Biomacromolecules
Article
the use of these natural peptides as they are expensive to
develop and produce in large quantities and are also subject to
proteolysis, which reduces their long-term stability in biological
environments.
water,53,54 and can indicate a preference for lipid-like
membranes.
Inspired by Kuroda’s work on binary copolymers,30 this
study aimed to evaluate the effect of the hydrophobic group of
ternary copolymers and develop a predictive tool for screening
the bioactivity of ternary copolymers prior to polymer
preparation. To accomplish this goal, calculated log P values
(C log P) of oligomeric models were utilized as a measure of
the amphiphilic balance by accounting for the hydrophobic
contribution of the polymer backbone as well as features of the
side chains such as hybridization, branching, and number of
carbon atoms. Using a library of eight hydrophobic monomers,
we synthesized a library of 36 statistical amphiphilic ternary
copolymers via reversible addition−fragmentation chain trans-
fer (RAFT) polymerization55,56 to systematically evaluate the
copolymer composition, degree of polymerization (DPn),
hydrophobic monomer carbon length (5, 7, and 9 carbons),
and chain type (cyclic, aromatic, linear, or branched) of the
hydrophobic monomer on antimicrobial and hemolytic
activity. Combining the data from this study with previous
work,33,57,58 an optimal C log P window for the prediction of
antimicrobial activity and biocompatibility for ternary copoly-
mers was determined.
Inspired by the structure of these peptides and thanks to
advancements in polymer chemistry, particularly controlled/
living polymerization, antimicrobial polymers have been
proposed as potential alternatives.6−13,20,22−26 Unlike their
peptide counterparts, these antimicrobial polymers can be
produced on a large scale and are less susceptible to
proteolysis. These advantages have led to a number of
statistical amphiphilic antimicrobial polymers being prepared
from a variety of monomers, such as (meth)acrylate,20,27−31
acrylamide,32,33 3-aminopropanoic acid,34,35 norbornene,36,37
phenyleneethynylene,38 maleimide,39 quaternary vinylpyri-
dine,40 urea,41 and oxetane.42 Although these polymers exhibit
high antimicrobial activity, they are often not specific to
bacterial cells and, therefore, kill mammalian and bacterial cells
without discrimination, resulting in high toxicity for the host
and reducing their potential applications. To overcome this
major limitation, researchers have tried to identify key factors
that allow reduction in their toxicity against mammalian cells
without affecting their antimicrobial performance.43,44 For
instance, the molecular weight of these polymers influences
their biocompatibility. An increase in molecular weight often
results in an increase in hemolytic activity but their
antimicrobial activity is not significantly impacted. More
importantly, the composition of these copolymers, such as
their amphiphilic balance,45,46 has a more significant impact on
their biocompatibility and antimicrobial activity.47−50 To
control the amphiphilic balance, these polymers are prepared
by copolymerization of hydrophobic and cationic monomers
and, in some instances, the inclusion of some hydrophilic
monomers. The number and type of cationic groups, as well as
the type of hydrophobic monomers, employed for their
syntheses exhibit a significant impact on their selectivity
toward bacterial cells.51 As the cationic groups are the ones
that facilitate the adsorption of polymers on the anionic
bacterial membrane via electrostatic interaction, different
cationic groups have been investigated to gain insight into
the structural effect of these components on overall
bioactivity.16,18,20 One of the most common choices for
antimicrobial cationic monomers is monomers functionalized
with an amino group, including primary, secondary, tertiary,
and quaternary groups. Judzewitsch33 and Palermo31 demon-
strated that amphiphilic copolymers containing primary amines
display high antimicrobial activity against Gram-negative
bacteria, whereas those containing quaternary ammonium
groups are more efficient against mycobacteria (Mycobacterium
smegmatis).33 Ragogna, Gillies, and co-workers proposed the
introduction of phosphonium groups as alternatives to amino
groups.7,8 As the hydrophobic groups disrupt bacterial and
mammalian membranes, a large range of hydrophobic
monomers have been tested to improve selectivity.52 For
instance, Kuroda and co-workers systematically investigated
the impact of hydrophobic groups of binary copolymers on
hemolysis and found that hemolytic activity increased as the
hydrophobicity increased. To rationalize this effect, Kur-
oda30,47 estimated partition coefficients (i.e., log P) by
counting the number of carbon atoms in the side chains and
found that high hemolytic activity was associated with a high
log P value. Effectively, log P characterizes the hydrophobicity
of a molecule using two immiscible layers, n-octanol and
MATERIALS AND METHODS
■
Materials. Ethylenediamine (Sigma-Aldrich, ≥99%), amylamine
(Sigma-Aldrich), isopentylamine (Sigma-Aldrich, 99%), heptylamine
(Sigma-Aldrich, 99%), N-propylbutylamine (Sigma-Aldrich, 98%),
cycloheptylamine (Sigma-Aldrich, 99%), cyclohexanemethylamine
(Sigma-Aldrich, 98%), nonylamine (Sigma-Aldrich, 98%), di-tert-
butyl dicarbonate (Sigma-Aldrich, 99%), acryloyl chloride (Merck,
≥96%), N-hydroxyethyl acrylamide (HEAm) (Sigma-Aldrich, 97%),
triethylamine (TEA) (Scharlau, 99%), trifluoroacetic acid (TFA)
(Sigma-Aldrich, 99%), RAFT agent (2-(n-butyltrithiocarbonate)-
propionic acid (BTPA), chloroform (Merck), dichloromethane
(DCM) (Merck), tetrahydrofuran (THF) (Merck), diethyl ether,
(Merck), hexane (Merck), dimethyl sulfoxide (DMSO) (Merck),
N,N′-dimethylacetamide (DMAc) (Sigma-Aldrich), and 5,10,15,20-
tetraphenyl-21H,23H-porphine zinc (ZnTPP) (Sigma-Aldrich) were
used as received. Deionized (DI) water was produced by a Milli-Q
water purification system and had a resistivity of 17.9 mΩ/cm.
Synthesis of Monomers. Synthesis of Cationic Monomer: tert-
Butyl (2-Acrylamidoethyl) Carbamate. tert-Butyl (2-
acrylamidoethyl)carbamate (Boc-AEAm) was prepared according to
a previously reported procedure.58 Ethylenediamine (0.33 mol) was
dissolved in chloroform (400 mL). 0.03 mol of di-tert-butyl
dicarbonate dissolved in 100 mL of chloroform was added dropwise
to this solution over 4 h at 0 °C while stirring and then the reaction
was continued overnight at room temperature. After filtering the
white precipitate, the organic phase was washed with 200 mL of DI
water six times and then dried using MgSO4. Solids were separated by
filtration, and the chloroform was evaporated resulting in a pale-
yellow oil product, which was used in the next step.
THF (100 mL) was added to dissolve the obtained oil. TEA (1.2
equiv) and acryloyl chloride (1.1 equiv) were added dropwise to the
solution at 0 °C with N2 bubbling. The reaction mixture was then
stirred at room temperature for 2 h. THF was then removed by rotary
evaporation. The crude product was then dissolved in chloroform
(150 mL) and washed against 0.1 M HCl solution (1 × 75 mL),
saturated NaHCO3 (1 × 75 mL), brine (1 × 75 mL), and DI water (1
× 75 mL). The organic phase was dried using MgSO4 and filtered,
and the remaining solvent was removed by rotary evaporation. The
product was further purified by repeated precipitation steps in hexane
to yield the Boc-protected monomer as a fine white powder, which
was dried in vacuo. The yield for the monomer was 38 mol %.
Synthesis of Hydrophobic Monomers. A standard procedure was
employed for the synthesis of eight hydrophobic monomers (N-
B
Biomacromolecules XXXX, XXX, XXX−XXX