Amphiphilic polymethacrylate derivatives as antimicrobial agents

Article abstract of DOI:10.1021/ja044205+

We have investigated the structure-activity relationship of cationic amphiphilic polymethacrylate derivatives in antimicrobial and hemolytic assays. The polymers were prepared by radical copolymerizations of N-(tert-butoxycarbonyl)aminoethyl methacrylate and butyl methacrylate in the presence of methyl 3-mercaptopropionate as a chain transfer agent to give precursor polymers protected with a tert-butoxycarbonyl (Boc) group. Subsequent treatment of the Boc-protected polymers with TFA affords the desired cationic random copolymers. We examined antimicrobial and hemolytic activities of a series of polymers having a wide range of mole percentage of butyl groups (0-60%) in three different molecular weight (MW) ranges. The smallest polymers (MW < 2000) showed the lowest MIC and reduced hemolytic activity compared to that of the higher MW ones. In addition, polymers containing a high percentage of butyl groups are less selective for bacterial cells than their less hydrophobic counterparts. Copyright

Full text of DOI:10.1021/ja044205+

Published on Web 03/03/2005
Amphiphilic Polymethacrylate Derivatives as Antimicrobial Agents
Kenichi Kuroda and William F. DeGrado*
Department of Biochemistry and Biophysics, and Department of Chemistry, UniVersity of PennsylVania,
Philadelphia, PennsylVania 19104
Received September 23, 2004; E-mail: wdegrado@mail.med.upenn.edu
Scheme 1. Synthesis of Amphiphilic Polymethacrylate
The membrane-disrupting ability of amphiphilic synthetic poly-
mers has been utilized in preparing chemical disinfectants and
biocides.1 A number of polymeric disinfectants have been prepared
using conventional synthetic polymers, including poly(vinyl py-
ridine)s,2 poly(vinyl alcohol)s,3 polyacrylates,4 and polystyrenes.5
Their amphiphilic structures disrupt cell membranes,1 causing
breakdown of the transmembrane potential, leakage of cytoplasmic
contents, and ultimately cell death. The cooperative action inherent
in polymeric structures enhances this disruption mechanism as
compared to small amphiphilic molecules, such as surfactants.6
However, one of the major drawbacks of polymeric disinfectants
is a lack of selectivity for bacterial over human cells, limiting their
clinical and medicinal utility.
Derivativesa
a Conditions: (i) methyl 3-mercaptopropionate, AIBN, acetonitrile, 60
°C, overnight; (ii) neat TFA, rt, 1 h.
Table 1. Characterization of Amphiphilic Polymethacrylates
b
series
[MMP]/[monomers]a
MPBu
DPb
MW rangec
1
2
3
0.05
0.10
0.50
0-57
0-53
0-47
32-46
19-31
5-9
7900-10100
4500-6000
1300-1900
In this report, we take steps toward the development of nontoxic
antimicrobial synthetic polymers in our investigation of the
structure-activity relationship of amphiphilic polymethacrylate
derivatives as measured via antimicrobial and hemolytic activities.
Free radical copolymerizations of N-(tert-butoxycarbonyl)amino-
ethyl methacrylate and butyl methacrylate (BMA) were conducted
using mole percentages of BMA from 0 to 60% in the presence of
methyl 3-mercaptopropionate (MMP) to give a precursor polymer
protected with tert-butoxycarbonyl (Boc) groups (Scheme 1). In
this polymerization, MMP served as a chain transfer agent to control
the degree of polymerization (DP)7 and allowed us to prepare low
molecular weight (MW) polymers with relatively high yields,
avoiding the necessity of time-intensive fractionation of polymers
by column chromatography. The subsequent treatment of the Boc-
protected polymer with TFA afforded the desired cationic random
copolymer. Natural host defense peptides are believed to selectively
target bacterial cells while remaining nontoxic to the host due to
preferable charge interactions between the dense population of
negatively charged lipids on bacterial cell surfaces and the cationic
side chains of the peptides;8,9 we have similarly incorporated
cationic functionality into the polymer framework to improve
selectivity. By alternating MMP concentrations, we obtained a series
of polymers of three different MW ranges displaying a wide range
of mole percentages of BMA (MPBu) (0-60%) (Table 1).10
Antimicrobial activity of the polymers was tested using turbidity-
based assays in Mueller-Hinton broth with Escherichia coli D31,
and the minimum inhibitory concentration (MIC) was determined
as the lowest polymer concentration to completely inhibit bacterial
growth after an 18 h incubation period at 37 °C. The MICs for all
series decreased as MPBu increased up to ∼30%, at which point
further increase of the butyl functionality did not affect the MIC
(Figure 1). The smallest polymer series, 3, further demonstrated
the lowest MIC of 16 µg/mL above ∼30% MPBu, which is
comparable with that of the bee venom toxin peptide, melittin (12.5
µg/mL). The increased activity with increasing MPBu suggests that
the hydrophobicity of polymers is a major consideration in the
antimicrobial action of polymers. As the polymers become more
hydrophobic, incorporation of polymers to lipid membranes is
a Concentration ratio of MMP to total amount of monomers in poly-
merizations. b Determined by 1H NMR. c Calculated from MPBu and DP.
Figure 1. Antimicrobial and hemolytic activities of polymethacrylate
derivatives: (A) polymer series 1; (B) 2; and (C) 3. MICs and HC50s
presented as opened and closed marks, respectively. (D) Selectivity index
(HC50/MIC).
enhanced, and thus the integrity of membrane is more efficiently
disrupted. However, further increases in MPBu create hydrophobic
polymers more likely to undergo a collapse of the polymer chain
in water or irreversible aggregation with components of the assay
medium, preventing antimicrobial action. It has also been reported
that aggregation of antimicrobial peptides reduces their antimicrobial
potency.11
To examine the effect of increasing hydrophobicity on the
solution properties of the polymers, their solubility in the assay
medium was measured using the growth media in the absence of
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C O M M U N I C A T I O N S
bacteria.10 The solubility limits of series 1 and 2 decreased from
60 to 16 µg/mL as MPBu increased from 20 to 60%. In contrast,
polymer series 3 is soluble in the medium up to at least 60 µg/mL
as MPBu varies from 0 to 50%. We speculate that the low molecular
weight of polymer series 3, coupled with its high solubility, not
only increases the molar concentration (the number of molecules
per unit volume) for any given weight concentration but also
promotes the availability of the polymers to act on the bacterial
inner membrane and cause cell death due to enhanced permeation
of the polymers through bacterial peptidoglycan layers.
peptides acquire selectivity in their antimicrobial activity.14,15 While
this seems to be a promising approach for the preparation of
nontoxic antimicrobial polymers, we demonstrate in this report that
copolymers consisting of flexible polymer backbones and random
amphiphilic sequences show antimicrobial activity comparable to
that of natural peptides and relatively reduced toxicity compared
to that of high MW polymers and the toxin melittin. Our results
suggest that preorganized facial amphiphilicity is not necessarily
required for antimicrobial activity in polymers, suggesting that the
polymer interface can induce an amphiphilic conformation in a large
enough population of the polymers to provide a potent antimicrobial
effect. Shai and co-workers have come to a similar conclusion
through the examination of diastereomeric peptides.12
In summary, antimicrobial and hemolytic activities of amphiphilic
polymethacrylate derivatives can be tailored by alternating the
content of hydrophobic groups and molecular weights. This class
of synthetic polymers is inexpensive and easy to prepare, allowing
the production of antimicrobial materials on industrial scales.
Determination of the specific elements in polymers, which affect
their biological activity, however, has previously been difficult due
to the broad molecular weight distributions and random sequences
characteristic of radical polymerization. Utilization of controlled
polymerization methods16 to produce well-defined polymers and
selection of amphiphilic block or alternating polymer structures will
provide us with greater insight into their antimicrobial mechanism.
Important features of polymers useful as disinfectants are not
only their antimicrobial activity but also the lack of toxicity to
human cells, particularly for medical and clinical utility. Toward
this end, their lytic activity against human red blood cells (hemolytic
activity) was evaluated as HC50 for each series of polymers, which
is the polymer concentration necessary for 50% lysis of cells. In
each series, the HC50 for the polymers decreased as MPBu increased
(Figure 1). In the high MPBu region (30-60%), the HC50 of the
high MW polymers (series 1 and 2) reached a plateau of <1 µg/
mL, which is lower than that of melittin (1.24 µg/mL) and likely
the minimum value (maximum toxicity) of the series of cationic
random copolymers studied here. In contrast, the HC50 values for
series 3 decrease monotonically with increasing MPBu and are 1
order of magnitude higher relative to those of larger MW polymer
series 1 and 2 for the same MPBu. This result provides a window
of efficacy from 10 to 30% MPBu in which series 3 is selectively
toxic to bacterial cells with a maximum selectivity (HC50/MIC) of
3 at 17% MPBu (Figure 1D). Previous studies on antimicrobial
peptides showed that a lack of selectivity arises when hydrophobic
lipid-peptide interactions overcome the electrostatic attraction to
the bacterial cell surface.9 Consistent with these studies, series 3
polymers showed selectivity (HC50 > MIC) in the low MPBu region,
where the polymers are less hydrophobic. One conclusion that could
be drawn from this result is that decreasing MPBu provides greater
selectivity for bacterial over human cells. However, in contrast to
the limited solubility of the polymers in the antimicrobial assay
medium, the polymers are highly soluble in the hemolysis assay
buffer (TBS ) Tris-buffered saline) up to at least 500 µg/mL at all
values of MPBu (0-60%), which may increase the number of
polymers able to interact with cell membranes. The solubility
difference observed in the assay media for the two experiments
indicates not only that the interactions of polymers with lipids must
be considered but also that environmental interferences and solution
properties (conformations) of polymers must also be evaluated to
correctly interpret the biological activities detected. Previous studies
on peptides and synthetic polymers proposed models of membrane
disruption mechanisms, in which polymer conformation and ag-
gregation play an important role in pore formation on mem-
branes.9,12,13 We are currently screening a variety of polymer
backbones and substituents to better understand the interplay of
these factors on the antimicrobial activities of polymers and the
mode of antimicrobial action.
Acknowledgment. We thank NIH for financial support.
Supporting Information Available: Synthesis of copolymers,
polymer characterization, solubility data in assay media, and assay
protocols. This material is available free of charge via the Internet at
http://pubs.acs.org.
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