Comparison of facially amphiphilic versus segregated monomers in the design of antibacterial copolymers

Article abstract of DOI:10.1002/chem.200801233

A direct comparison of two strategies for designing antimicrobial polymers is presented. Previously, we published several reports on the use of facially amphiphilic (FA) monomers which led to polynorbornenes with excellent antimicrobial activities and sel

Full text of DOI:10.1002/chem.200801233

FULL PAPER
DOI: 10.1002/chem.200801233
Comparison of Facially Amphiphilic versus Segregated Monomers in the
Design of Antibacterial Copolymers
Gregory J. Gabriel,[a] Janet A. Maegerlein,[a] Christopher F. Nelson,[b]
Jeffrey M. Dabkowski,[b] Tarik Eren,[a] Klaus Nꢀsslein,[b] and Gregory N. Tew*[a]
Abstract: A direct comparison of two
strategies for designing antimicrobial
polymers is presented. Previously, we
published several reports on the use of
facially amphiphilic (FA) monomers
which led to polynorbornenes with ex-
cellent antimicrobial activities and se-
lectivities. Our polymers obtained by
copolymerization of structurally similar
segregated monomers, in which cation-
ic and non-polar moieties reside on
separate repeat units, led to polymers
with less pronounced activities. A wide
range of polymer amphiphilicities was
surveyed by pairing a cationic oxanor-
bornene with eleven different non-
polar monomers and varying the como-
nomer feed ratios. Their properties
were tested using antimicrobial assays
and copolymers possessing intermedi-
ate hydrophobicities were the most
active. Polymer-induced leakage of
dye-filled liposomes and microscopy of
with the phospholipid bilayer com-
pared with copolymers from segregated
monomers. We conclude that a well-de-
fined spatial relationship of the whole
polymer is crucial to obtain synthetic
mimics of antimicrobial peptides
(SMAMPs): charged and non-polar
moieties need to be balanced locally,
for example, at the monomer level, and
not just globally. We advocate the use
of FA monomers for better control of
biological properties. It is expected
that this principle will be usefully ap-
plied to other backbones such as the
polyacrylates, polystyrenes, and non-
natural polyamides.
polymer-treated bacteria support
a
membrane-based mode of action. From
these results there appears to be pro-
found differences in how a polymer
made from FA monomers interacts
Keywords:
amphiphiles
·
antimicrobial polymers · peptides ·
polynorbornene
polymerization
·
ring-opening
Introduction
number of antimicrobial polymers and peptidomimetics,[9–25]
most of which draw structural and functional inspiration
from a class of natural cationic macromolecules called anti-
microbial peptides (AMPs),[26,27] has grown in recent years.
In this vein, the physicochemical properties of a range of
potent small molecules and polymers, collectively known as
synthetic mimics of antimicrobial peptides (SMAMPs), have
been extensively studied.[2,9–25,28,29]
In examining the structural and biological activities of
SMAMPs, synthetic polymer chemists have focused on
tuning the hydrophobic/hydrophilic balance of their poly-
mers. Altering the amphiphilicity of antimicrobial polymers
has been accomplished most commonly through two meth-
ods (Table 1). First, in what we have termed the “segregat-
ed” route, relatively non-polar monomers are polymerized
with a masked cationic monomer to produce positively
charged, amphiphilic random copolymers. Using structurally
different non-polar monomers that have a range of hydro-
phobicities or by adjusting feed ratios, the amphiphilicities
of the copolymers can be straightforwardly varied. This
strategy is exemplified by DeGrado[17] and Gellman,[12] inde-
There is increasing interest in selective antimicrobial poly-
mers whose potency against bacteria and non-toxicity to-
wards mammalian cells distinguishes them from most poly-
meric biocides that are broadly poisonous.[1–6] This selectivity
is especially important for both the development of new an-
tibiotics, as well as for the incorporation of polymers into
materials that will be in intimate contact with human tissue
such as cardiovascular and orthopedic implants.[7,8] Thus, the
[a] Dr. G. J. Gabriel, M. Sc. J. A. Maegerlein, Dr. T. Eren,
Prof. Dr. G. N. Tew
Department of Polymer Science and Engineering
University of Massachusetts, Amherst, MA 01003 (USA)
Fax : (+1)413-545-0082
E-mail: tew@mail.pse.umass.edu
[b] C. F. Nelson, J. M. Dabkowski, Prof. Dr. K. Nꢀsslein
Department of Microbiology, University of Massachusetts
Amherst, MA 01003 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200801233.
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433

pendently, and by us in this paper (structures A–C respec-
tively, Table 1).
to interact with the phospholipid bilayer. This design has led
to the discovery of several very selective polymers in which
the molecules preferentially target specific bacteria over
mammalian red blood cells (Table 1, polymers E and
F).[20,25]
The second method, using “facially amphiphilic” (FA)
repeat units has been a focus of our laboratory for some
time.[15,20,24,25,32,33,35,36] In this design, every repeat unit carries
a charged and non-polar moiety. Most commonly, it involves
the synthesis of monomers with both a masked cationic and
a tunable non-polar group. The spatial arrangement of poly-
mer E (Table 1), or the linker flexibilities of polymer F,
allows the charged and non-polar moieties to be positioned
on opposite sides of the polymer backbone as it rearranges
Within this definition, alkylated pyridinium homopoly-
mers would be included since each repeat unit carries a cat-
ionic charge and non-polar alkylating side chain. Interesting-
ly Tiller et al. have extensively used alkylated pyridinium
homopolymers, to successfully endow surfaces with antimi-
crobial activity, in which every unit is FA and therefore the
charge density was essentially
equal for all chains.[6] Recently,
Table 1. Representative structures of antimicrobial polymers obtained by using “segregated” monomers (A, B,
and this work, C) and “facially amphiphilic” monomers (D–F).[a]
Sen and co-workers (polymer D
in Table 1)[9] synthesized two
series of alkylated pyridinium
copolymers. What they termed
Schematic
General structure
Base
MIC [mgmLÀ1
]
representation
selectivity
“same-centered”
copolymers,
with FA units, carried both a
cationic charge and a non-polar
alkyl chain while “different-
centered” copolymers separated
the charged pyridinium group
and the alkyl group. Notably,
the most selective copolymers
were in the “same-centered”
set although these are statistical
random copolymers using both
FA and neutral repeat units
meaning that the charge density
and spatial arrangement cannot
be as carefully controlled com-
pared to when all units are FA.
In addition, this also does not
allow them to be strictly placed
within this second category but
since they are based on alkylat-
ed polypyridines, which can be
considered FA, we have placed
them here. Similarly, Young-
blood and co-workers used
non-ionic hydrophilic groups to
modify the hemolytic activity of
alkylated pyridium polymers.[23]
Thus, with two successful
studies on the use of FA mono-
mers (Table 1, polymers E and
F),[20,25] it was speculated that
this method may be a more ef-
fective way to produce selective
antibacterial polymers than the
commonly employed segregated
monomer route. In this report,
we put this speculation to the
test with a set of copolymers
synthesized from masked cat-
ionic and non-polar alkyl sub-
A
B
C
ref. [17]
3
300 (Ec)
ref. [12]
32
3.1 (Bs)
20
75 (Sa)
D
E
ref. [9]
34
50 (Bs)
ref. [20]
>100
40 (Ec, Sa)
F
ref. [25]
>533
<3.8 (Sa)
[a] For each example, a series of polymers were synthesized and the best selectivities as defined by HC/MIC
are noted above (HC=hemolytic concentration, MIC=minimum inhibitory concentration). All hemolytic
values are based on an HC50 standard except for B which used a MHC (minimum hemolytic concentration).[12]
The bacterial types tested giving these values are also shown (Ec=Escherichia coli, Bs=Bacillus subtilis, Sa=
Staphylococcus aureus).
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Design of Antibacterial Copolymers
FULL PAPER
stituted norbornene monomers (polymer C, Table 1). Most
importantly, we believe this work presents a fair comparison
between the two strategies (polymers C versus polymers E,
Table 1) in which both methods use a norbornene backbone,
the charged groups are always primary amines, and the non-
polar alkyl groups are structurally similar. By convention, in
the field of antimicrobial polymers, selectivity has been de-
fined in this paper as the ratio between HC and MIC (he-
molytic concentration defined as HC50 and minimum inhibi-
tory concentration defined as MIC90, both typically reported
in mgmLÀ1). Within the polynorbornene framework, this
study provides a direct comparison between polymers com-
posed of FA repeat units and polymers in which the charged
and non-polar moieties are segregated on different mono-
mers (Figure 1). Starting with the polyamine oxanorbornene
homopolymer (PAON), little antibacterial and hemolytic ac-
tivity was discovered. To test these ideas, new copolymers
from segregated monomers were designed to survey a wide
range of amphiphilicities. The strategy of copolymerizing
segregated monomers converted PAON into active poly-
mers though none were found with selectivities superior to
those constructed of very comparable FA monomers. This
implies that the FA monomer design is superior to the seg-
regated monomer approach within the norbornene frame-
work.
targeted at two molecular weights (number average molecu-
lar weight, Mn) using ring-opening metathesis polymeri-
zation with Grubbs’ 3rd generation catalyst (low Mn =2.7–
4.2 kDa and high Mn =12.2–15.9 kDa of the Boc-protected
polymers).[30,31] Gel-permeation chromatography gave mon-
omodal signals and narrow molecular weight indices (1.05–
1.15). Using this strategy, copolymers having a broad range
of amphiphilicities, but with approximately the same
number of amines, and thus charges, could be easily ob-
tained just by varying the alkyl comonomers (Figure 2).
Results and Discussion
Design and synthesis: With previous reports illustrating how
antibacterial and hemolytic activities can be tuned by vary-
ing the amphiphilicity of membrane-disruptive polynorbor-
nenes, we set forth to synthesize random copolymers sepa-
rating monomers splitting the charged and non-polar groups
(Figure 2). Instead of installing hydrophobicity early in the
synthesis as was done in the previous system using FA mon-
omers, a universal oxanorbornene imide precursor was used
and derivatized with different alkyl chains (Figure S1).[30]
One Boc-protected amine oxanorbornene and eleven other
monomers carrying various alkyl chains were synthesized.
As a starting point, 50:50 mol% random copolymers were
Figure 2. 50:50 random copolymers synthesized at two different molecu-
lar weights. PAON and several non-50:50 compositions were also synthe-
sized for a total of thirty-two polymers. Notation examples: PAON =the
homo-amine polymer, A5’=50:50 random copolymer of the amine and
the 5’ non-polar monomer.
Biological activities: MIC assays were used to evaluate the
antibacterial properties of these polymers against Gram-
negative E. coli and Gram-positive S. aureus (Table 2). Low
Mn PAON was found to be inactive with an MIC of
400 mgmLÀ1 against both bacteria while the MIC values of
Figure 1. Two methods to endow polynorbornenes, based on polyamine oxanorbornene, PAON, with antibacterial properties. The center structure was
previously reported.[20] The right structure, A5’, synthesized for this study, shows a five-carbon branched chain as the non-polar moiety (one of eleven
alkyl chains tested). Presumably, rotation about the single bonds (red curved arrows) in the polymer backbone allows for the orientation of these copoly-
mers into a globally FA conformation.
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435

G. N. Tew et al.
the copolymers against E. coli showed a clear trend with the
polymers of intermediate hydrophobicity (A4, A4’, A5, and
A5’) having the most potent activities with MICs of
50 mgmLÀ1 (Table 2 and Figure 3). The most hydrophilic co-
the AMP, Magainin, which was shown to possess an MIC of
12 mgmLÀ1 against E. coli.[24]
As expected, incorporating more hydrophobic groups
onto the polyamine resulted in copolymers with increased
ability to lyse human red blood cells.[20] A high hemolytic
concentration (HC) is one measure of non-toxicity against
mammalian cells and is essential for accurately capturing
AMP activity. For the low Mn copolymers, undesirable he-
molytic activities (HC ꢀ 50 mgmLÀ1) were measured for A4
and all copolymers that were more hydrophobic. The higher
Mn copolymers displayed undesirable hemolytic activities
starting even sooner at A3. Selectivity values (HC/MIC) of
20 were observed for low Mn A1 and A3; selectivity for S.
aureus was 20 for both copolymers. These values are better
than those of Magainin, which has a selectivity of 10 for
both E. coli and S. aureus.[24] Notably, these values are far
below those observed for the most selective polymers in the
previous studies utilizing FA monomers which were >100
and >533 (Table 1).[20,25]
Nonetheless, the ability to adjust biological activities
using segregated monomers that split the charged and non-
polar moieties was encouraging and thus feed ratios deviat-
ing from 50:50 were also explored. Non 50:50 copolymer an-
alogues of low Mn A1, which was inactive, and A5’ which
was active but not selective were tested (Table 3). Unexpect-
edly, the biological activities did not improve dramatically.
In particular, incorporating more hydrophobicity into A1
hardly improved the activity as much as anticipated while
increasing the charge density of A5’ did not eliminate the
copolymerꢂs high hemolytic activity.
The inability to tune the biological properties was surpris-
ing given earlier findings.[20,25] It was thought that adding hy-
drophobicity to A1 via increasing the ratio of the non-polar
unit should theoretically produce a copolymer with a similar
amphiphilicity as the more hydrophobic copolymers A4 and
A5, both of which were active (Table 2). In other words, if
global amphiphilicity was a key determinant of activity then
an A1 copolymer with a high enough ratio of non-polar
groups (>50%) should approach a hydrophobicity, and
therefore activity, of A4 or A5 both of which were modestly
Figure 3. MICs for E. coli of selected polymers illustrating optimal hydro-
phobicity for the most active ones A4, A5 (low Mn) and A3 (high Mn).
polymer A1 and the most hydrophobic copolymer A12 were
inactive similar to PAON. A similar trend was observed
with S. aureus, although these copolymers performed slightly
better against Gram-positive bacteria in general. Higher Mn
copolymers were fairly inactive compared to their shorter
analogues. In comparison, the previous study on polynorbor-
nenes constructed from FA monomers identified a polymer
with an MIC as low as 3.8 mgmLÀ1.[25] Therefore, while the
route using segregated monomers was successful in endow-
ing the inactive PAON with modest antibacterial properties
by adding hydrophobicity, the MIC values were not as
potent in general as the previously reported polynorbor-
nenes.[20,25] Antimicrobial activity was also not as efficient as
Table 2. Antibacterial and hemolytic activities.
Low Mn polymers[a]
PAON
A1
A2
A3
A4
A4’
A5
A5’
A6
A6’
A9
A12
MIC (Ec)
MIC (Sa)
HC
selectivity (Ec)
selectivity (Sa)
400
400
2000
–
400
100
2000
–
200
50
250
1.3
5.0
75
25
500
6.7
20.0
50
50
<50
<1.0
<1.0
50
50
<50
<1.0
<1.0
50
75
<50
<1.0
<0.7
50
50
<50
<1.0
<1.0
100
100
<50
<05
<0.5
100
50
<50
<05
<1.0
200
125
<50
<0.3
<0.4
400
100
<50
<0.1
<0.5
–
20.0
High Mn polymers
PAON
A1
A2
A3
A4
A4’
A5
A5’
A6
A6’
A9
A12
MIC (Ec)
MIC (Sa)
HC
selectivity (Ec)
selectivity (Sa)
ꢁ400
400
2000
–
ꢁ400
200
250
–
200
200
250
1.3
50
200
50
1.0
0.3
100
100
<50
<0.5
<0.5
200
200
<50
<0.3
<0.3
400
200
<50
–
400
400
<50
–
ꢁ400
200
<50
–
ꢁ400
400
<50
–
ꢁ400
200
<50
–
ꢁ400
400
<50
–
–
1.3
1.3
<0.3
–
<0.3
–
<0.3
–
[a] MIC and HC values are reported in mgmLÀ1. Ec=Escherichia coli, Sa=Staphylococcus aureus, Selectivity=HC/MIC. Selectivity values are not given
for inactive polymers (MIC ꢁ400 mgmLÀ1).
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Design of Antibacterial Copolymers
FULL PAPER
Table 3. Biological data for non-50:50 copolymers (low Mn).
A1 Copolymers
duced leakage studies on dye-
filled liposomes composed of E.
coli lipid extracts were per-
formed and showed that the
most active copolymers caused
the most dye release (Figure 5).
These E. coli vesicles seemed to
model membranes quite well
A5’ Copolymers
A0.810.2
A0.610.4
A0.410.6
A0.210.8
A0.85’0.2
A0.65’0.4
A0.45’0.6
A0.25’0.8
MIC (Ec)
MIC (Sa)
HC
selectivity (Ec)
selectivity (Sa)
400
200
250
–
400
100
2000
–
250
200
2000
8.0
200
250
500
2.5
50
50
<50
<1.0
<1.0
25
100
250
250
<50
0.6
50
100
<50
<2.0
<1.0
<50
<0.5
<0.5
1.3
8.0
10.0
2.0
0.6
A0.8
1
0.2 =a random copolymer with ꢂ80:20 molar ratio of monomers.
antibacterial. Similarly, making A5’ more hydrophilic
through feed ratio adjustments unpredictably did not de-
crease its hemolytic activity even though it was expected
that a higher amine to non-polar monomer ratio could im-
prove the HC value of A5’ (HC ꢀ 50 mgmLÀ1) closer to
that of the relatively less hydrophobic A1 (HC
2000 mgmLÀ1).
=
It has been previously demonstrated that preformed sec-
ondary structures are not necessary for AMP-like activi-
ty.[13,17,24,32–34] Collectively, these studies imply that globally
amphiphilic conformations, induced by the membrane bilay-
er, are able to effectively capture AMP activity which was
recently supported by Gellmanꢂs observations.[12] At the
same time, Sen recently demonstrated that “same centered,”
FA repeat units were superior to “different centered,” segre-
gated ones.[9] All of our previous designs were based on the
supposition that local amphiphilicity was critical to generat-
ing highly potent yet non-toxic SMAMPs. For the first time,
we have directly compared our FA design to segregated
ones and the inability to tune the biological activity of the
copolymers argues that the spatial arrangement of charge
and non-polar groups is important and not just the overall
global amphiphilicity. In fact there may be profound advan-
tages in how a polymer made from FA monomers interacts
with the phospholipid bilayer compared to copolymerized
segregated monomers.
Figure 4. Cartoon proposing that polymer interactions with the polar
head group (black circles) and the non-polar lipid tails (squiggly lines) of
the membrane are significantly different for polymers from segregated
monomers (A) versus that of polymers from FA monomers (B).
The imperfect statistical nature of random copolymeriza-
tion can lead to runs of polar and non-polar units, at least
for some percentage of the polymer population (Figure 4A,
shows a run of three cationic units as well as sections of ad-
jacent non-polar units). This arrangement may not be opti-
mal for disrupting the lipid membrane bilayer especially if
the polymer loops in and out of the membrane rather than
having close contacts with the membrane as postulated here
for the polymers containing FA units (Figure 4B). With
polymers from FA monomers, 100% of the polymer popula-
tion has every monomer containing the identical ratio of
cationic to hydrophobic character. The local charge density
and the consistency of that density throughout the polymer
are extremely important in the polynorbornene systems it
appears. More structural studies of these polymers in the
presence of lipid bilayers, such as in our previous work with
antimicrobial oligomers (AMOs), are underway.[35,36]
Figure 5. Graph of polymer-induced dye leakage.
with the dye-leakage results tracking approximately with the
MIC values. Still, they were not perfect mimics of E. coli as
was noticed when comparing the induced-leakage abilities
of PAON and A12. Both PAON and A12 had an MIC of
400 mgmLÀ1 against E. coli yet A12 caused much more dye
release. Presumably, in the complex interactions between
these polymers and bacteria, more hydrophobic polymers
can interact better with a liposome than with intact bacteria.
Still, A12 did not cause as much leakage as the less hydro-
phobic but more antibacterial A4 and A5, showing that an
optimum balance was critical to capture AMP-like activity.
Membrane studies: Two experiments were used to probe
the membrane-disruption activity of these copolymers; vesi-
cle dye leakage and fluorescence microscopy. Polymer-in-
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G. N. Tew et al.
Lastly, live E. coli bacteria were incubated with a two-
component stain and treated with and without polymer
(Figure 6). The stain was made up of a green-emitting dye
(SYTO9 from Invitrogen) which has the ability to stain all
cells and a red-emitting propidium iodide dye that can only
enter cells with compromised membranes. Fluorescence mi-
croscopy showed that PAON essentially had the same effect
on E. coli as the negative control. It was unclear though
whether there was no significant interaction between the
cationic PAON and the net anionic membrane of E. coli, or
if there are interactions just not destructive ones. In con-
trast, antibacterial A4 not only displayed more intense red-
emission but also agglutination of the cells possibly due to
the aggregation of patches of torn membrane.
charged and non-polar units (in other words not perfectly al-
ternating) along the polymer backbone when the “segregat-
ed” monomer route is followed. This arrangement of repeat
units was shown to play a significant role in the copolymerꢂs
activity, especially for the low Mn copolymers which have an
average degree of polymerization of 8. Important trends for
the segregated copolymers were identified. In particular an
optimum hydrophobicity was observed for the most active
50:50 copolymers.
Surprisingly, investigation of non-50:50 copolymers did
not lead to improved activities or selectivities. This result
argues that the balance of hydrophobic/hydrophilic areas at
the local monomer level is much more critical to attain
highly active and selective polymers rather than just the
global amphiphilicity or overall charge density. This report
further clarifies the importance of the spatial relationship of
the charged and non-polar moieties in antimicrobial poly-
mers and advocates the use of FA monomers for better con-
trol of biological properties. It is expected that this principle
will be usefully applied to other polymeric backbones such
as the polyacrylates, polystyrenes, and non-natural polyam-
ides. A nice model system including strictly alternating co-
polymers would be an ideal further test.
Acknowledgements
We acknowledge the National Institutes of Health (RO1-GM-65803) and
the Office of Naval Research (N00014-07-1-0520) for their generous sup-
port. We are also grateful for early contributions to the project by Jason
Rennie and Courtney McConoghy.
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Conclusion
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Design of Antibacterial Copolymers
FULL PAPER
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Received: June 22, 2008
Published online: November 19, 2008
Chem. Eur. J. 2009, 15, 433 – 439
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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