. Typhimurium.
Statistical Analysis. Statistical analyses were carried out using one-
way ANOVA with Bonferroni posthoc tests. The significance level was
set at R ) 0.05 (95% confidence intervals).
Results
Figure 1. Time-kill curve of unconjugated pDMAEMA (12.8 kDa)
againstS. epidermidis1457. 9 ) Control, O) 0.05 mg/mL, 1 )
0.1 mg/mL, • ) 0.2 mg/mL, × ) 0.4 mg/mL and 0 ) 0.8 mg/mL.
Characterization of pDMAEMA Polymers. SEC-HPLC
analysis produced a single dominant, clear peak for each
polymer, confirming purity (data not shown). The molecular
weight (Mn), as determined by SEC-HPLC and the polydisper-
sity index (PDi) of pDMAEMA polymers used in this report
are described in the Supporting Information section. The Mn
can also be calculated from 1H NMR, however, the peaks from
the polymer are broad and difficult to determine, see Supporting
Information section. This was carried out for the reducible
polymer to give two different values, depending on the peaks
chosen and as both values are within reasonable agreement with
the SEC results (SEC is usually quoted to give up to 10% error)
SEC data was used. It is also noted that the apparent molecular
weight of the polymers decreases upon quaternisation whereas
one would expect an increase in mass, this is ascribed again to
the differences in hydrodynamic volumes. The pKa of uncon-
jugated pDMAEMA was 7. This is in agreement with other
reports in the literature where it is reported to be 7 - 7.5.13,56
The pKa of the 50% quaternized pDMAEMA was found to be
approximately 11. The pKa of the hostasol-conjugated and
reducible polymers was not determined as these molecules were
not used in pH determination experiments.
Figure 2. Time-kill curve of unconjugated pDMAEMA (12.8 kDa)
againstSalmonella ser. Typhimurium IMD 574. 9 ) Control, O
0.5 mg/mL, 1 ) 1 mg/mL, • ) 2 mg/mL, × ) 4 mg/mL and 0 ) 8
mg/mL.
positive bacterium as it is a catheter-related clinical isolate that
could represent a target organism for pDMAEMA as a coating
for indwelling medical devices, such as catheters.42 All of the
polymers gave comparable MIC values (Table 1), except for
50% quaternized pDMAEMA against Salmonella ser. Typh-
imurium, which was found to be much higher. It is possible
that the more positive charge on the 50% quaternized polymer
may influence its ability to inhibit the growth of Gram-negative
bacteria. Hostasol alone was also tested for its antimicrobial
activity (Table 1). It was not found to show any antimicrobial
activity at concentrations up to 0.016 mg/mL. This concentration
coincides to 8 times its concentration at MIC in hostasol-
conjugated pDMAEMA, against S. epidermidis and 0.2 times
its concentration at MIC in hostasol-conjugated pDMAEMA,
against Salmonella ser. Typhimurium. Higher concentrations
were not able to be tested due to insolubility in aqueous media.
Time-kill Curves. Figures 1 and 2 show time-kill curves of
the effect of unconjugated pDMAEMA against S. epidermidis
1457 and Salmonella ser. Typhimurium IMD 574, respectively.
In both instances the polymer was bacteriostatic against the
MIC’s. In order to obtain an overview of how pDMAEMA
interacts with bacteria, a screen of 13 bacteria and 1 yeast strain
was carried out. pDMAEMA was found to have an antimicrobial
effect against all of the Gram-negative bacteria tested in the
range of 0.1 - 1 mg/mL. It had variable effects on the Gram-
positive bacteria tested (MIC values range from 0.1 - > 18 mg/
mL) and did not effect the growth of the yeast Candida albicans
up to 10 mg/mL. The MIC screen results are described in the
Supporting Information section.
From the results of the MIC screen, it appeared that
pDMAEMA may generally be more effective against Gram-
negative bacteria than Gram-positive. In order to investigate
this further, we examined a number of different derivatives of
pDMAEMA against two representative strains of Gram-positive
and Gram-negative bacteria (Table 1). Salmonella ser. Typh-
imurium IMD 574 was chosen as a model Gram-negative
bacterium as it is an infection-related food isolate that could
represent a target organism for pDMAEMA as a food packaging
coating.41 S. epidermidis 1457 was chosen as a model Gram-

448 Biomacromolecules, Vol. 11, No. 2, 2010
Rawlinson et al.
Figure 3. Effect of pH on antimicrobial activity. pH values 9 ) 5, O) 6, 1 ) 7, • ) 7.5, × ) 8 and 0 ) 9. A ) unconjugated pDMAEMA
(12.8 kDa) against Salmonella ser. Typhimurium IMD 574. B ) unconjugated pDMAEMA (12.8 kDa) againstS. epidermidis1457. % Growth
is compared to untreated control and is designated at 100%.
Figure 4. Fluorescent pictures of hostasol-conjugated pDMAEMA (10.7 kDa) interactions with bacteria. A ) Salmonella ser. Typhimurium
IMD 574, B ). S. epidermidis1457 bar ) 5 µM. Ex wavelength ) 465-495 nm, Em wavelength ) 515-555 nm.
bacteria, as after 24 h persister bacteria had regrown and there
was no evidence of a ·3 log10 reduction in CFU/mL.
incubated alone or with hostasol-conjugated pDMAEMA for
30 min, 2 and 4 h. The bacteria were analyzed by flow cytometry
and live cells were selected based on PI staining. Trypan blue
was added to quench the fluorescence outside the bacteria and
the samples were reanalyzed. When the bacteria were incubated
with the fluorescently conjugated pDMAEMA, the fluorescence
peak shifted to the right compared with peaks for the bacteria
alone (Figures 5A and 6A). This is due to increased fluorescence
of the bound polymer. When trypan blue was added to the S.
epidermidis samples, the increased fluorescence of the polymer
was completely quenched, compared with control, i.e. all
samples showed similar fluorescence peaks to bacteria with no
polymer suggesting that the polymer is not internalized (Figure
5B). However, when trypan blue was added to the Salmonella
ser. Typhimurium samples, some bacteria retained fluorescence
(Figure 6B). This showed that some of the polymer is able to
penetrate the Gram-negative bacteria.
Outer Membrane Permeabilisation. In order to test if
pDMAEMA’s ability to penetrate Salmonella ser. Typhimurium
is due to the polymer’s ability to permeabilise the outer
membrane of Gram-negative bacteria, we incubated unconju-
gated pDMAEMA (12.8 kDa) with Salmonella ser. Typhimu-
rium IMD 574 and used the fluorescent probe NPN to measure
membrane permeabilisation. At pH 7.5 pDMAEMA permeabi-
lized the outer membrane of this bacteria within 1 min of
incubation (Figure 7). The outer membranes of E. coli ATCC
10536 and Salmonella ser. Enteritidis ATCC 13076 were also
permeabilized within 1 min (data not shown). Controls of: 1)
bacteria incubated with polymer, but no NPN, and 2) polymer
incubated with NPN, but no bacteria, did not produce any
increase in fluorescence (data not shown). At pH 7, the
permeabilisation of Salmonella ser. Typhimurium by
pDMAEMA was decreased and at pH 6 it was even lower
compared to that at pH 7.5 (Figure 7).
Optimum pH and Temperature for Antimicrobial
Activity. As was suggested by the high MIC of 50% quaternized
pDMAEMA (Table 1), the charge on the polymer may influence
its ability to inhibit bacterial growth. Therefore MIC’s were
carried out at different pH values. The optimum pH for
antimicrobial activity for the two selected pathogens (Figure
3) showed a difference in the effect of unconjugated
pDMAEMA against the Gram-positive compared with Gram-
negative bacteria. Despite both bacteria showing an optimum
for antimicrobial activity, of 7.5 - 8, against the Gram-negative
bacteria, Salmonella ser. Typhimurium, pDMAEMA was ef-
fective at pH ) 7.5 and above, while against the Gram-positive
bacteria, S. epidermidis, pDMAEMA was only effective at pH
) 8 and below. As mentioned previously, the pKa of
pDMAEMA is approximately 7 - 7.5 and for both bacteria tested,
optimum antibacterial activity is around the pKa. The effect of pH
was also tested against the 50% quaternized pDMAEMA (9.7 kDa)
using Salmonella ser. Typhimurium. We found that even at pH
9, 50% quaternized pDMAEMA did not have any effect against
Salmonella ser. Typhimurium (data not shown). The bacteria
did not grow above the pH values tested.
The optimum temperature for activity for unconjugated
pDMAEMA (12.8 kDa) was 37 - 43 °C. At ambient temper-
ature, the activity was reduced against Gram-negative Salmo-
nella ser. Typhimurium (data not shown).
Hostasol-conjugated pDMAEMA Binding to Bacteria.
Hostasol-conjugated pDMAEMA (10.7 kDa) was used to
visualize the polymer binding to bacteria. In Figure 4,
pDMAEMA can be seen binding to each of Salmonella ser.
Typhimurium and S. epidermidis after 30 min incubation. A
similar result was seen after 2 h incubation (data not shown).
Internalisation of pDMAEMA by Bacteria. In order to test
whether pDMAEMA was able to be internalized, bacteria were

Poly(2-(dimethylamino ethyl)methacrylate)
Biomacromolecules, Vol. 11, No. 2, 2010 449
Figure 5. Flow cytometric analysis of S. epidermidis 1457 treated with 0.1 mg/mL hostasol-conjugated pDMAEMA (10.7 kDa) (+100
µg/mL PI to gate for live cells). A ) No trypan blue, B ) Cell treated with 1 mg/mL trypan blue. Red line ) control, blue line ) 30 min
treatment, green line ) 2 h treatment, purple line ) 4 h treatment. FL 1: 530/40 nm filter. Results gated for single alive bacteria.
Figure 6. Flow cytometric analysis of Salmonella ser. Typhimurium IMD 574 treated with 1 mg/mL hostasol-conjugated pDMAEMA
(10.7 kDa) (+100 µg/mL PI to gate for live cells). A ) No trypan blue, B ) Cell treated with 1 mg/mL trypan blue. Red line ) control,
blue line ) 30 min treatment, green line ) 2 h treatment, purple line ) 4 h treatment. FL 1: 530/40 nm filter. Results gated for single
alive bacteria.
Figure 7. Permeabilisation of the outer membrane of Salmonel-
laser. Typhimurium IMD 574 by mg/mL unconjugated
pDMAEMA (12.8 kDa). 9 ) pH 6, O) pH 7, 1 ) pH 7.5. RFU )
relative fluorescence units. Values compared to fluorescence of
bacteria incubated with NPN, but no polymer (0 RFU).
Figure 8. Permeabilisation of the cytoplasmic membrane of
Salmonellaser. Typhimurium IMD 574 by unconjugated
pDMAEMA (12.8 kDa). 9 ) PBS, O) 2 mg/mL pDMAEMA, 1 )
0.1% Triton X-100, • ) 1% Triton X-100.
2
increase. This increase can be an indicator of bacterial cell
growth.57 Figures 10 and 11 demonstrate the effect of
pDMAEMA on cell growth of S. epidermidis and Salmonella
ser. Typhimurium, respectively. After incubation of the polymer
with bacteria for both 30 min and 4 h a shift to the left of the
peaks was observed when the bacteria were incubated with
pDMAEMA, i.e. the N2 (replicating bacteria) peaks are reduced
and N1 (unreplicating bacteria) peaks are larger in treated
samples compared to untreated amples. This indicates that cell
growth was inhibited, as the cellular DNA of the bacteria was
not replicating as much as untreated controls, suggesting that
the cells were not progressing through their normal cell cycle
pattern.
Cytoplasmic Membrane Permeabilisation. pDMAEMA
may also have an effect on the cytoplasmic (inner) membrane
of bacteria. In order to test this hypothesis, both Gram-negative,
Salmonella ser. Typhimurium and Gram-positive, S. epidermidis,
were loaded with the dye calcein-AM and then incubated with
unconjugated pDMAEMA. Permeabilisation of the cytoplasmic
membrane was observed with both bacteria based on a decrease
in calcein fluorescence relative to controls containing no polymer
(Figures 8 and 9).
Effect of pDMAEMA on Bacterial Cell Growth. The DNA
content of bacteria fluctuates as it goes through its life cycle.
The DNA fluorescence, when stained with fluorescent dyes, is
an indication of the number of chromosomes inside individual
cells.57 As the bacteria grow they replicate their chromosome
in replicates of N1, N2, N3 etc., therefore, DNA staining will
Enhancement of the Efficacy of Erythromycin by
pDMAEMA. Against all of the Gram-negative bacteria tested
(Salmonella ser. Typhimurium IMD 574, Salmonella ser.

450 Biomacromolecules, Vol. 11, No. 2, 2010
Rawlinson et al.
positive bacteria and no effect on the yeast. The selectivity for
Gram-negative over Gram-positive bacteria has also been
previously observed with other polymers.58,59
As pDMAEMA is a thermo- and pH-responsive polymer,19,60
pH and temperature may influence its ability to inhibit the
growth of bacteria. At higher temperatures, pDMAEMA phase-
transitions out of solution. We therefore tested to see if it would
retain its effectiveness at temperatures slightly above body
temperature. pDMAEMA was effective at inhibiting growth up
to 43 °C and remained in solution. This effect was seen with
both the Gram-negative and Gram-positive bacteria. However,
the effect of pH on growth inhibition was quite different for
the Gram-negative compared with the Gram-positive bacteria.
pDMAEMA only inhibited the Gram-negative bacterium, Sal-
monella ser. Typhimurium, when the polymer was around its
pKa or when in a more hydrophobic state, at higher pH values.
When the polymer is more hydrophobic, less protonated, it may
be able to interact more effectively with the outer membrane
of the Gram-negative bacteria. This was confirmed by a decrease
in permeabilisation of the outer membrane observed at pH 7
and 6 compared with pH 7.5. Against the Gram-positive
bacteria, S. epidermidis, however, pDMAEMA was only ef-
fective at around its pKa and when it is more highly protonated
and more hydrophilic at lower pH values. The lack of the outer
membrane, in Gram-positive bacteria, reduces the barriers for
the polymer to access the cytoplasmic membrane. Therefore,
although the polymer must retain some hydrophobic portions,
as the ability to inhibit growth decreased with decreasing pH,
hydrophobicity does not appear to be as important in the
inhibition of the Gram-positive bacterium.
Figure 9. Permeabilisation of the cytoplasmic membrane of S.
epidermidis 1457 by unconjugated pDMAEMA (12.8 kDa). 9 )
PBS, O) 0.1 mg/mL pDMAEMA, 1 ) 1 mg/mL pDMAEMA, × )
0.1% Triton X-100, • ) 1% Triton X-100.
Enteritidis ATCC 13076 and E. coli ATCC 10536), incubation
with unconjugated pDMAEMA, at concentrations 4 and 10
times lower than its MIC, reduced the MIC of erythromycin by
7 - 59 fold (Table 2). This effect was also seen as a 2-fold
reduction in erythromycin MIC, using 4 times lower than MIC
pDMAEMA concentration, against S. epidermidis 1457.
Cytotoxicity Analysis. The cytotoxicity of pDMAEMA
polymers was tested against sheep erythrocytes. Up to 10 mg/
mL, none of the pDMAEMA polymers tested were found to
cause significant hemolysis (Table 3). Cytotoxicity against the
human intestinal epithelial cell line, Caco-2, and the human
monocytic cell line, U937, was assessed by MTT assay (Table
3). All polymers produced similar levels of cytotoxicity to the
human cell lines, though all were more cytotoxic toward U937
cells than toward Caco-2 cells. Surprisingly the reducible
polymer, that has previously been reported to be less toxic to
U937 cells,39 did not cause less cytotoxicity than unconjugated
pDMAEMA.
A range of pDMAEMA polymers were tested in order to
investigate effects of modifications to the polymer on antimi-
crobial activity. Unconjugated pDMAEMA was tested to ensure
that, without any modifications, the polymer retained activity.
A polymer with reportedly lower cytotoxicity,39 reducible
pDMAEMA, was tested to investigate if the disulfide bond in
this molecule interferes with antimicrobial activity. A more
positively charged polymer, the 50% quaternized pDMAEMA,
was tested to investigate the effect of charge on the polymer.
All polymers had similar MIC values to each other except for
the 50% quaternized pDMAEMA against Salmonella ser.
Typhimurium (Table 1). Up to a pH value of 9, 50% quaternized
pDMAEMA remained unable to inhibit growth of the Gram-
negative bacteria. As the pKa of this polymer is approximately
11, it is highly protonated at pH 9 and consequently hydrophilic.
The results for the unconjugated polymer showed that pD-
MAEMA was only able to inhibit the growth of the Gram-
negative bacteria when presented in a more hydrophobic state,
at pH values around or above its pKa. Therefore, as pH 9 is
lower than the pKa of quaternized pDMAEMA, the result for
the 50% quaternized polymer against Salmonella ser. Typh-
imurium correlates with the unconjugated polymer data.
Discussion
We have previously shown that pDMAEMA is able to inhibit
the binding and uptake of Salmonella ser. Typhimurium to the
human epithelial intestinal cell line, HT29-MTX-E12, and
reduce the, in Vitro, inflammatory response to bacterial cell and
toxin challenge.36 We also reported that pDMAEMA conjugated
with hostasol, was bactericidal against Salmonella ser. Typh-
imurium IMD 574,36 however, in the present work unconjugated
pDMAEMA was found to be bacteriostatic. The hostasol
conjugated to pDMAEMA does not appear to adjust the MIC
concentration compared to other pDMAEMA derivatives in-
cluding the unconjugated polymer, but it does confer a
bactericidal ability on it, while having no effect on its own. It
is unclear as to why this occurs, however, one hypothesis is
that it is due to the increased size of the side arm when hostasol
is attached to pDMAEMA, which may allow for the polymer
to reach the cytoplasmic membrane of the bacteria more
effectively.30 Another hypothesis is that the increased hydro-
phobicity of the hostasol may increase the polymer’s ability to
enter bacterial cell membranes. Hostasol alone was not cytotoxic
to sheep red blood cells, nor the human cell lines U937 and
Caco-2 at concentrations up to 0.016 mg/mL. Using the hostasol-
conjugated pDMAEMA, we screened an array of Gram-negative
and Gram-positive bacteria as well as one yeast strain, to
investigate the scope of pDMAEMA’s antimicrobial activity.
pDMAEMA was found to inhibit the growth of all the Gram-
negative bacteria tested, with variable effects on the Gram-
To date, most reports with pDMAEMA as an antimicrobial
agent have used a quaternized derivative. The majority of these
reports also use pDMAEMA as part of a copolymer with other
polymers, which may be contributing to its antimicrobial
activity. In contrast to our data, some studies have found
quaternized pDMAEMA to be effective against the Gram-
negative bacteria, E. coli.30,32 In these reports the bacteria were
only incubated with pDMAEMA for 1 h. However, in the
present study pDMAEMA was incubated with the bacteria for
18 h. While it is possible that the quaternized polymer is able
to inhibit growth of Gram-negative bacteria over short periods,
over longer periods the bacteria are able to grow back. In

Poly(2-(dimethylamino ethyl)methacrylate)
Biomacromolecules, Vol. 11, No. 2, 2010 451
Figure 10. Effect of unconjugated pDMAEMA (12.8 kDa) onS. epidermidis 1457 cell cycling. A ) cell growth after 30 min, B ) cell
growth after 4 h. Red line ) control untreated bacteria, blue line ) bacteria treated with 0.1 mg/mL pDMAEMA. FL 3: 613/20 nm filter.
Results gated for single bacteria. Arrows represent N1 and N2 DNA replicates of actively growing bacteria.
Figure 11. Effect of unconjugated pDMAEMA (12.8 kDa) on Salmonella ser. Typhimurium IMD 574 cell cycling. A ) cell growth after
30 min, B ) cell growth after 4 h. Red line ) control untreated bacteria, blue line ) bacteria treated with 1 mg/mL pDMAEMA. FL 3:
613/20 nm filter. Results gated for single bacteria. Arrows represent N1 and N2 DNA replicates of actively growing bacteria.
Table 2. Enhanced Antimicrobial Activity of Erythromycin against Bacteria by Addition of Unconjugated pDMAEMA. MIC’s are Expressed in
µg/mL
Salmonella ser. Typhimurium Salmonella ser. Enteritidis
E. coli
S. epidermidis
Antimicrobial agent
MIC
Fold reduction
MIC
Fold reduction
MIC Fold reduction MIC Fold reduction
Erythromycin (Ery)
125
2000
3.9
-
-
32
8
23.4
2000
2.32
3.17
-
-
10
7
5.86
0.5
-
0.24
0.1
0.12
0.24
-
-
2
0
pDMAEMA
-
Ery + pDMAEMA (0.25 × MIC)
0.1
59
45
Ery + pDMAEMA (0.1 × MIC)
15.6
0.13
Table 3. Cytotoxicity of pDMAEMA Polymers and Hostasol (mg/mL). IC50 ) Concentration that Produces 50% Cytotoxicity Compared to
100% Trition X-100 Control
RBC
Caco-2
U937
Polymer
1 h IC50
1 h IC50
24 h IC50
1 h IC50
24 h IC50
Unconjugated
Reducible
50% Quaternized
Hostasol-conjugated
Hostasold
>10
7.6 ( 0.37
>10
2.1 ( 1.2
0.68 ( 0.08
5.2 ( 1.2
0.27 ( 0.03c
>0.016
0.38 ( 0.03
0.05 ( 0.004
1.1 ( 0.14a
0.05 ( 0.02
>0.016
0.19 ( 0.06
0.05 ( 0.01
1.0 ( 0.26b
0.06 ( 0.01
>0.016
>10
>10
>10
>10
4.9 ( 2.1
>0.016
>0.016
a P < 0.05. b P < 0.001 as compared to unconjugated polymer in the same column. c P < 0.01. d Maximum solubility of hostasol was obtained at 0.016
mg/mL.
addition, Yancheva et al.33 found that unconjugated and 50%
a fluorescent tag, bound to both the Gram-negative, Salmonella
ser. Typhimurium, and Gram-positive, S. epidermidis. To test
if the polymer was gaining internal access to the bacteria, the
fluorescence was quenched with trypan blue. All fluorescence
around the Gram-positive bacteria was quenched showing that
the polymer is acting at the cell surface. However, some
fluorescence remained around the Gram-negative bacteria after
quenching, suggesting that the polymer is getting inside the
bacteria. pDMAEMA was shown to permeabilise the outer
membrane of Salmonella ser. Typhimurium, Salmonella ser.
quaternized pDMAEMA had a similar inhibitory effect against
E. coli, producing an MIC of 0.3 mg/mL. This value is similar
to the MIC we found for the unconjugated polymer against E.
coli ATCC 10536 (0.5 mg/mL). However, again, the bacteria
were only exposed to the polymers for a short time (30 min).
A longer time point may have produced a different result for
the quaternized molecule.
The initial step in the mode of action of cationic biocides is
adsorption onto the bacterial cell surface.37 pDMAEMA, with

452 Biomacromolecules, Vol. 11, No. 2, 2010
Rawlinson et al.
Enteritidis, and E. coli. It is possible that the remaining
fluorescence seen is the polymer located inside the outer
membrane.
To use pDMAEMA as an antimicrobial coating or treatment,
it must be shown to be safe to humans. Reports on the
cytotoxicity of pDMAEMA vary depending on the method of
administration, quaternization of the polymer, other polymers
attached, and size.65-68 Moreau et al.65 found that pDMAEMA
caused little or no hemolysis to human red blood cells, however,
when injected intravenously into the tail vein of rats, caused
death at 5.1 mg/kg, but was tolerated at 2.1 mg/kg. Yancheva
et al.68 showed that despite the fact that pDMAEMA did not
cause hemolysis of red blood cells it did encourage hemeaglut-
tination. Here we have found that none of the pDMAEMA
polymers caused any substantial hemolysis against sheep red
blood cells. Cytotoxicity against Caco-2 and U937 cells was
similar to concentrations found for 43 kDa pDMAEMA against
human brain microvascular endothelial cells.66 Cytotoxicity
against the human cell lines was also similar to the antimicrobial
polymer, chitosan, against B16F10 murine melanoma cell line
and the cationic reference polymer, poly(L-lysine).69 Against
Caco-2 cells none of the polymers produced significantly
different cytotoxicity concentrations than the unconjugated
polymer, except for the hostasol-tagged polymer after 24 h.
Against U937 cells the hostasol-tagged and reducible polymers
produced similar cytotoxicity to the unconjugated polymer,
while the 50% quaternized polymer was slightly less toxic.
Reducible pDMAEMA has previously been reported to be less
cytotoxic than unconjugated pDMAEMA.39 Here the reducible
polymer was not less cytotoxic than the unconjugated polymer.
This may be due to slight variations in the structure of the
polymer used here and in previous reports.
Cytotoxicity against the human intestinal cell line Caco-2 was
found to be 5-fold greater than that found for the human
monocyte-like cell line U937. Together with the lack of affect
on red blood cells, these results show great variability depending
on the cell type tested. In addition, pDMAEMA has been
previously shown to have similar cytotoxicity to the natural
polymer chitosan, which is considered nontoxic,70 and when
added to human epithelial cell layers, pDMAEMA prevents
disruption of the cells after challenge with bacterial toxins.36
Therefore, conclusions about the toxicity of this polymer must
be made with caution, and although the in vivo work of Moreau
et al.65 showed a fairly low tolerance for intravenous injection
of the polymer, perhaps as a topical or oral formulation, it could
prove to be safe and effective.
The next steps in the mode of action of cationic biocides are
the diffusion through the cell wall and binding to the cytoplasmic
membrane. The variation in the effect on Gram-positive
compared with Gram-negative bacteria may be partially ex-
plained at this step. Lienkamp et al.58 synthesized an array of
polymers to mimic the effects of antimicrobial peptides. They
found that some of their polymers were also more effective
against Gram-negative than Gram-positive bacteria. They related
this effect to the molecular weight of the polymer. They
suggested that a polymer of 10 kDa may be less effective against
Gram-positive bacteria than a polymer of 3 kDa, as the larger
polymer can form polyion complexes in the thick murein layer
(cell wall) around the cytoplasmic membrane, which are more
difficult to dissociate at higher molecular weights. Therefore
the larger polymer may be unable to transfer across the cell
wall to the membrane and therefore not able to disrupt the
membrane and cause cell death. In the case of the Gram-negative
bacteria, the murein layer is a thin layer between the outer
membrane and the cytoplasmic membrane so it is potentially
easier for the polymer to cross. This may also be the case with
pDMAEMA against some of the Gram-positive bacteria as the
polymers used here are large, approximately 7-18 kDa in size.
In addition to size, the charge on the bacteria may also contribute
to the difference in activity. Chung et al.61 suggested that the
antimicrobial polymer, chitosan, is more effective against Gram-
negative bacteria than Gram-positive as the Gram-negative cells
are more hydrophilic and have a more negative charge so the
polymer is able to interact with them more than the Gram-
positive. The reason for the variation in activity within the Gram-
positive group is unclear, but it may also be related to charge,
hydrophobicity and hydrodynamic volume.
The final steps in the mode of action of cationic biocides are
the disruption of the cytoplasmic membrane, release of cytoplasmic
constituents and cell death. pDMAEMA permeabilized the cyto-
plasmic membrane of both the Gram-positive and the Gram-
negative bacteria. In addition, the polymer interfered with the
growth cell cycle of the bacteria. Therefore, it is not only able to
kill the bacteria, typical of cationic biocides, but it may also slow
their growth. Due to the ability of pDMAEMA to disrupt the outer
membrane of Gram-negative bacteria it may have potential to
increase the activity of other antimicrobial agents that are less
effective against these bacteria. Erythromycin is a hydrophobic,
macrolide, antibiotic that is extensively used in treating Gram-
positive bacterial infections.62,63 However, it has only very limited
use in the treatment of Gram-negative infections due to the outer
membrane of these bacteria acting as a permeability barrier.64 For
this reason, agents that sensitize the outer membrane of Gram-
negative bacteria are useful to increase the antimicrobial activity
of antibiotics, including erythromycin.62 In this study we found
that, due to pDMAEMA’s ability to disrupt the outer membrane
of bacteria, it was able to increase the antibacterial activity of
erythromycin by up to 59-fold at concentrations where pDMAEMA
was not inhibitory by itself. The permeabilization of the outer
membrane by pDMAEMA appears to remove the barrier for the
antibiotic, allowing it to enter the bacteria and inhibit growth. This
erythromycin-pDMAEMA mix may have use in clinical settings,
particularly in the treatment of resistant bacteria where other
antibiotics have become less effective. Further investigations into
the use of pDMAEMA in combinatorial therapies are necessary
to fully understand the potential of the polymer in this context.
Conclusions
The mode of action of the quaternized and protonated polymer
pDMAEMA was similar to other cationic biocides and involved
direct binding to bacteria, diffusion through the cell wall,
disruption of the cytoplasmic membrane, and cell death. This
effect was dependent on the bacteria type, as Gram-negative
bacteria require the polymer to be in a less-charged/hydrophobic
state to permeabilize the outer membrane. This antimicrobial
effect was bacteriostatic in nature and may be useful for
enhancing the efficacy of another antibiotic, erythromycin. The
mucoadhesive polymer, pDMAEMA, therefore, appears to show
promise as a potential antimicrobial coating, when formulated
with other antimicrobial agents, due to its effective inhibitory
action against S. epidermidis.
Acknowledgment. This study was funded by a grant from
Science Foundation Ireland (Investigator Grant 04 IN3 B575).
We thank Rebecca O’Mahony, Siobhan McClean, Denise
Drudy, Michael Folan, and James P. O’Gara for the bacterial
strains used in this study. R. K. Randev assisted with polymer

Poly(2-(dimethylamino ethyl)methacrylate)
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synthesis. We also acknowledge the help of Alfonso Blanco
Ferna´ndez with flow cytometry analysis.
Supporting Information Available. Molecular weight (Mn)
and polydispersity index (PDi) values for pDMAEMA polymers,
as determined by SEC-HPLC. NMR trace of reducible pD-
MAEMA. A table showing MIC values of pDMAEMA against
numerous bacteria and yeast is also presented. This material is
available free of charge via the Internet at http://pubs.acs.org.
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