The role of hydrophobicity in the antimicrobial and hemolytic activities of polymethacrylate derivatives

Article abstract of DOI:10.1002/chem.200801523

We synthesized cationic random amphiphilic copolymers by radical copolymerization of methacrylate monomers with cationic or hydrophobic groups and evaluated their antimicrobial and hemolytic activities. The nature of the hydrophobic groups, and polymer co

Full text of DOI:10.1002/chem.200801523

FULL PAPER
DOI: 10.1002/chem.200801523
The Role of Hydrophobicity in the Antimicrobial and Hemolytic Activities of
Polymethacrylate Derivatives
[
a]
[b]
[c]
Kenichi Kuroda,* Gregory A. Caputo,* and William F. DeGrado
Abstract: We synthesized cationic
random amphiphilic copolymers by
radical copolymerization of methacry-
late monomers with cationic or hydro-
phobic groups and evaluated their anti-
microbial and hemolytic activities. The
nature of the hydrophobic groups, and
polymer composition and length were
systematically varied to investigate
how structural parameters affect poly-
mer activity. This allowed us to obtain
the optimal composition of polymers
suitable to act as non-toxic antimicrobi-
als as well as non-selective polymeric
biocides. The antimicrobial activity de-
pends sigmoidally on the mole fraction
of hydrophobic groups (fHB). The he-
molytic activity increases as fHB in-
creases and levels off at high values of
fHB, especially for the high-molecular-
weight polymers. Plots of HC50 values
versus the number of hydrophobic side
chains in a polymer chain for each po-
lymer series showed a good correlation
and linear relationship in the log–log
plots. We also developed a theoretical
model to analyze the hemolytic activity
of polymers and demonstrated that the
hemolytic activity can be described as
a balance of membrane binding of
polymers through partitioning of hy-
drophobic side chains into lipid layers
and the hydrophobic collapsing of po-
lymer chains. The study on the mem-
brane binding of dye-labeled polymers
to large, unilamellar vesicles showed
that the hydrophobicity of polymers
enhances their binding to lipid bilayers
and induces collapse of the polymer
chain in solution, reducing the appar-
ent affinity of polymers for the mem-
branes.
Keywords: antibiotics · biocides ·
hemolysis · liposomes · polymers
Introduction
was found initially in hospitals, however, the drug-resistant
strains rapidly spread to other public places as well as com-
munities across countries.
[1]
The development of synthetic antimicrobial agents is an
emerging need due to antibiotic resistance of bacteria caus-
ing fatal infectious diseases, which significantly threaten
public health in global regions. The resistance of bacteria
New antimicrobial agents are urgently needed, which re-
quires new design concepts and approaches to combat drug-
resistant bacteria. Considering the wide use of agents as
drugs and in consumer products, it would be ideal to create
antimicrobials that can meet the following criteria: selective
toxicity to bacteria versus human cells and low susceptibility
to the development of resistance in bacteria. In this context,
host-defense peptides have been extensively studied and
their potential as alternatives for conventional antibiotics
has been explored. These peptides consist of a relatively
small number of residues (<40 aa). Upon binding to cell
membranes, the peptides form facially amphiphilic helical
structures with cationic and hydrophobic side chains regular-
ly distributed on different sides of the helix surface. The am-
phiphilic features of peptides function by disrupting bacteri-
al cell membranes, causing breakdown of the transmem-
brane potential, leakage of cytoplasmic components, and ul-
timately cell death. Natural antimicrobial peptides are be-
lieved to selectively target bacteria due to the preferable
charge interaction between the cationic side chains of the
[
a] Prof. K. Kuroda
Department of Biologic and Materials Sciences
University of Michigan School of Dentistry
011 N. University Ave., Ann Arbor, MI 48109 (USA)
1
Fax : (+1)734-647-2110
E-mail: kkuroda@umich.edu
[
b] Prof. G. A. Caputo
Department of Chemistry and Biochemistry, Rowan University
01 Mullica Hill Rd., Glassboro, NJ 08028 (USA)
2
Fax : (+1)856-256-5453
E-mail: Caputo@rowan.edu
[
c] Prof. W. F. DeGrado
Department of Biochemistry and Biophysics
University of Pennsylvania School of Medicine
6th & Hamilton Walk, Philadelphia, PA 19104-6059 (USA)
3
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800xxx.
Chem. Eur. J. 2009, 15, 1123 – 1133
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1123

peptides and the greater density of negative charges on the
bacterial cell surface relative to eukaryotic cells. The appli-
cation of peptide-based antimicrobials, however, is limited
in practical terms by the high costs associated with their
preparation and manufacturing, preventing their ubiquitous
use in pharmaceutical and biomedical applications.
molecular weight to investigate the optimal balance of cat-
ionic side chains and hydrophobic groups, as well as the role
of hydrophobic groups in the antimicrobial and hemolytic
activities of random copolymers. We also examined the
effect of the hydrophobic nature of polymers and lipid prop-
erties in membrane binding by using unilamellar vesicles
with different lipid compositions.
To this end, design and preparation of synthetic antimi-
crobial agents and the corresponding mechanistic studies
have been conducted to gain a better understanding of how
to create new agents including non-biological peptide
Results and Discussion
[
16]
mimics and synthetic polymers. Synthetic foldamers,
cluding b-peptides and peptoids,
in-
[21]
have been utilized to
Polymer design and synthesis: For the preparation of con-
ventional antimicrobial polymers, quaternary amines are
perhaps the most used cationic groups, and polymers are
often of high molecular weights (MWs). In contrast, we
chose primary amine groups as the cationic functionality
and relatively low-MW polymethacrylate backbones (MW=
1000–10000), mimicking structural features of antimicrobial
peptides (MW=2500–3000 for magainin and melittin)
(Scheme 1). Previously, we established that hydrophobic and
mimic the amphiphilic structures and helical conformations
of natural antimicrobial peptides. Facially amphiphilic well-
defined oligomers of aryl amides and phenylene ethynylene
also have been previously explored. These peptide mimics
exhibited higher activity and selectivity than the natural an-
timicrobial sequences. Although these peptide mimics and
oligomers can provide well-defined sequences and structures
suitable for structure–activity studies, their preparation re-
mains time- and cost-intensive.
In contrast to peptide mimics,
polymeric biocides/disinfectants
are easy to make and are inex-
pensive. A number of polymer-
ic antimicrobial agents have
been prepared using scaffolds
of conventional synthetic poly-
mers including polystyrenes,
polypryridines,
and
poly-
ACHTUNGTRENNUNG( meth)acrylates. The polymer
structures are often modified
with cationic and hydrophobic
side chains, which are randomly
distributed in a polymer chain
Scheme 1. Synthesis of amphiphilic polymethacrylate derivatives.
and thus display random am-
phiphilicity. Although the poly-
mers display high antimicrobial activity, they often suffer
from little or no selectivity to cell types or high levels of tox-
icity to human cells. However, a series of amphiphilic poly-
charged side chains are required for antimicrobial activity,
[40]
although the optimal balance had not been defined. Here,
we systematically varied the length and mole fraction of the
hydrophobic groups (fHB) and degree of polymerization
(DP). The polymers were synthesized by copolymerization
of the appropriate monomers in the presence of methyl-3-
mercaptopropionate as a chain-transfer agent, followed by
deprotection of tert-butoxycarbonyl (Boc) groups in tri-
fluoroacetic acid (TFA). The composition and molecular
weight of the final polymers were determined by comparing
[38]
norbones have been prepared, and their random copoly-
mers displayed selective toxicity to bacteria over human red
[38]
blood cells. Recently, Mowery et. al hypothesized that the
global amphiphilic conformation of random amphiphilic co-
polymers can be induced by a bacterial membrane sur-
[39]
face. Random copolymers of b-peptides with cationic and
hydrophobic groups displayed cell-type selectivity in their
antimicrobial and hemolytic activities. We previously report-
ed antimicrobial and hemolytic activities of random copoly-
mers based on polymethacrylates containing hydrophobic
1
the integrated intensities of the H NMR resonances from
the terminal chain-transfer agent relative to the side chains
(Table 1). The results were confirmed by size-exclusion
chromatography using the Boc-protected precursors of se-
lected polymers. In general, there was reasonable but not
exact agreement between the mole ratio of the side chains
used in the synthesis, and that obtained in the products. The
lack of exact agreement reflects the selective enrichment of
the polar products during the final ether-precipitation step.
The polymers labeled with a dansyl group at the polymer
[40]
butyl and cationic side chains. Although these polymers
have a random sequence of hydrophobic and cationic
groups and thus lack of secondary rigid structures, they dis-
played high antimicrobial activity and modest selectivity for
bacteria over human cells.
Here, we systematically varied the nature of the hydro-
phobic side chains, the composition of the polymers, and the
1124
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Chem. Eur. J. 2009, 15, 1123 – 1133

Polymethacrylate Derivatives
FULL PAPER
Table 1. Characterization of amphiphilic random copolymers.
As a prelude to evaluating the antimicrobial copolymers,
we examined the activity of the homopolymer with 100%
[
b]
3
Polymer Side chain
series
f
HB
DP
range
M
n
range
n
Average M /10
[
a]
[c]
ꢁ1 [d]
ꢁ1
A[ g mol
H
U
G
R
N
N
]
ACHTUNGTNRENUNG[ g mol ]
aminoethyl side chains and 0% hydrophobic groups (fHB
0) (Figure 1). The highest-molecular-weight polymer tested
DP=30) showed the greatest activity, and activity de-
=
C
C
C
1
1
1
-3.3
-2.0
-1.6
0.16–0.74 14–31
0.13–0.70 8–20
0.12–0.64 7–11
3000–3200
1700–2100
1300–1900
3.3
2.0
1.6
methyl
ethyl
(
creased as MW decreased.
C
C
C
C
2
2
2
2
-5.0
0.10–0.55 20–32
0.11–0.70 11–22
0–0.68 11–16
0–0.50 7–9
3600–6100
2300–3500
2300–3600
1600–2200
5.0
2.9
2.9
1.8
-2.9(1)
-2.9(2)
-1.8
[
[
[
e]
e]
e]
C
C
C
4
4
4
-8.7
-5.0
-1.6
0–0.57 32–46
0–0.53 19–31
0–0.47 5–9
7900–10100
4500–6000
1300–1900
8.7
5.0
1.6
butyl
C
C
C
6
6
6
-4.8
-3.3
-2.0
0.09–0.51 20–24
0–0.36 11–19
0–0.30 8–10
4400–5600
2700–4100
1900–2300
4.8
3.3
2.0
hexyl
benzyl
Bz-3.5
Bz-2.2
Bz-1.8
0.09–0.80 13–17
0.08–0.51 8–10
0.08–0.40 6–9
2700–4100
2000–2400
1600–2000
3.5
2.2
1.8
Figure 1. Antimicrobial activity of cationic homopolymers.
[
n
a] The series of polymers having alkyl groups were denoted by C -mo-
lecular weight averaged over the series, in which n is the number of car-
bons in the alkyl side chains. [b] fHB =mole fraction of monomers with
hydrophobic groups in a polymer chain. [c] DP=degree of polymeri-
zation (number of monomers in a polymer chain). [d] The number-aver-
The antimicrobial activity of the copolymers depended
critically on their hydrophobic content and polymer length.
In general, the antimicrobial activity for a given series of hy-
drophobic side chains depends sigmoidally on fHB (Figure 2).
At low values of fHB, the activity of a series of polymers with
a given molecular weight will level off at the value expected
for the homopolymer. As the mole fraction of the hydro-
phobic substituents is increased, the activity increases, reach-
ing approximately the same value for all polymers. The
amount of data obtainable for polymers with high values of
fHB was limited by the limited
age molecular weight of polymers (M
n
) was calculated from fHB, DP, and
the molecular weights of monomers and methyl 3-mercaptopropionate.
[
40]
[
e] Data reported previously.
end were prepared by the same procedure using a chain-
transfer agent 2 modified with a dansyl group (Scheme 2
Table 2).
solubility of the polymers.
The sigmoidal shapes of the
fHB-versus-MIC curves is well
described by the empirical Hill
Equation (1):
c2
logðMICÞ ¼ c þ
Scheme 2. General scheme for interactions of polymers with lipid bilayers.
Table 2. Characterization of dansyl-labeled random copolymers.
1
1
þðfHB=fmid n
Þ
ð1Þ
in which fmid is the midpoint of the curve, n relates to the
steepness of the curve, and c and c relate to the asymptotic
limits (see Supporting Information for the fitting parame-
ters). This treatment was used for the C -, C -, and C -poly-
3
ꢁ1
Polymer
Side chain
f
HB
DP
M
n
/10 [g mol
]
1
2
D
D
D
0
–
0
0.27
0.49
13
16
12
3.4
3.7
2.6
27
49
butyl
butyl
1
2
4
mer series, although the hexyl and benzyl polymers were
not soluble over a sufficient range to allow fitting of the pa-
rameters. From an examination of the C through C -poly-
1
4
Antimicrobial activity of homopolymers and random co-
polymers: We determined antimicrobial activity as the mini-
mal inhibitory concentration (MIC) using E. coli as a test
organism. Because we were interested in evaluating the ac-
tivity of a single polymer chain to investigate the correlation
between biological activity and the structural parameters of
polymers including fHB and polymer length, the MIC data
are given in molar concentration.
mer series, we observed that as the hydrophobic group be-
comes smaller, the curves become steeper, with a “coopera-
tivity value” of n ranging from approximately 28ꢀ10 to
3.5ꢀ2 (based on the quality of the fits). Furthermore, as the
alkyl-chain length increased the curves shifted to the left, al-
though there was also some dependence on MW; polymers
with lower MW and smaller alkyl chains required a higher
fHB to achieve maximal efficacy.
Chem. Eur. J. 2009, 15, 1123 – 1133
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1125

K. Kuroda, G. A. Caputo, and DeGrado
Figure 2. Antimicrobial activity of cationic random copolymers of various
molecular weights with A) methyl, B) ethyl, C) butyl, D) hexyls, and
E) benzyl groups. The average molecular weights for a given polymer
series are indicated in the legend. The Hill Equation [Eq. (1)] was used
to fit smooth curves to the data in panels A–C. Fitting parameters are
Figure 3. Hemolytic activity of cationic random copolymers with
A) methyl, B) ethyl, C) butyl, D) hexyls, and E) benzyl groups. The aver-
age molecular weights for a given polymer series are indicated in the
[
40]
legend. [a] Data reported previously.
[
40]
provided in the Supporting Information. [a] Data reported previously.
increases and levels off at high values of fHB, especially for
the high-MW polymers. The polymers with the largest MW
in a given series of hydrophobic groups displayed the high-
ꢁ
1
On the other hand, the MIC values given in mgmL dis-
played little or no dependence on polymer length (Support-
ing Information). The exception was the C -polymer series
est activity, except the C series, in which the activity of the
4
1
that has a wide range of average molecular weights (1600–
smallest-MW polymers is highest. As the alkyl-chain length
increased, the curves shifted to the smaller fHB region, indi-
cating that the polymers with longer alkyl chains are more
8700 MWs) compared to those of other series, which is
likely to create the evident dependence of maximum effica-
cy (lowest MIC values) on the MWs; the lowest-MW poly-
hemolytic at a given f . In contrast to the MICs, the HC50
HB
ꢁ
1
mers in the C series displayed the lowest MIC values. This
values given in mgmL displayed the same trends as those
in mm (see the Supporting information).
4
is probably because the low MW of the polymers increases
the molar concentration (the number of polymer molecules
per volume) for any given weight concentration, which de-
The plots of HC50 values versus the number of hydropho-
bic side chains in a polymer chain (Nside chains =fHB ꢁDP) for
each polymer series showed good correlation (Figure 4,
panel A) and linear relationship in the log–log plots
(Figure 4, panel B). The data of the C - and C -polymer
ꢁ
1
creases the MICs in mgmL because the MICs in mm (an ac-
tivity of each polymer molecule) are almost the same for all
members of the C -polymer series at high f values.
4
HB
4
6
series fit well to a simple power equation, and the data sets
and curves of each series of polymers seem to be in parallel
with each other. These results suggest that the hemolytic ac-
tivity of polymers reflects the hydrophobic nature of poly-
mer side chains.
Hemolytic activity of random copolymers: The hemolytic
activity is dependent on the hydrophobic properties of poly-
mers and MW (Figure 3). The HC50 values are limited at
low fHB because the hemolytic activity of polymers with low
values of fHB was too low to give entire hemolysis curves for
HC50 determination in the range of polymer concentrations
used here. In general, the hemolytic activity increases as fHB
Theoretical analysis of hemolytic activity: We further ex-
plored the hemolytic activity of polymers using our theoreti-
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Polymethacrylate Derivatives
FULL PAPER
chains decreases the apparent affinity of polymers to the
membranes or reduces the number of polymers interacting
with the membranes, resulting in a decrease in hemolytic ac-
tivity. It is, however, beyond the scope of this report to de-
termine the active species in the mechanism of the hemolyt-
ic activity of polymers, and we assume all polymers bound
to the membranes are involved
in the pore formation causing
lysis of red blood cells (RBCs).
For this reason, we introduce Scheme 4. Synthetic scheme of
here a more simplified scheme a chain-transfer agent with a
dansyl group.
for creating a theoretical model
(
Scheme 4).
The dissociation constants Kc and K ’ are defined by
d
Equation (2):
½
P ꢂ
½Pꢂ½Sꢂ
½P ꢃ Sꢂ
c
0
K ¼
c
and Kd
¼
ð2Þ
½
Pꢂ
HC50 can be presented as the total polymer concentration
Eq. (3)]:
[
Figure 4. The HC50 of copolymers as a function of the number of side
chains (Nside chains) (A and B) or ꢀlogP (C and D). Nside chains is defined as
HC50 ¼ ½Pꢂþ½P ꢃ Sꢂþ½Pcꢂ
ð3Þ
f
HB ꢁDP. The data for polymers having butyl and hexyl groups were fit to
c4
the equation HC50 =c
3
(Nside chains
)
in which c
3
and c
4
are constant. The
Here, we define HC50 as the polymer concentration neces-
sary for 50% lysis, at which 50% of the binding sites are oc-
cupied by polymers, and thus let [P·S] can be replaced by
[S]total ꢁ0.5=[S]1/2 [Eq. (4)]:
HC50 data for C - and C
4
6
-polymer series in panels C and D were fit to
Equation (10). For the calculation of logP using Equation (7), the
number of carbon atoms in benzyl groups was set as n=7. See Support-
ing Information for the fitting parameters.
HC50 ¼ ½Pꢂþ½Sꢂ1=2þ½P ꢂ ¼ ½Sꢂ þ½Pꢂð1þK Þ or
c
1=2
c
ð4Þ
cal model to investigate the effect of the hydrophobic
nature of polymers on their hemolytic activity. The polymers
probably exist in multiple states during the binding and for-
mation of polymer–lipid complexes. Because of its amphi-
philic nature, the polymer chain undergoes a hydrophobic
collapse or aggregation in an aqueous environment. The
proposed models for the antimicrobial action of host-de-
fense peptides suggest that the peptides bind to cell mem-
brane surfaces by combination of electrostatic and hydro-
phobic interactions, following the insertion of peptides into
the hydrophobic region of lipid bilayers. Based on this, the
general scheme for the binding and insertion of polymers
into lipid membranes and collapse of a polymer chain in so-
lution is shown in Scheme 3.
HC50ꢁ½Sꢂ1=2
½Pꢂ ¼
1þK
c
Because [S]=[P·S]=[S]1/2 at the polymer concentration=
HC50, K ’ is defined as [Eq. (5)]:
d
HC ꢁ½Sꢂ
½Sꢂ
½Sꢂ
HC ꢁ½Sꢂ
0
50
1=2
1=2
1=2
50
1=2
Kd
¼
ꢃ
¼
ð5Þ
1
þK
1þK
c
c
Here, we assume that the binding of polymers to the cell
membranes can be described in terms of partitioning of hy-
drophobic side chains from the aqueous phase to the hydro-
phobic regions of lipid layers. The octanol/water partition
coefficient of substances (P=[substance]octanol/[substan-
ce]water, or its decadic logarithm, logP) has been used to pre-
dict partitioning of substances or drugs to cells and organs
[42]
by translocation through cell membranes. Here, we simi-
larly use logP for estimation of partitioning of alkyl side
chains into hydrophobic regions of lipid layers. As the logP
values for substances are experimentally obtained and previ-
Scheme 3. Membrane binding and collapsing of polymers.
[
43]
In this scheme, P and P are defined as free polymers and
ously reported,
those of methanol (ꢁ0.77), ethanol
c
collapsed polymers inactive to the membranes, respectively.
S, P·Sbound, and P·Sinserted relate to binding sites on the mem-
branes, and polymers bound to the sites and inserted into
the lipid bilayers, respectively. We assume that the hemolytic
activity of polymers reflects the polymers bound to cell
membranes, and the hydrophobic collapsing of polymer
(ꢁ0.31), butanol (0.88), and hexanol (2.03) give the linear
relationship defined in Equation (6):
logP ¼ 0:567nꢁ1:3851
ð6Þ
in which n is the number of carbon atoms in the alkyl
Chem. Eur. J. 2009, 15, 1123 – 1133
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1127

K. Kuroda, G. A. Caputo, and DeGrado
groups of alcohols. Based on this relationship, we have a rel-
ative partition coefficient [logP(n)] for alkyl side chains
Membrane binding of polymers: We also examined the
binding behavior of the polymers to lipid bilayers using
large unilamellar vesicles (LUVs). The main lipid compo-
nent of the outer leaflet of human RBCs is neutral phospha-
[Eq. (7)]:
[44,45]
logPðnÞ ¼ 0:567n
ð7Þ
tidylcholine (PC) lipids.
of E. coli consist mainly of neutral phospatidyethylamine
PE) (75–85%) with anionic phospatidylglycerol (PG) (10–
0%) and cardiolipin (CL) (ꢅ5%), and liposaccharides
LPS) (in the outer membrane). The abundance of anionic
PG, CL, and LPS in bacterial membranes results in a high
In contrast, the cell membranes
The total partition coefficient of hydrophobic side groups
in a polymer chain is given by the summation of logP of
alkyl side chains in a polymer, defined as ꢀlogP=logP(n)ꢁ
(
2
(
Nside chains. The coefficient K ’ is further converted as shown
d
[46,47]
[Eq. (8)]:
density of negative charges on the bacterial cell surface.
Based on this, we prepared LUVs from POPC as well as
mixtures of POPE/POPG (8:2), POPC/POPG (8:2), or
POPE/POPC (8:2) to investigate the effects of charge pair-
ing of cationic polymers and anionic lipids and to examine
the response to different chemical structures in the lipids,
mimicking the surface properties of cell membranes. (Note
that POPE cannot be used as a neutral control as it does
not form LUVs in the absence of other lipids, and even in
the presence of 20% POPC the vesicles are only stable for
a short time.) The fluorescence properties of the dansyl
group are sensitive to the surrounding environment: the
emission spectrum undergoes a blue-shift, exhibits a narrow-
ing in spectral width, and the quantum yield (overall emis-
sion intensity) increases upon transfer from a polar (aque-
X
0
Kd ¼ expðDG=kTÞ ¼ 2:3026 ꢄ 10ðꢁm ꢄ
logPþC Þ ¼
0
X
1
0ðꢁm
logPꢁC00
ð8Þ
in which m is a variable to scale the relative logP values to
the experimental data, and C ’ refers to the standard state.
On the other hand, collapse of the polymer chain reflects
the hydrophobicity of side chains, and we attribute the hy-
drophobicity of polymers to ꢁꢀlogP [Eq. (9):
0
X
0
0
K ¼ 10ðl
logPꢁC Þ
ð9Þ
c
0
[48]
ous) to a nonpolar (hydrophobic) environment. The MIC
of dansyl-labeled polymers was close to those of polymers
in which l is a variable for scaling the curves to the experi-
mental data, and C ’’ refers to the standard state. From
Equations (5), (8), and (9), we obtain an equation describing
the relationship between HC50 and total partitioning coeffi-
cient (hydrophobicity) of polymers [Eq. (10)]:
0
with corresponding fHB and DP in the C -polymer series in
4
Figure 2, indicating the dye likely has little or no effect on
the antimicrobial activity of the compounds.
We first examined the inherent effect of polymer chains
on the emission properties of the dansyl fluorophore. In the
absence of any LUVs, as the fraction of butyl groups in-
creased the fluorescence-emission intensity increased, and
the spectra exhibited a blue-shift and narrowing (Figure 5,
panel A). This is probably the result of the polymer chain
adopting a collapsed conformation by association of butyl
X
0
HC50 ¼ ½Sꢂ1=2þ10ðꢁm
logP þ C Þ
0
X
X
ð10Þ
0
00
þð10ðꢁm
logP þ C Þ ꢄ 10ðl
logPꢁC ÞÞ
0
0
Equation (10) fits well to the HC50 data of the C - and C -
4
6
polymer series, if [S]1/2 is fixed to 0.05 (Figure 4, panels C
and D). Although we were unable to obtain good fitting to
the data for C and C polymers, these data points were also
side chains in aqueous solution, which was denoted P in
c
Schemes 3 and 4, and the dansyl group becoming seques-
tered in a more hydrophobic pocket of the collapsed poly-
mer chain. We, however, observed a precipitate in the aque-
ous solution of the most hydrophobic polymer D49 over time
(several weeks) and a significant decrease in its fluorescence
intensity, whereas other hydrophilic polymers D and D
1
2
found in the vicinity of the fitting curves for the C -and C -
4
6
polymer series. These results indicate that lysis of RBCs re-
flects the membrane binding of polymers through partition-
ing of hydrophobic side chains of polymers into lipid layers.
In addition, the hemolytic activity of polymers is described
by the simple parameter of ꢀlogP, which is irrelevant to the
identity of alkyl groups, suggesting that the hemolytic activi-
ty of polymers depends on the total hydrophobicity of poly-
mers rather than the chemical structure of the side-chain
groups. The fitting curves display a turnover at high ꢀlogP
regions, at which the polymer chains are likely strongly col-
lapsed or irreversibly aggregated, resulting in large Kc
values or a decrease in the apparent affinity of polymers to
the membranes. In addition, both C -and C -polymer series
0
27
appear to be stable in solution.
In the presence of lipid (POPC), both the overall fluores-
cence intensity increased and the spectra narrowed as the
lipid concentration increased for all polymers tested. Both
observations are consistent with the dansyl group being
more shielded from the aqueous environment (Figure 5,
panels B–D). The spectra of D0 and D27 blue-shifted in
higher lipid concentrations as the dansyl groups were trans-
ferred to the hydrophobic region of lipid layers from aque-
ous solutions or the hydrophobic environment provided by
the polymers. In contrast, D49 exhibited a small red-shift in
the spectra as lipid concentration increased, indicating that
the environment around the dansyl group in the polymer is
4
6
reached the same HC50 of about 0.07 mm after the turnover.
These results indicate that this is the highest hemolytic ac-
tivity that this polymer system can possibly access by in-
creasing the hydrophobicity of polymers.
1128
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Chem. Eur. J. 2009, 15, 1123 – 1133

Polymethacrylate Derivatives
FULL PAPER
POPG. Interestingly, the differ-
ence between neutral lipids
(
POPC and POPE), which are
the major components of
LUVs, seems to be a primary
factor in the binding of cationic
polymers rather than the popu-
lation of negative charges in the
membrane
surfaces,
even
though the effect of charge cou-
pling between cationic side
chains of polymer and anionic
lipids is expected to positively
contribute to the formation of
polymer–lipid complex. These
data suggest that the properties
of lipids play an important role
in the affinity of polymers as
well as their specificity.
In general, the K ’’ values for
d
all lipid types except POPC/
POPG decrease as the mole
fraction of butyl groups of poly-
mers increases, indicating that
hydrophobicity of polymers en-
hances the binding of polymers
Figure 5. Emission spectra of dansyl-labeled polymers in the absence or presence of POPC LUVs. A) Normal-
ized emission spectra of D
D) D49 in the presence of 0, 15, and 100-mm POPC.
0
, D27, and D49 in the absence of LUVs. Emission spectra of B) D
0
, C) D27, and to lipid bilayers. The isotherms
of D27 and D49 for POPE/
POPC displayed at least two
different modes of binding in
slightly less polar (more hydrophobic) than that in the poly-
mer–lipid complex. The spectra of all polymers exhibited
lmax of similar wavelength (ꢅ530 nm) at 100-mm lipid, sug-
gesting that the dansyl groups are eventually located in a
similar hydrophobic environment of polymer–lipid complex
in high lipid concentrations. The spectra of polymers for
LUVs consisting of POPC/POPG and POPE/POPC dis-
played a trend similar to that of POPC and exhibited lmax
values within the same wavelength region (ꢅ530 nm) at
high lipid concentrations (see Supporting Information).
We obtained binding isotherms of polymers by monitoring
an increase in emission intensity at 500 nm from the dansyl
group by titrating LUVs into the sample (Figure 6). The
fluorescence-emission data were fit to a single site-binding
isotherm [Eq. (13)], Experimental Section) to give the over-
the low (0–20 mm) and high (>20 mm) lipid concentrations
that yield a relatively large K ’’ value of 15.85 for D , which
deviates from that of other polymers. We were unable to
d
27
obtain good fitting to the data for D49 due to the multiple
modes of binding. The polymers displayed lower K ’’ values
d
for POPC than that for POPE/POPG, indicating that these
polymers bind RBC-type membranes in preference to bacte-
rial cell membranes. In addition, the differences in K ’’ di-
d
minishes as hydrophobicity of the polymers increases, imply-
ing that their binding to lipids is no longer dependent on the
lipid contents when the hydrophobic polymer–lipid interac-
tion is dominant in the complex formation. Polymers D27
and D49 displayed similar K ’’ values for their binding to
d
POPC, suggesting a further increase in hydrophobic content
does not affect the affinity of polymers to lipid bilayers. This
is likely due to a hydrophobic collapse of the polymer chain
in aqueous media or irreversible aggregation, preventing in-
teractions with lipid layers. This binding behavior of poly-
mers is consistent with the trends in the antimicrobial and
hemolytic activities and suggests that the membrane-disrup-
tion mechanism correlates to the hydrophobic binding of
all dissociation constant, K ’’, in the equilibrium between
d
the polymers in solution and the polymer–lipid complex, as-
suming equilibrium between P and P·S (Scheme 4). The
c
binding behavior of the polymers to LUVs depends on both
the hydrophobic content of the polymers and the chemical
structures of lipids. Polymer D displayed higher K ’’ values
0
d
for LUVs containing an anionic POPG lipid than those for
their neutral controls (Table 3). This result indicates that the
affinity of the polymer to neutral lipid bilayers is higher
than that to negatively charged bilayers. On the other hand,
the LUVs containing a POPE lipid showed relatively larger
polymers to lipid bilayers. It is interesting that the K ’’ dis-
d
played a maximum increase of only four-fold as the fraction
of hydrophobic (butyl) groups of polymers increased to
from 0 to 0.49, whereas the HC50 varies by up to 100-fold.
This indicates that the activity of polymers should be inter-
preted as the result of a combination of both the binding of
K ’’ values than those for LUVs of POPC and POPC/
d
Chem. Eur. J. 2009, 15, 1123 – 1133
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1129

K. Kuroda, G. A. Caputo, and DeGrado
hibited the greatest enhancement, and those bound to
POPC LUVs showed the least enhancement. These results
indicate that the microenvironments around the dansyl
groups in the polymer–lipid complex seem to reflect the
membrane-bound conformation of polymers, that is, how
the polymers physically interact with the membrane surface
(
P·Sbound) and insert into the bilayer (P·Sinserted) (Scheme 3).
The presence or absence of charged groups on both the
membrane surface and the polymer chain can affect the
exact location at which the polymer and, therefore, the
dansyl group is oriented in the membrane. Small changes in
fluorophore depth have been shown to affect fluorescence
properties; for example, a dansyl residing in the headgroup
region could show slightly different emission properties to a
dansyl group either oriented at the interfacial region or that
becomes buried in the nonpolar core of the bilayer upon
binding the vesicle. Additionally, as the polymers become
hydrophobic, the value of F /F decreases (see Table S11 in
inf
0
the Supporting Information). It could be possible that for
more hydrophobic polymers F is larger than F , resulting
0
inf
in a decrease in F /F .
inf
0
In summary, the hydrophobicity of polymers enhances
binding to lipid bilayers and induces a collapse of polymer
chains in solution, causing a decrease in the apparent affini-
ty of polymers to the LUVs. In addition, the binding of
polymers to lipid bilayers depends on the overall hydropho-
bicity of polymers as well as the chemical properties of the
lipid headgroups. The activity of polymers is due to both
membrane binding of polymers and pore formation induced
by polymers.
Membrane binding and antimicrobial activity of polymers:
According to the results of membrane binding of polymers,
the hydrophobic interaction between polymers and lipid bi-
layers drives the binding of polymers to both bacteria and
erythrocytes. However, the affinity of polymers to POPC
LUVs is higher than that to POPE/POPG LUVs (Table 3),
indicating bacteria would be more resistant than erythro-
cytes to the binding of polymers. Because the antimicrobial
activity of polymers levels off for highly hydrophobic poly-
mers, displaying a MIC of 5–10 mm (Figure 2), the antimicro-
bial action also likely depends on the balance between the
membrane binding and collapsing of polymers driven by the
polymer hydrophobicity as shown in the hemolysis. There-
fore, the bacterial resistance to the binding of polymers
could cause a turnover in MIC at lower hydrophobicity of
polymers, which is seen in the HC50 for more hydrophobic
polymers (Figure 4, panels C and D). This earlier turnover
in the MIC of polymers increases the minimum MIC values
(decreases the maximum efficacy) and reduces the selectivi-
ty of activity towards bacteria over erythrocytes. Consider-
ing these results, the rational design of polymer structures
and a fine tuning of their properties are necessary for the
further development of non-toxic synthetic polymers.
Figure 6. Binding isotherms of dansyl-labeled polymers A) D
and C) D49
0
, B) D27
,
.
Table 3. Dissociation constants of the membrane binding of polymers.
K
d
’’ [mm]
Polymer
POPC
POPC/POPG
POPE/POPC
POPE/POPG
D
D
D
0
2.27
0.83
1.08
3.30
3.27
0.87
5.00
5.59
1.76
n.d.
[
a]
27
49
15.85
[
a],[b]
[b]
n.d.
[
a] The isotherms displayed at least two different modes of binding at
lower (0–20 mm) and higher lipid concentrations. [b] Not determined due
to low quality of fitting.
polymers and pore formation, a mechanism that also likely
reflects the amount of polymers bound to the membranes as
well as the hydrophobic nature of polymers.
We also examined the effect of the formation of the poly-
mer–lipid complex on fluorescence intensity by comparing
emission-intensity values in the absence of lipid and once
the polymer was fully bound to LUVs. The level of enhance-
ment in fluorescence intensity shows a dependence on lipid
composition (Figure 6), although the fluorescence spectra of
polymers in high lipid concentrations are similar (Figure 5).
In all cases the polymers bound to POPE/POPG vesicles ex-
MIC and HC50 of polymers: Although the hemolytic activity
is driven by the hydrophobic nature of polymers, the amphi-
1130
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1123 – 1133

Polymethacrylate Derivatives
FULL PAPER
philic balance seems to be important in their antimicrobial
activity (Figure 2) and selectivity towards bacteria over
RBCs. To examine a relationship between these activities
of the polymers, the MIC and HC50 values were plotted in
Figure 7. The MIC values are mostly within the range of 5–
Conclusion
[40]
We have investigated the optimal balance of hydrophobic
and cationic side chains for the antimicrobial and hemolytic
activities of amphiphilic polymethacrylate copolymers. The
polymers vary systematically in the nature of their hydro-
phobic groups, composition, and length. Analysis of mem-
brane binding and efficacy indicates that the hydrophobic
nature of polymers is a key factor in controlling the antimi-
crobial and hemolytic activities. This reflects the balance be-
tween the polymers bound to lipid bilayers, which are mem-
brane-active and cause lysis, and hydrophobically collapsed
polymers, which are not interacting with membranes. In ad-
dition, the membrane binding of polymers strongly depends
on the lipid properties, and bacteria could be more resistant
to the binding of polymers than erythrocytes, resulting in a
lack of selective activity towards bacteria over erythrocytes.
This may be an inherent property of these synthetic poly-
mers and reflects the need for careful engineering in the
design of non-toxic antimicrobial polymers.
Figure 7. Relationship between HC50 and MIC of copolymers. Selectivity
index (SI) is defined as HC50/MIC. Polymers with MIC<10 mm and SI<1
are suitable for preparation of biocides due to their high activity against
bacteria and human cells. Those with MIC<10 mm and SI>1 display effi-
cacy selective towards bacterial cells and are useful as non-toxic antimi-
crobials.
Experimental Section
Materials: The bee-venom toxin melittin (85%) was purchased from
Sigma–Aldrich. All other chemicals and solvents were obtained from
Sigma–Aldrich and Fisher Scientific and used without further purifica-
tion.
20 mm for all polymers, whereas the HC50 values range from
about 0.03 to 100 mm. This is reflected in the different de-
pendencies of MIC and HC50 on the fHB values of the poly-
mers: the HC50 values for most of the polymer series de-
crease as fHB increases, whereas the MICs of all polymers
reached approximately the same value at high fHB (Figures 2
and 3). Thus, polymers with methyl side chains show selec-
tivity towards E. coli over RBCs with a maximum selectivity
index (HC /MIC) of 10 and a high antimicrobial activity
Polymer synthesis: The polymers, except those having methyl (C
1
-3.3,
2
.0, and 1.6) and ethyl groups (C -2.9(2), and 1.8) were prepared accord-
2
[40]
ing to procedures reported previously, in which the Boc-protected pre-
cursor polymers were isolated by precipitation in diethyl ether, followed
by Boc-deprotection by TFA, yielding cationic polymers. For the poly-
mers with methyl (C -3.3, 2.0, and 1.6) and ethyl groups (C -2.9(2) and
1
2
1.8), the crude polymers were deprotected by treatment with TFA with-
out isolation by precipitation in diethyl ether of the Boc-protected pre-
cursor polymers. The experimental procedure is described briefly as fol-
lows: N-(tert-butoxycarbonyl)aminoethyl methacrylate and alkyl or
benzyl methacrylate (0.435 mmol total) were polymerized in acetonitrile
(0.5 mL) containing 2,2’-azobisisobutyronitrile (AIBN) (0.716 mg,
50
(
MIC=5–10 mm).
Important properties of synthetic antimicrobials are not
4
.35mmol) and methyl 3-mercaptopropionate (MMP) as a chain-transfer
only high activity against bacteria but also the lack of toxici-
ty to human cells. To this end, the activity profile in Figure 7
provides general information on the optimal composition of
polymers for a given application, which is useful for the
future design and manufacturing of polymers. In the case of
preparation of polymeric biocides for cleaning or sanitizing
surfaces, polymers are required to have high antimicrobial
activity (lowest MIC). Because their toxicity to human cells
is not crucial, the polymers at the bottom left region (MIC<
agent at 60–708C. The solvents were removed under reduced pressure,
and the obtained crude polymer was dissolved in TFA and stirred for
3
2
0 min. After removing TFA by N gas perfusion, the oily product was
dissolved in a small amount of methanol and added into diethyl ether.
The resultant precipitate was collected by centrifugation and lyophilized
to give cationic copolymers as a white powder. Note that polymers
having high MWs and a high percentage of cationic groups selectively
precipitate in diethyl ether. As a result, the MWs of the collected poly-
mers are higher than those of the Boc-protected precursors, and the hy-
drophobic content is less than 60–70% in general, whereas the hydropho-
1
bic content of the Boc-protected precursors is greater. H NMR analysis
1
0 mm and selectivity <1), which contain mainly C - and C -
2 4
provided determination of the degree of polymerization (DP) and the
mole percentage of alkyl methacrylate, fHB, of the polymers (see Support-
ing Information for more details).
polymer series, will be useful for this purpose. On the other
hand, for polymers used as antibacterial agents in cosmetics,
food, and house paints, their toxicity to human cells should
be avoided. To this end, polymers displaying possibly lowest
MIC and high HC50 or high selectivity (>1) will be available
in the polymer series with methyl side chains (top left
region).
Synthesis of a chain-transfer agent with a dansyl group: Dansyl chloride
(500 mg, 1.85 mmol) was added to cystamine dihydrochloride (209 mg,
0.97 mmol) in DMF (15 mL) and triethylamine (0.78 mL, 5.6 mmol). The
reaction mixture was stirred at RT overnight. After the solvent was
evaporated under reduced pressure, the residue was taken up by ethyla-
cetate and washed in water. The aqueous layer was back-washed in ethyl-
acetate. The combined organic layer was washed in 1n NaOH, 10%
3
citric acid, sat. NaHCO , and brine. The organic layer was dried over
Chem. Eur. J. 2009, 15, 1123 – 1133
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
1131

K. Kuroda, G. A. Caputo, and DeGrado
Na
2
SO
4
, and the solvent was evaporated under reduced pressure. The
½OD414ðpolymerÞꢁOD414ðsolventÞꢂ
P ¼
ð11Þ
product was purified by silica-gel column chromatography (EtOAc/
hexane 1:1) to give product 1 (450 mg, 75%); H NMR (500 MHz,
½OD414ðmelittinÞꢁOD414ðsolventÞꢂ
1
CDCl
brm, 1H), 3.11 (m, 2H), 2.89 (s, 6H), 2.50 ppm (t, 2H).
Tris-(2-carboxyethyl)phosphine (TCEP) (38 mg, 0.13 mmol) was added to
(60 mg, 0.097 mmol) in methanol (1 mL). After addition of 3 drops of
3
): d=8.56 (d, 1H), 8.25 (m, 2H), 7.53 (m, 2H), 7.19 (d, 1H), 5.10
HC50 was obtained as the polymer concentration at 50% hemolysis,
which was estimated by curve fitting using Equation (12):
(
1
water with a glass pipette, the reaction mixture was stirred at RT over-
night. The solvent was evaporated under reduced pressure, and the resi-
n
PðC
p
Þ ¼ 100=½lþðK=C
p
Þ ꢂ
ð12Þ
due was dissolved in CH
layer was dried over Na
duced pressure. The crude product was purified by silica-gel column
2
Cl
SO
2
and washed by water and brine. The organic
4
, and the solvent was evaporated under re-
2
in which P(C
centration (C
p
) and K form a hemolysis curve for a given polymer con-
), and HC50, respectively. K and n are variable parameters
p
1
chromatography (EtOAc/hexane 1:1) to give product 2. H NMR
in the curve fitting. The polymers with a low percentage of hydrophobic
groups displayed relatively low hemolytic activity, and provided HC50
values around or larger than the highest polymer concentration used
here (1 mgmL ), which is not presented in Figure 3 because the limited
data points did not present reliable intra- or extrapolate estimation.
(
500 MHz, CDCl
3
): d=8.57 (d, 1H), 8.28 (m, 2H), 7.59 (t, 1H), 7.53 (t,
1
H), 7.21 (d, 1H), 5.13 (brm, 1H), 3.10 (m, 2H), 2.90 (s, 6H), 2.53 ppm
+
(
3
m, 2H); HRMS (ESI): calcd for C14
10.0805.
H
18
N
2
O
4
S
2
: 310.0810 [M] ; found:
ꢁ1
Synthesis of copolymers labeled with a dansyl group: Cationic homo-/co-
polymers labeled with a dansyl group at the polymer end were prepared
by the procedure used for the synthesis of copolymers previously de-
scribed. The monomers (0, 30, 50 mol% of butyl methacrylate relative to
total amount of monomers, 0.161 mmol) were polymerized by using
AIBN in the presence of a chain-transfer agent 2 with a dansy group
Under these assay conditions, the HC50 of melittin was 1.25ꢀ
ꢁ
1
0.64 mgmL (n=10). The HC50 reported is the average value of at least
two independent experiments, except the data for C
only one experiment was performed.
2
-2.9(2), for which
Steady-state fluorescence spectroscopy: Fluorescence spectra and intensi-
ty measurements were recorded by using an Aviv Automated Titrating
Differential/Ratio Spectrofluorometer (Aviv Biomedical, Lakewood, NJ).
For liposome-binding experiments, the excitation and emission bandpass
were both set to 2 nm, whereas for emission-spectrum measurements, the
excitation bandpass was set to 3 nm and the emission bandpass was set to
1.5 nm. For liposome-binding experiments the excitation and emission
wavelengths were 353 and 500 nm, respectively, for dansyl-labeled poly-
mers. Dansyl-labeled-polymer fluorescence-emission spectra were record-
ed by using an excitation wavelength of 353 nm and monitoring the emis-
sion spectra from 450–600 nm. All experiments were performed in 1ꢁ1-
cm quartz semi-microcuvettes with constant stirring by a magnetic stir-
flea. Background intensities or spectra were subtracted from all samples.
(
5 mg, 0.016 mmol). The resultant polymers were treated by TFA to
remove Boc groups. After removing TFA by N gas perfusion, the oily
2
residue was dissolved in a small amount of methanol and added into di-
ethyl ether. The resultant precipitate was collected by centrifugation and
1
lyophilized to give cationic copolymers as a white powder. H NMR anal-
ysis provided determination of the degree of polymerization (DP) and
1
the mole fraction of alkyl methacrylate, fHB, of the polymers. H NMR
(
500 MHz, CD
3
OD): for the representative polymer (fHB =49 and DP=
1
7
2
1.8): d=8.62 (brs, 1H), 8.35 (brs, 1H), 8.24 (brs, 1H), 7.63 (brs, 2H),
.32 (brs, 1H), 3.67 (brs, 12.07H), 4.02 (brs, 11.53H), 3.01 (brs, 1.46H),
.92 (s, 6H), 2.3–1.8 (brm, 20.32H), 1.8–0.8 ppm (brm, 85.73H).
Liposome preparation: Lipids were purchased from Avanti Polar Lipids,
Inc. (Alabaster, Al) and used without further purification. Liposomes
were composed of either 100% 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-
phocholine (POPC); POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phos-
pho-rac-(1-glycerol)] (POPG) (8:2 mol/mol); 1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphoethanolamine (POPE) and POPC (8:2 mol/mol); or
POPE and POPG (8:2 mol/mol). Lipids dissolved in chloroform were
Antimicrobial testing: A protocol for an antimicrobial test was reported
[
40]
previously.
A bacterial strain, Escherichia coli D31 (ampicillin- and
streptomycin-resistant) was grown in Mueller–Hinton broth (MH broth)
at 378C overnight. Turbidity of the E. coli culture was measured as opti-
cal density at l=600 nm (OD600) in a 1-mL plastic disposable cuvette (1-
cm path length) using an Eppendorf BioPhotometer. This cell culture
was diluted with MH broth to give a cell suspension (20 mL) with
OD600 =0.1, which was incubated at 378C for 1.5 h. Healthy cell growth
was confirmed by measuring OD600 (valued between 0.5 and 0.6). This
cell suspension was diluted to give a bacterial stock solution with
OD600 =0.001, and this E. coli stock (90 mL) was added to each well in a
mixed in the appropriate ratios and first dried under a stream of N
further dessicated under vacuum for at least 3 h to remove any remaining
chloroform. The dried lipid film was then stored under N
at ꢁ208C until
used. To form liposomes, the lipid film was rehydrated in HBS buffer
10 mm HEPES, 100 mm NaCl, pH 7.1) to obtain a final lipid concentra-
2
, then
2
(
9
6-well, sterile assay palate (Costar Clear Polystyrene 3370, Corning).
tion of 10 mm (a final volume of 0.5 mL was typically used). The sample
was vortexed for at least 3 min to ensure complete resuspension of the
lipid film. The sample was then subjected to five rounds of freezing in a
dry-ice/acetone bath and thawing at 378C in a heated water bath. The
sample was then passed 21 times through a Liposofast extruder (Avestin,
Inc., Ottowa, Canada) with two stacked polycarbonate membranes, each
with a pore size of 400 nm. POPC and POPC/POPG liposomes were
stored at 48C and used for up to 1 week after preparation, whereas
POPE/POPG and POPE/POPC liposomes were used immediately after
extrusion.
The assay plate was incubated at 378C for 18 h without shaking. Bacterial
growth was detected at OD595 using ThermoLabsystems Multiskan Spec-
trum and was compared to that of MH broth without copolymers and E.
coli. MIC was defined as the lowest polymer concentration to completely
inhibit bacterial growth in at least two samples of triplicate measure-
ments.
[
40]
Hemolysis assay: A hemolysis-assay protocol was reported previously.
Human red blood cells (RBCs) were obtained by centrifuging a whole-
blood sample and removing plasma and white blood cells. The RBCs
(
1
1 mL) were diluted with TBS buffer (9 mL, 10 mm Tris buffer, pH 7.0,
50 mm NaCl) and this suspension (0.75 mL) was further diluted by TBS
buffer (33 mL) to give a RBC stock suspension. This RBC stock (90 mL),
Polymer-binding assay: Polymer binding to liposomes was assayed by
monitoring changes in the emission spectrum of dansyl. An aliquot of po-
lymer from a concentrated stock in water was added to HBS buffer to a
final concentration of 2 mm polymer in 800 L, and was allowed to equili-
brate for 5 min at which point the dansyl fluorescence-emission intensity
was recorded. The sample was then titrated with aliquots of liposomes
(typically 1–5 mL), allowed to equilibrate for 5 min with constant mixing
by means of a microstir bar, and fluorescence emission was recorded
again. Intensities were corrected for dilution and for the inner filter
effect at the highest lipid concentrations. For binding-constant analysis,
fluorescence-emission data were fit to a single site-binding isotherm
[Eq. (13)] by using the KaleidaGraph software package:
ꢁ
1
and the polymer stock solutions (10 mL, [polymer]=10–0.0003 mgmL
were added to a 96-well, round-bottomed microplate and incubated at
78C for 1 h with shaking. The range of final polymer concentrations in
)
3
ꢁ
1
ꢁ1
the microplate was 1 mgmL –0.03 mgmL . The microplate was centri-
fuged at 4000 ppm for 5 min. Supernatant (30 mL) was diluted with TBS
buffer (100 mL), and OD414 of the solution was measured as hemoglobin
concentration. Melittin was used as a positive control, and the most con-
centrated sample (100 mgmL ) was used as a reference for 100% hemol-
ysis. Percentage of hemolysis (P) was calculated from Equation (11):
ꢁ
1
1132
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 1123 – 1133

Polymethacrylate Derivatives
FULL PAPER
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
[23] D. H. Liu, S. Choi, B. Chen, R. J. Doerksen, D. J. Clements, J. D.
K
d
00þ½Pꢂ
T
þ ½Lꢂ
T
=nꢁ ðK
d
00þ½Pꢂ
T
þ½Lꢂ
T
=nÞ ꢁ4½Pꢂ
T
½Lꢂ
T
=n
F ¼ F þDF
0
2
½Pꢂ
T
Winkler, M. L. Klein, W. F. DeGrado, Angew. Chem. 2004, 116,
1
178–1182; Angew. Chem. Int. Ed. 2004, 43, 1158–1162.
24] L. Arnt, K. Nusslein, G. N. Tew, J. Polym. Sci. Polym. Chem. Ed.
004, 42, 3860–3864.
25] K. Nusslein, L. Arnt, J. Rennie, C. Owens, G. N. Tew, Microbiology-
Sgm 2006, 152, 1913–1918.
ð13Þ
[
[
2
in which [P]
T T
and [L] are total polymer and lipid concentrations, respec-
tively, F is fluorescence intensity at [L]=0, and n is the number of lipids
per polymer-binding site, which was held fixed at n=6. This was chosen
to allow good data fitting for all tested polymers, but similarly good fits
0
[
[
26] T. Tashiro, Macromol. Mater. Eng. 2001, 286, 63–87.
27] G. J. Gabriel, A. Som, A. E. Madkour, T. Eren, G. N. Tew, Mater.
Sci. Eng. R 2007, 57, 28–64.
were obtained using n=4–9. DF is defined as DF=FinfꢁF
0
, in which Finf
[
[
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Published online: December 15, 2008
Chem. Eur. J. 2009, 15, 1123 – 1133
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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