Modulating Antimicrobial Activity and Mammalian Cell Biocompatibility with Glucosamine-Functionalized Star Polymers

Article abstract of DOI:10.1021/acs.biomac.5b01766

The development of novel reagents and antibiotics for combating multidrug resistance bacteria has received significant attention in recent years. In this study, new antimicrobial star polymers (14-26 nm in diameter) that consist of mixtures of polylysine and glycopolymer arms were developed and were shown to possess antimicrobial efficacy toward Gram positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) (with MIC values as low as 16 μ mL^-1) while being non-hemolytic (HC₅₀ > 10 000 μ mL^-1) and exhibit excellent mammalian cell biocompatibility. Structure function analysis indicated that the antimicrobial activity and mammalian cell biocompatibility of the star nanoparticles could be optimized by modifying the molar ratio of polylysine to glycopolymers arms. The technology described herein thus represents an innovative approach that could be used to fight deadly infectious diseases.

Full text of DOI:10.1021/acs.biomac.5b01766

Article
pubs.acs.org/Biomac
Modulating Antimicrobial Activity and Mammalian Cell
Biocompatibility with Glucosamine-Functionalized Star Polymers
Edgar H. H. Wong,*,†,‡,⊥ Mya Mya Khin,†,‡ Vikashini Ravikumar,§ Zhangyong Si,†,‡ Scott A. Rice,§,∥
and Mary B. Chan-Park*,†,‡
†School of Chemical and Biomedical Engineering, and ‡Centre for Antimicrobial Bioengineering, Nanyang Technological University,
Singapore 637459
∥
§The Singapore Centre for Environmental Life Sciences Engineering, and School of Biological Sciences, Nanyang Technological
University, Singapore 637551
S
* Supporting Information
ABSTRACT: The development of novel reagents and
antibiotics for combating multidrug resistance bacteria has
received significant attention in recent years. In this study, new
antimicrobial star polymers (14−26 nm in diameter) that
consist of mixtures of polylysine and glycopolymer arms were
developed and were shown to possess antimicrobial efficacy
toward Gram positive bacteria including methicillin-resistant
Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterococcus (VRE) (with MIC values as low as 16 μg
mL−1) while being non-hemolytic (HC50 > 10 000 μg mL−1)
and exhibit excellent mammalian cell biocompatibility. Structure function analysis indicated that the antimicrobial activity and
mammalian cell biocompatibility of the star nanoparticles could be optimized by modifying the molar ratio of polylysine to
glycopolymers arms. The technology described herein thus represents an innovative approach that could be used to fight deadly
infectious diseases.
An alternative approach in the development of new
antimicrobial agents lies in the use of synthetic polymer
chemistry. Over the last few decades, advances in controlled
polymerization have led to a paradigm shift in the widespread
application of well-defined synthetic biomaterials using func-
tional macromolecules and nanoparticles produced by
techniques such as (N-carboxyanhydride (NCA)) ring-opening
polymerization (ROP)7,8 and reversible addition−fragmenta-
tion chain transfer (RAFT) polymerization9 which have found
their way into various applications, including drug or gene
delivery systems,10−13 tissue engineering,14,15 and biosens-
ing.16,17 The ability to efficiently tune the chemical and physical
characteristics (such as the functionality, size, and shape), and
ultimately the biological properties, of the bionanomaterial by
simply controlling the polymerization feed compositions and
reaction conditions makes polymeric systems highly attractive
and applicable in the biomedical field. There are various types
of antimicrobial polymer systems that have been previously
developed,18−22 mostly mimicking the amphiphilic feature of
AMPs. While these nanomaterials have excellent antimicrobial
activity, most are inherently cytotoxic toward human cells, as
was observed, which limits their potential clinical application.
Thus, it is essential that the antimicrobial agent not only is
INTRODUCTION
■
The rise of multidrug resistance in bacteria is now considered a
critical healthcare issue worldwide, and the development of
alternative antimicrobial agents that kill bacteria by new
mechanisms is of paramount importance for overcoming this
global healthcare challenge. Hospital-acquired infections such
as those caused by Gram positive pathogens, including the
methicillin-resistant Staphylococcus aureus (MRSA) and vanco-
mycin-resistant Enterococcus (VRE), have resulted in an
estimated 23 000 deaths annually in the United States alone.1
In Europe, MRSA infections affect more than 150 000 patients
annually, incurring extra in-hospitable costs worth 380 million
EUR per year.2 Several strategies have been exploited in recent
years to identify more effective alternative antimicrobial agents,
including the development of the iChip3 (isolation chip) which
allows the screening of soil microorganisms that were
previously “unculturable” in the laboratory. The use of this
technology led to the discovery of teixobactin,4 a highly potent
antibiotic in fighting Gram positive pathogens such as MRSA
by inhibiting the bacteria cell wall synthesis. In addition,
antimicrobial peptides (AMPs), which exert their bactericidal
properties by physically damaging the bacterial cytoplasmic
membrane instead of targeting intracellular targets, have been
shown to be effective against a wide spectrum of bacteria.5,6
However, the synthesis of AMPs usually involves laborious
multistep procedures. In addition, AMPs are generally known
to be hemolytic, thus limiting their application.
Received: December 30, 2015
Revised: February 9, 2016
Published: February 9, 2016
© 2016 American Chemical Society
1170
DOI: 10.1021/acs.biomac.5b01766
Biomacromolecules 2016, 17, 1170−1178

Biomacromolecules
Article
using DMF (with 1 wt % LiBr) as the eluent at a flow rate of 1 mL
min−1. Dynamic light scattering (DLS) and zeta potential measure-
ments were measured using a Malvern Zetasizer Nano ZS apparatus
equipped with a He−Ne laser operated at 633 nm. All samples were
measured at a polymer concentration of 1−3 mg mL−1 and at a
scattering angle of 173°.
effective in combating pathogens but also must demonstrate
minimal toxicity toward human cells.
Herein, the development of a new antimicrobial agent with
good biocompatibility in the form of glucosamine-function-
alized star polymers is described. This star polymer, which is
comprised of polylysine and polyglucosamine-based arms
radiating from a central cross-linked core, was generated
using a combination of various modern synthetic polymer
chemistry protocols including RAFT polymerization,9 NCA-
ROP,7 and click chemistry.23−25 The compounds produced
exhibited selective killing of Gram positive over Gram negative
pathogens, were non-hemolytic and exhibited low cytotoxicity
toward mammalian cells. The rationale behind the design of
this nanoparticle is based on the ability of polylysine to induce
bacteria death, whereas the glycopolymer may provide
biocompatibility to human cells and the capability to infiltrate
the peptidoglycan layer found only in bacteria (because of its
resemblance to the peptidoglycan structure). By adjusting the
chemical composition, specifically the ratio of polylysine to
polyglucosamine-based arms, the antimicrobial activity and
mammalian cell biocompatibility can be modulated. It is
hypothesized that the star architecture offers a distinct
advantage over linear analogues in terms of having better
mammalian cell biocompatibility as the polyglucosamine-based
arms can effectively shield and reduce the propensity of the
cationic polylysine arms from interacting with other cells,
thereby decrease cytotoxicity when the arms are “locked” in a
nanoparticle form. The results from the compounds developed
here suggest that these compounds represent a structure that
can be further modified and optimized for the subsequent
development of antimicrobial agents for clinical applications.
Synthesis of 5-Hexynyl Acrylate, 1. A solution containing 5-
hexyn-1-ol (5.1 g, 50.9 mmol) in THF (200 mL) was degassed with Ar
for 45 min at 0 °C, prior to the sequential addition of triethylamine
(8.5 mL, 61.1 mmol) and acryloyl chloride (4.6 mL, 56.0 mmol). The
reaction mixture was allowed to warm to room temperature, and
stirring was continued for another 5 h. Precipitated urea was filtered
off, and the solvent was removed in vacuo. The contents were
redissolved in EtOAc (150 mL) and washed against 0.1 M HCl
aqueous solution (75 mL × 2), saturated NaHCO3 solution (75 mL ×
2), and brine (75 mL × 2) in the following order. The organic phase
was dehydrated over MgSO4, filtered, and dried in vacuo to give 1 as a
1
pale yellow oil (5.7 g, 37.7 mmol, 74 mol %). H NMR (300 MHz,
CDCl3, 25 °C): δH (ppm) = 6.40−6.36 (d, 1H, CHHCH), 6.13−
6.06 (dd, 1H, CHCH2), 5.81−5.79 (d, 1H, CHHCH), 4.18−4.15
(t, 2H, CH2O−C(O)), 2.24−2.21 (m, 2H, CH2−C ≡ H), 1.95 (s,
1H, −CH), 1.82−1.75 (m, 2H, −CH2−), 1.64−1.57 (m, 2H,
−CH2−). 13C NMR (300 MHz, CDCl3, 25 °C): δC (ppm) = 166.1,
130.5, 128.4, 83.8, 68.7, 63.9, 27.6, 24.9, 18.0.
Synthesis of Acrylate-Functionalized N-Acetyl D-Glucos-
amine Peracetate Monomer, 4. In total, the synthesis of the
acrylate-functionalized N-acetyl D-glucosamine peracetate required
four steps. First, to a 100 mL round-bottom flask containing acetyl
chloride (50 mL) was added N-acetyl-D-glucosamine (10 g) under
strong stirring. The suspension was stirred for 1 day at 25 °C in a
closed system after which the solids were completely dissolved at the
end of the reaction. (Caution! HCl fumes evolve during the reaction.)
CHCl3 (100 mL) was then added to the light brown solution, and the
mixture was poured into ice water (125 mL) under strong stirring. The
organic phase was separated and washed against cold, saturated
NaHCO3 solution (100 mL × 3), dehydrated over MgSO4, filtered,
and concentrated in vacuo. The crude product was purified by silica gel
column chromatography to give 2 as a white solid product (8.2 g, 22.4
mmol, yield = 50 mol %); Rf = 0.286 (hexane/EtOAc = 2/3).
The chloro-functionalized sugar 2 (4.8 g, 13.1 mmol) was dissolved
in DMF (80 mL). Sodium azide (6.4 g, 98.3 mmol) was added to the
solution, and the reaction mixture was stirred at 40 °C for 1 day.
Insoluble salts were filtered off, and the solvent was removed in vacuo.
EtOAc (100 mL) and water (100 mL) were added to the flask and
stirred for 10 min. The organic phase was separated and washed
against water (50 mL × 2), dehydrated over MgSO4, filtered, and dried
in vacuo to give 3 as a white solid product (4.2 g, 11.3 mmol, yield =
87 mol %).
EXPERIMENTAL SECTION
■
Materials. 5-Hexyn-1-ol (Aldrich, 96%), triethylamine (Sigma-
Aldrich, ≥99%), acryloyl chloride (Merck, ≥96%), N-acetyl-D-
glucosamine (Sinopharm Chemical Reagent), acetyl chloride (Aldrich,
98%), sodium azide (Aldrich, ≥99%), copper iodide (CuI) (Sigma-
Aldrich, 98%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDE-
TA) (Aldrich, 99%), anhydrous dimethylformamide (DMF) (Sigma-
Aldrich, 99.8%), anhydrous tetrahydrofuran (THF) (Sigma-Aldrich,
≥99.9%), benzylamine (Aldrich, 99%), ethylenediaminetetraacetic acid
(EDTA) (Sigma-Aldrich, ≥99%), N-(2-hydroxyethyl)acrylamide
(HEAm) (Aldrich, 97%), N,N′-methylenebis(acrylamide) (Aldrich,
97%), trifluoroacetic acid (TFA) (Sigma-Aldrich, 99%), thioanisole
(Sigma-Aldrich, ≥99%), trifluoromethanesulfonic acid (TFMSA) (Alfa
Aesar, 98%), hydrazine hydrate solution (Sigma-Aldrich, 78−82%),
sodium hydrogen carbonate (NaHCO3) (Vetec, 99%), and
magnesium sulfate (MgSO4) (Sigma-Aldrich, ≥97%) were used as
received. Chloroform (CHCl3), hexane, ethyl acetate (EtOAc), and
diethyl ether (DEE) were purchased from Aik Moh Paints and
Chemicals and used as received. Deuterated solvents (CDCl3, DMSO-
d6, and D2O) were obtained from Cambridge Isotope Laboratories and
used as received. Lys NCA monomer,13 N-succinimidyl-5-hexynoate,26
1-((3-azidopropoxy)carbonyl)ethyl butyl carbonotrithioate,27 and
benzyl dodecyl carbonotrithioate28 were synthesized according to
literature procedures. High-purity water with a resistivity of >15 MΩ
cm was obtained from a Merck Millipore Integral 3 water purification
system.
Azido-functionalized sugar 3 (2.9 g, 7.8 mmol) was dissolved in
EtOAc (60 mL), and the solution was cooled to 0 °C in an ice−water
bath and degassed with Ar for 45 min. CuI (300 mg, 1.6 mmol),
PMDETA (651 μL, 3.1 mmol), and the synthesized 5-hexynyl acrylate
1 (1.3 g, 8.6 mmol) were then sequentially added to the solution. The
reaction mixture was stirred at 25 °C under positive Ar atmosphere for
6 h. The solution was then washed with EDTA solution (ca. 0.05 M in
water, 40 mL × 2), followed by water (40 mL × 2). The organic phase
was dehydrated over MgSO4, filtered, and concentrated in vacuo. The
concentrated solution was reprecipitated twice into DEE/hexane 7:3
solvent mixture (100 mL) and dried in vacuo to yield 4 as a white
1
sticky solid (3.5 g, 6.7 mmol, yield =86 mol %). H NMR (300 MHz,
CDCl3, 25 °C): δH (ppm) = 7.67 (s, 1H, triazole), 6.76−6.73 (d, 1H,
amide), 6.41−6.36 (d, 1H, CHHCH), 6.15−6.06 (m, 2H, CH
CH2 and CH−N−NN overlap), 5.83−5.79 (d, 1H, CHHCH),
5.56−5.49 (t, 1H, sugar ring), 5.25−5.18 (t, 1H, sugar ring), 4.60−
4.54 (q, 1H, sugar ring), 4.31−4.04 (m, 5H, CH2O−C(O) and
−CHNH−C(O) overlap), 2.79−2.75 (m, CH2−C(C)N), 2.06
(s, 6H, methyl), 1.75 (s, 6H methyl), 2.06−1.75 (m, 4H,
−CH2CH2−). 13C NMR (300 MHz, CDCl3, 25 °C): δC (ppm) =
170.7, 170.6, 169.4, 166.9, 147.8, 130.8, 128.5, 120.4, 85.9, 74.8, 72.3,
68.1, 64.2, 61.8, 53.3, 28.1, 25.5, 25.1, 22.8, 21.4, 20.7, 20.6.
1
Characterization of Synthetic (Macro)molecules. H and 13C
nuclear magnetic resonance (NMR) spectroscopy was conducted on a
Bruker Avance DPX-300 spectrometer using deuterated solvents
(obtained from Cambridge Isotope Laboratories) as reference solvents
and at a sample concentration of ca. 10−20 mg mL−1. Gel permeation
chromatography (GPC) was carried out on a Shimadzu liquid
chromatography system equipped with a Shimadzu refractive index
detector (RID-10A) and two Polargel columns operating at 40 °C
1171
DOI: 10.1021/acs.biomac.5b01766
Biomacromolecules 2016, 17, 1170−1178

Biomacromolecules
Article
Scheme 1. Synthesis Pathway for the Formation of Glucosamine-Functionalized Star Polymers
Synthesis of ω-RAFT End-Group-Functionalized Linear
Poly(Z-L-lysine). The synthesis of RAFT-functionalized polypeptide
proceeded in two steps via tandem NCA-ROP and copper-mediated
click chemistry. First, Lys NCA monomer (1.35 g, 4.4 mmol) was
dissolved in anhydrous DMF (7 mL) under Ar atmosphere. To this
solution was added benzylamine (24 μL, 0.22 mmol in 2 mL
anhydrous DMF), and the mixture was stirred at 25 °C under positive
Ar atmosphere for 1 day. At the end of the ROP process, N-
succinimidyl-5-hexynoate (260 mg, 2,2 mmol in 4 mL anhydrous
THF) was added into the reaction for the in situ conversion of
terminal amines to alkyne groups. The reaction mixture was stirred for
another day prior to repeated precipitation in DEE (100 mL). The
alkyne-functionalized polymer was dried in vacuo and used directly in
the next step (1.21 g, yield = 90 wt %); GPC-DRI (DMF): Mn = 3800
g mol−1, Đ = 1.5.
The alkyne-terminated peptide (840 mg, 0.21 mmol yne) and azido
RAFT agent, 1-((3-azidopropoxy)carbonyl)ethyl butyl carbonotri-
thioate (202 mg, 0.63 mmol), were dissolved in DMF (10 mL), and
the solution was purged with Ar for 30 min prior to the addition of
CuI (16 mg, 0.08 mmol) and PMDETA (35 μL, 0.16 mmol). The
solution was stirred for 3 h at 25 °C and under positive Ar atmosphere.
The polymer was then precipitated into DEE (100 mL) and the excess
RAFT agent remained in the supernatant. The polymer was
redissolved in CHCl3 (40 mL) and washed with EDTA solution (ca.
0.05 M in water, 40 mL × 2), followed by water (40 mL × 2). The
organic phase was dehydrated over MgSO4, filtered, and concentrated
in vacuo. The concentrated solution was reprecipitated twice into DEE
(100 mL) and dried in vacuo to yield the RAFT-functionalized
poly(Z-L-lysine) as a yellow solid (620 mg, 69 wt %); GPC-DRI
(DMF): Mn = 4100 g mol−1, Đ = 1.6.
General Linear Polymer Formation via RAFT-Based Photo-
polymerization. Typically, the (macro)RAFT agent and monomer
were dissolved with DMSO in a Schlenk tube where the monomer to
solvent (wt/vol) ratio is 1:5. The solution was subjected to three
freeze−pump−thaw cycles and last backfilled with Ar. The Schlenk
tube was then placed in the middle of a self-constructed photoreactor
and irradiated for a day. (Near) full monomer conversion (>90%) was
attained in all cases. The LED light source was a commercial strip
lighting (300 LEDs, 60W total).
General Core Cross-Linked Star Polymer Formation via
RAFT-Based Photopolymerization. In a similar fashion to the
synthesis of linear polymers, the preformed macroinitiators and cross-
linker N,N′-methylenebis(acrylamide) (15 mol eq with respect to the
RAFT group) were dissolved with DMSO in a Schlenk tube where the
macroinitiator to solvent (wt/vol) ratio is 1:8. The solution was
subjected to three freeze−pump−thaw cycles and last backfilled with
Ar. The Schlenk tube was then placed in the middle of the
photoreactor and irradiated for 2 days. Full conversion of the vinyl
groups was observed. The crude sample was reprecipitated twice into
DEE and dried in vacuo prior to the removal of protecting groups.
General Procedure for Removal of Protecting Groups. The
carboxybenzyl groups were first removed, followed by the acetyl
groups. Generally, the polymer (ca. 3 wt %) was dissolved in TFA
followed by the addition of thioanisole and TFMSA (5 mol eq each
with respect to Cbz). The solution was stirred at 0 °C for 2 h. The
contents were reprecipitated thrice into DEE, washed with DEE, and
dried in vacuo prior to the next step. For the removal of acetyl groups,
the polymer (ca. 2 wt %) was dissolved in DMSO followed by the
addition of hydrazine (11 mol eq with respect to the acetyl group) and
the solution was stirred at 25 °C for 12 h. The polymer was
reprecipitated twice into DEE, dried in vacuo, redissolved in water, and
further purified using a Vivaspin Centrifugal Concentrator (molecular
weight cutoff 3 kDa). The purified solution was then lyophilized to
yield a white fluffy product.
Mammalian Cell Biocompatibility Studies. The mammalian
cell biocompatibility study was carried out with human aortic smooth
muscle cells (AoSMC, CC-2571 Lonza). Mammalian cells were
cultured in 96-well plates from initial inocula of 1 × 105 cells in each
well. A graded concentration series of polymer solutions in culture
medium was prepared, 200 μL of which was added to the cell cultures.
The cells were incubated at 37 °C for 24 h, then the culture medium
containing polymer was removed, and each well was washed with PBS
prior to the addition of MTT solution (100 μL, 1 mg mL−1 in DMEM;
initial MTT solution concentration was 5 mg mL−1 in PBS). After
another 4 h of incubation, the MTT medium was removed. DMSO
(100 μL) was added, and the plate was shaken at 100 rpm for 15 min.
The cell viability was measured based on the absorbance of each well
at 570 nm.
Hemolysis Studies. The hemolytic activity was determined
against human erythrocytes. Erythrocytes were isolated from freshly
drawn human blood (approval number: IRB-2015-03-040). Human
blood was drawn directly into K2-EDTA-coated Vacutainer tubes to
prevent coagulation of blood. The blood was stored at 4 °C for 1 h and
centrifuged at 1000g for 10 min to get red blood cells (RBC). RBC
were washed with PBS for three times (vortex mixing with PBS and
centrifugation at 1000g for 10 min to remove supernatant) and RBC
1172
DOI: 10.1021/acs.biomac.5b01766
Biomacromolecules 2016, 17, 1170−1178

Biomacromolecules
Article
were resuspended to achieve 5% (v/v) in PBS (pH 7.4). Serial dilution
was done in sterilized tubes by mixing 60 μL of polymer solution
(20 000 μg mL−1) with 60 μL of PBS. Serial dilution of the polymers
in PBS was performed followed by the addition of 60 μL of
erythrocyte suspension to the polymer solutions. PBS buffer (pH 7.4)
was used as a negative hemolysis control, and Triton X-100 (1% v/v in
PBS) was used as a positive hemolysis control. The tubes containing
polymer solution and erythrocyte suspension were incubated for 2 h at
37 °C and 150 rpm in an inoculation shaker. Then, RBC with polymer
solutions were centrifuged at 1000g for 5 min. The supernatants (60
μL) were then transferred to each well of a fresh microtiter plate (96-
well microplate) followed by the addition of 60 μL of PBS, and
absorbance was determined at 540 nm by using a Tecan InfinitePro
series M200 Micro plate Reader. The hemolysis percentage (H) was
calculated from the following equation:
hemolysis = [(Op − Ob)/(Ot − Ob)] × 100%
where Op is the absorbance for the polymer, Ob is the absorbance for
the negative control (PBS), and Ot is the absorbance for the positive
control of Triton X-100.
Minimum Inhibitory Concentration (MIC) Determination.
Bacteria cells were grown overnight at 37 °C in Mueller−Hinton broth
(MHB) to mid log phase and diluted to 104−105 CFU mL−1 in MHB.
A 2-fold dilution series of 100 μL of polymer solution in medium was
made in 96-well microplates, followed by the addition of 100 μL of the
bacterial suspension (104 to 105 CFU mL−1). The plates were
incubated at 37 °C for 18−24 h, and the absorbance at 600 nm was
measured with a microplate reader (BIO-RAD Benchmark Plus). A
positive control without polymer and a negative control without
bacteria were included. MICs were determined as the lowest
concentration that inhibited cell growth by more than 90%.
RESULTS AND DISCUSSION
■
Overall, the synthesis of the glucosamine-functionalized star
polymer nanoparticle can be divided into three key stages: (i)
synthesis of glycopolymer macroinitiator (L-pGSA), (ii)
synthesis of polylysine macroinitiator (L-pLYS), and (iii) star
formation followed by deprotection (Scheme 1). To synthesize
the polyglucosamine-based macroinitiator L-pGSA, an acrylate-
functionalized sugar monomer 4 was first made via copper-
catalyzed alkyne azide cycloaddition (CuAAC)23,24 where the
premade azido-functionalized N-acetyl D-glucosamine perace-
tate 3 was clicked with 5-hexynyl acrylate 1 to introduce a
polymerizable group onto the sugar compound (Figure 1). The
1H NMR spectra in Figure 1 depicts the successful trans-
formation of the sugar precursors into the final acrylate-
functionalized sugar monomer 4 where all the functional
groups (such as the methine proton of the triazole ring at
resonance g′, δH 7.67 ppm and the vinyl protons at resonances
a′ and b′, δH 5.79−6.41) were accounted for. The chemical
structure of the acrylate-functionalized glucosamine monomer
4 was further confirmed by 13C NMR analysis (Supporting
Information (SI), Figure S1).
1
Figure 1. H NMR spectra of the synthesized 5-hexynyl acrylate 1 as
well as the sugar monomer precursors leading to the final acrylate-
functionalized N-acetyl D-glucosamine peracetate monomer 4.
mer L-pGSA showed the chemical compositions of the
polymers as expected (SI, Figures S2 and S3). The targeted
(and also obtained) number-averaged degree of polymerization
(DPn) for the glucosamine and HEAm blocks in L-pGSA are 30
and 50, respectively. GPC differential refractive index (DRI)
chromatograms elucidate the formation of a well-defined
glycopolymer PGSA with a dispersity (Đ) value of 1.2 (SI,
Table S1), and the subsequent poly(HEAm) chain extended
product, L-pGSA (note that L denotes for linear morphology
and p indicates that the functional (hydroxyl) groups are in
their protected form), was evident based on the shift in
molecular weight distribution to shorter retention time (SI,
Figure S4). Noteworthy, the inclusion of a poly(HEAm) spacer
block was necessary to ensure a higher macroinitiator-to-star
conversion under the current reaction conditions as we have
observed poor star formation (<20%) when no or shorter (DPn
= 20) spacer block was used. HEAm was judiciously chosen for
our system as poly(HEAm) are known for their excellent
biocompatibility with mammalian cells and low-fouling
nature.31,32
The sugar monomer 4 was polymerized using a recently
developed RAFT-based photopolymerization technique that
enables excellent control over molecular weight and end-group
retention even at full monomer conversion.29,30 The polymer-
ization of 4 was taken to 100% monomer conversion after 24 h
and without any further purification (Scheme 1, step (v), the
glycopolymer (PGSA) block was chain extended with a spacer
block in a one-pot approach upon the addition of N-(2-
hydroxyethyl)acrylamide (HEAm) (Scheme 1, step vi).
Polymerization of the second block was also taken to full
Meanwhile, the synthesis of the polylysine macroinitiator L-
pLYS initially entails the NCA-ROP of ε-carboxybenzyl(Z)-L-
lysine (Lys NCA) monomer with benzylamine (Scheme 1, step
1
monomer conversion after 24 h. H NMR analysis of the
glucosamine-based homopolymer PGSA and diblock copoly-
1173
DOI: 10.1021/acs.biomac.5b01766
Biomacromolecules 2016, 17, 1170−1178

Biomacromolecules
Article
Figure 2. (a) GPC-DRI chromatograms of the protected macroinitiators and the corresponding (glucosamine-functionalized) star polymers
prepared via the arm-first approach using RAFT-based photopolymerization. (b) DLS normalized volume distribution of the final deprotected forms
as measured in water.
i), followed by in situ modification of the amino end-group into
clickable terminal alkyne via nucleophilic substitution reaction
with N-succinimidyl-5-hexynoate (Scheme 1, step ii). The
linear alkyne-terminated poly(Z-L-lysine) (PZLL-yne) was then
converted into a macroRAFT agent (PZLL-RAFT) after
CuAAC reaction with an azido-functionalized trithiocarbonate
RAFT agent (Scheme 1, step iii). Finally, the polymer was
chain extended with HEAm via the same RAFT-based
photopolymerization as above (Scheme 1, step iv), yielding
12 to 19 (Table 1 and SI, Figure S13 and Table S1).
Noteworthy, the Narm values represent estimates and not the
Table 1. Physical Characterization of the Macroinitiators
and Star Polymers via GPC, DLS, and Zeta Potential
Analysis
a
a
b
c
entry
Mn (g mol‑1)
Đ
% conv Narm dH (nm) ζ (mV)
S-GSA 25
S-GSA 50
S-GSA 65
S-GSA 100
L-LYS
168 000
231 000
167 000
157 000
13 800
1.5
1.7
1.4
1.3
1.6
1.4
60
65
61
63
13
19
13
12
23.7
25.9
15.7
13.7
7.2
33.6
33.4
23.2
8.3
1
the polylysine macroinitiator L-pLYS. The H NMR spectra of
PZLL-RAFT and L-pLYS revealed their anticipated chemical
compositions, with L-pLYS having ca. 15 residues of amino acid
and a DPn of 50 for the poly(HEAm) spacer block (which is
the same number of spacer units compared to the L-pGSA
macroinitiator) (SI, Figures S5 and S6). GPC analysis of PZLL-
yne and PZLL-RAFT however, revealed bimodal distributions
(SI, Figure S7), which is not entirely surprising given that PZLL
experiences a coil-to-helix transition at around a DPn of 15 and
potentially produces a bimodal distribution as an artifact during
GPC analysis. This phenomenon has recently been docu-
mented in the literature.33 The shift in molecular weight
distribution after chain extension with HEAm indicated that
PZLL-RAFT was indeed still active.
For the star formation step, the synthesized macroinitiators
were mixed as a cocktail mixture at different molar ratios of L-
pGSA to L-pLYS and subjected to RAFT-based photo-
polymerization in the presence of a cross-linkable monomer,
N,N′-methylenebis(acrylamide), for 48 h (Scheme 1, step vii).
This approach in forming star polymers, termed the arm-first
approach, permits the incorporation of various types of
macroinitiators (that go on to form the arms of the star) into
a single nanoparticle construct as they converge together during
the cross-linking step.34,35 1H NMR analysis of the star
polymers in Figures S8−S12 (SI) revealed their exact chemical
compositional structures (S-pLYS, S-pGSA 25, S-pGSA 50, S-
pGSA 65, and S-pGSA 100; where S denotes for star polymer
and the number indicates the average mol percentage of
glycopolymer arm with respect to the total number of arms). In
general, GPC analysis of Figure 2a revealed that the
polymerizations were well-moderated, with star polymers
having Đ < 1.7, except for S-pLYS (Đ = 3.5) because it
contains star−star coupled products, while the estimated
average number of arms per star particle (Narm) was between
25.3
1.2
L-GSA
15 200
5.5
a
Determined based on the protected forms via a GPC-DMF system
using narrow polystyrene standards as the reference. The Mn (and
hence Narm) values are relative to a polystyrene calibration and are
b
estimates of the actual molecular weights. Refers to the conversion of
c
macroinitiator to star. Estimated based on literature protocol.34c
actual molecular weights and were determined relative to
polystyrene standards. Given that the star polymer usually
adopts a smaller hydrodynamic volume compared to a linear
counterpart of equal molecular weight, the Narm values reported
in Table 1 most likely underestimate the actual values.34a While
the GPC DRI chromatograms in Figure 2a clearly illustrate the
evolution of linear macroinitiators into higher molecular weight
species, only moderate macroinitiator-to-star conversion values
(60−65%) were achieved. The star conversion did not improve
even after extended polymerization times (72 h).
There are two possible explanations for the moderate star
conversions. We hypothesized that the moderate macro-
initiator-to-star conversion is due to steric effects. Both L-
pGSA and L-pLYS contain bulky side groups that may have
created a steric congestion during star formation, thereby
preventing the macroinitiators from fully inserting into the
nanoparticle form. Furthermore, some of the PZLL chains
adopt α-helix structure and occupy larger hydrodynamic
volume than random coil configuration, which adds more
steric congestion to the already-sterically congested system.
The addition of the spacer block poly(HEAm) relieved the
steric hindrance (vide supra) but was insufficient to induce a
higher macroinitiator-to-star conversion. Another possible
1174
DOI: 10.1021/acs.biomac.5b01766
Biomacromolecules 2016, 17, 1170−1178

Biomacromolecules
Article
reason is that L-pGSA and L-pLYS contain some dead chains
that were unable to participate in the cross-linking step and
hence were “left behind” as observed via GPC analysis.
Although possible, it is unlikely that this reason alone
contributed to such star conversion values as the RAFT-based
photopolymerization method was previously shown to proceed
with high efficiency.27 We believe that the obtained moderate
star conversion values were primarily caused by steric effects,
and to a lesser extent due to the contamination of dead chains
in L-pGSA and L-pLYS. It is worth mentioning that although
the star polymer samples consist partially of linear polymer
impurities, as shown below, the glucosamine-functionalized star
polymer samples have significantly different (improved)
biological properties compared to linear macromolecules. As
a proof of concept, this demonstrates the advantage and
difference between star and linear polymers even though the
current star production pathway could potentially be further
optimized to eliminate any linear polymer impurities to obtain
pure star products for better comparison.
which is expected given that the glucosamine moieties are not
as cationic compared to polylysine.
To test the mammalian cell biocompatibility of the star
polymers, human aortic smooth muscle cells (AoSMC) were
used as a model cell line and the metabolic activity, an indicator
of viability, was determined using MTT assay. At 100 μg mL−1,
the cell viability was high (>80%) for all glucosamine-
functionalized star polymers (even for S-GSA 25 with the
lowest degree of glucosamine functionalization) (Figure 3a). S-
Proving that the star polymers truly comprise of mixtures of
polylysine and glycopolymers is extremely challenging.
However, considering that both L-pGSA and L-pLYS were
composed of identical spacer lengths and types (poly(HEAm))
and used the same family of RAFT agent (trithiocarbonate), it
is safe to assume that heteroarm stars were formed and not just
mixtures of two homoarm stars. This is especially true given
that controlled radical polymerization is a statistical process.
Other studies have indirectly shown the successful formation of
heteroarm stars via stimuli-responsive hierarchical self-assem-
bly34,36 but this method of verification of heteroarm stars
formation is not possible with our system. Nevertheless, the
indiscriminate incorporation of various macroinitiators to form
heteroarm stars using controlled radical polymerization
protocols similar to ours, as demonstrated in these reports,
provides strong evidence that heteroarm glucosamine-function-
alized stars were formed.
The protecting groups (carboxybenzyl and acetyl) of the star
and linear polymers were removed sequentially prior to
biological testing (Scheme 1, step viii). First, the carboxybenzyl
groups of PZLL blocks were removed in the presence of
trifluoroacetic acid, trifluoromethanesulfonic acid and thioani-
sole, followed by the removal of acetyl protecting groups on the
glycopolymer using hydrazine. 1H NMR analysis of the star and
linear polymers confirmed the quantitative removal of all
protecting groups (including the RAFT groups as indicated by
the decoloration of the samples from yellow to white) (SI,
Figures S8−12, S14, and S15). The samples were then purified
by dialysis and lyophilized. Dynamic light scattering (DLS)
distributions of the deprotected star and linear polymers (S-
GSA 25, S-GSA50, S-GSA 65, S-GSA 100, L-LYS, and L-GSA)
are shown in Figure 2b. The average hydrodynamic diameter
(dH) values of the water-soluble stars are ca. between 14 and 26
nm, which are distinctively different to the dH of the linear
macroinitiators (≤7.2 nm) (Table 1). The size range of the star
polymers was comparable to other star polymers that were
made via the arm-first approach. Although the star samples
consisted of some linear polymer impurities, the DLS results
indicated monomodal distributions. We postulate that the
linear impurities in the star samples may be entangled with the
star species when dissolved in solution, and hence were not
detectable by DLS. Zeta potential measurements showed that
the stars are less cationic with increasing glycopolymer content
(with zeta potential (ζ) values decreasing from 33.6 to 8.3 mV),
Figure 3. (a) Percentage of living AoSMC after 24 h incubation with
various star polymers at sample concentrations of 100 and 500 μg
mL−1. (b) Determination of the SMC IC50 for S-GSA 25 and S-GSA
50. The horizontal dashed line indicates the cutoff point for 50% cell
viability.
LYS which does not contain any glycopolymer units was
cytotoxic. At a higher sample concentration of 500 μg mL−1,
the cell viability decreased but improved with higher glucos-
amine content. The half maximal inhibitory concentration
(IC50) values for S-GSA 25 and S-GSA 50 were determined to
be 337 and 369 μg mL−1, respectively (Figure 3b and Table 2).
The trend observed in Figure 3 was correlated with the charge
density of the star polymer nanoparticle as polymers with
higher zeta potential values were more cytotoxic, most likely
because of increased electrostatic interactions with the
negatively charged cell surfaces.
An additional experiment was performed to ascertain the
effect of the star architecture over mammalian cell biocompat-
ibility. For this, a control sample which is a physical blend of L-
LYS and L-GSA (75:25 molar ratio) that mimics the chemical
composition of S-GSA 25 was subjected to similar MTT assay
as above (SI, Figure S16). Interestingly, at 100 μg mL−1, the
1175
DOI: 10.1021/acs.biomac.5b01766
Biomacromolecules 2016, 17, 1170−1178

Biomacromolecules
Article
Table 2. Antimicrobial and Hemolytic Activity as Well as Mammalian Cell Biocompatibility of Glucosamine-Functionalized Star
Polymers, and Their Comparison to Other Synthetic Antimicrobial Peptides
a
MIC (μg mL−1
)
b
Gram negative
Gram positive
selectivity
HC50 (μg mL−1
)
IC50 (μg mL−1
)
HC50/
MIC
IC50/
MIC
entry
E. coli P. aeruginosa
S. aureus
MRSA E. faecalis VRE
RBC
SMC
ref
S-LYS
32
256
16−32
32−64
64−128
256
16
32
8
16
64
32
64
>10 000
>10 000
>10 000
>10 000
∼0
>417
>208
>104
>39
∼0
7.0
3.8
2.4
S-GSA 25
S-GSA 50
S-GSA 65
S-GSA 100
L-LYS
>512
>512
>512
>512
64
>512
>512
>512
>512
256
337
369
610
128
256
>512
16
∼0
∼0
L-GSA
>512
31.3
11.5
2
>512
31.3
8.3
>512
31.3
c
indolicidin
tachyplesin I
melittin
31.3
∼250
∼8
∼41
∼8
5
39
40
41
42
c
d
100
∼340
c
2
0.5
9
∼4
magainin
analogue
1.5
45
protegrin
analogue
3
3
9
3
42
43
c
d
polymyxin B
1−2
∼211
∼141
a
The strains used in this study were: E. coli (ATCC 8739), P. aeruginosa (ATCC 27853), S. aureus (ATCC 29213 and ATCC 25923), methicillin-
resistant S. aureus (BAA-40), E. faecalis (OG1RF), and vancomycin-resistant E. faecalis (V583). Note that some of the strains used for testing in this
study differ from those used in the testing of other synthetic antimicrobial peptides listed in the table−please consult the appropriate references for
b
c
d
the exact strains used. Values were determined based on the MIC against S. aureus. Extrapolated values. Selectivity was determined using the
lowest MIC value when no MIC values were recorded for S. aureus.
cell viability of S-GSA 25 was significantly higher (85%) than
the control sample (41%) even though it consists of linear
polymers of similar chemical composition. This result highlights
the unique property of the star architecture in providing better
biocompatibility. When locked in a nanoparticle form, it is
hypothesized that the polyglucosamine-based arms shield and
reduce the probability of adjacent polylysine arms from
nonspecific interactions with cell surfaces, giving the star
polymer better human cell biocompatibility over free linear
chains. It is worthwhile noting that both S-LYS and L-LYS were
equally cytotoxic toward AoSMC, and the mammalian cell
viability only improved with the addition of polyglucosamine-
based arms.
The sample concentration at which 50% of red blood cells
were lysed, defined as the HC50, was used as the metric for
determining the hemolytic activity of the polymers. Remark-
ably, even at the highest polymer concentration tested (10 000
μg mL−1), none of the star polymers were hemolytic, including
S-LYS which has poor cytocompability with SMC (Table 2).
The good hemocompatibility for the star polymers may be
attributed to the absence of hydrophobic groups, which are
known to be responsible for inducing hemolytic activity.37,38
The antimicrobial efficacy of the synthesized nanomaterials
against a range of Gram negative and Gram positive pathogens
was determined based on their minimum inhibitory concen-
trations (MICs) (Table 2). Polymers that do not contain sugar
molecules (i.e., S-LYS and L-LYS) had good antimicrobial
activity against both Escherichia coli (Gram negative) and
Staphylococcus aureus (Gram positive), with MIC values
between 16 and 64 μg mL−1. On the other hand, polymers
that do not contain cationic polylysine (i.e., S-GSA 100 and L-
GSA) were not active against all bacteria tested. Interestingly,
the glucosamine-functionalized heteroarm stars selectively
killed Gram positive pathogens and not Gram negative strains.
This level of specificity is most likely attributed to the difference
in chemical structure between the two families. For Gram
positive bacteria, a thick peptidoglycan layer constitutes as the
outermost layer whereas for the Gram negative family, the
outer membrane consists of a lipid bilayer. Given that the
glucosamine moieties on the stars resemble the peptidoglycan
structure, we hypothesize that the polyglucosamine-based arms
assist the infiltration of the nanoparticles into the Gram positive
bacteria wall, subsequently enabling the polylysine arms to
induce cell death. In contrast, the polyglucosamine-based arms
act as “shields” (vide supra) instead of “infiltrators”, which
hinder the adjacent polylysine arms from interacting with the
Gram negative bacteria outer membrane, rendering the
nanoparticles inactive toward Gram negative pathogens. The
decrease in antimicrobial activity with decreasing polylysine
content supports the notion that the polylysine arm is the
primary cause of bacteria death. Of note, all of the materials
were considered nonactive against Pseudomonas aeruginosa. S-
LYS, S-GSA 25 and S-GSA 50 were further assessed with more
Gram positive pathogens including E. faecalis, and clinical
strains MRSA and VRE. These stars were most effective against
E. faecalis, with MICs between 8 and 64 μg mL−1. Barring S-
LYS which is potent against all Gram positive strains tested, the
only heteroarm star that was considered to be effective against
MRSA and VRE was S-GSA 25, with MICs of 32 and 64 μg
mL−1, respectively.
The selectivity (also known as therapeutic index) of the star
polymers can be defined as the ratio of HC50 to MIC or IC50 to
MIC. The MIC against S. aureus was used as the reference. For
HC50/MIC index, S-LYS yields the best result (>417), followed
by S-GSA 25 (>208), S-GSA 50 (>104), and S-GSA 65 (>39)
in the order of increasing glucosamine functionalization.
However, as shown in Figure 3, the cytocompatibility of S-
LYS with a human model cell line SMC was extremely poor
(<30% at 100 μg mL−1). Among the heteroarm stars, S-GSA 25
showed the best IC50/MIC (7.0). Taking into account both the
HC50/MIC and IC50/MIC values, S-GSA 25 was considered to
be the best performing antimicrobial agent that exhibited
1176
DOI: 10.1021/acs.biomac.5b01766
Biomacromolecules 2016, 17, 1170−1178

Biomacromolecules
Article
Author Contributions
minimal toxicity to human cells in this study. To get an
indication on the selectivity of S-GSA 25, its HC50/MIC value
was compared to other antimicrobial agents like AMPs. S-GSA
25 outperforms all the AMPs listed in Table 2 in terms of
selectivity. Although the AMPs in general have lower MIC
values than S-GSA 25, they inherently suffer from having low
HC50 (and possibly IC50) values. The good selectivity of S-GSA
25 thus highlights the attractiveness of using star polymers as
effective antimicrobial agents.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Singapore Ministry of Education Tier 3 grant (MOE2013-T3-1-
002). Singapore NMRC Ministry of Health Industry Alignment
Fund Category 2 (MOH IAF CAT Application No.
MOHIAFCAT2006).
Notes
CONCLUSIONS
■
The authors declare no competing financial interest.
In conclusion, novel glucosamine-functionalized core cross-
linked star polymers with polylysine and polyglucosamine-
based arms were made via a recently developed RAFT-based
photopolymerization technique and the arm-first approach. The
glycosylated star polymer nanoparticles (14−26 nm in
diameter) demonstrate good cytocompatibility with a model
mammalian cell line (human aortic smooth muscle cells),
yielding >80% cell viability. In addition, the star polymer
samples show superior biocompatibility compared to free linear
polymers as the star architecture provide the platform in
shielding the particles effectively from nonspecific interactions
between the polylysine arms with cell surfaces. Furthermore,
the star polymers are non-hemolytic even at 10 000 μg mL−1.
Antimicrobial studies reveal that the glucosamine-function-
alized stars target specifically Gram positive pathogens and not
Gram negative species, most likely because of the resemblance
between the glucosamine moieties with the peptidoglycan layer
of the bacteria. The stars were also effective against clinical
strains such as MRSA and VRE with MICs as low as 32 μg
mL−1. Given the good biocompatibility of the glycosylated
stars, they demonstrate excellent selectivity (or therapeutic
index), with the most optimum being the star having 25% of
polyglucosamine-based arms, S-GSA 25. The antimicrobial
activity and mammalian cell biocompatibility are tunable
depending on the molar ratio of polylysine to glycopolymer
arms, where stars with lesser glucosamine functionalization
exhibit better antimicrobial efficacy but poorer human
cytocompatibility and vice versa. This crucially demonstrates
that an intricate balance between antimicrobial activity and
biocompatibility needs to be struck to achieve the best
biological outcome. The technology described in here could
lay the foundation for the development of more advanced
antimicrobial agents in the future.
ACKNOWLEDGMENTS
■
This work was funded and supported by a Singapore Ministry
of Education Tier 3 grant (MOE2013-T3-1-002) and a
Singapore NMRC Ministry of Health Industry Alignment
Fund Category 2 (Project Number: NMRC/MOHIAFCAT2/
003/2014 (Application No.: MOHIAFCAT2005)). S.A.R. and
V.R. acknowledge financial support from the Singapore Centre
on Environment Life Sciences Engineering, whose research is
supported by the National Research Foundation Singapore,
Ministry of Education, Nanyang Technological University and
National University of Singapore, under its Research Centre of
Excellence Programme.
REFERENCES
■
(1) The White House. https://www.whitehouse.gov/the-press-
office/2014/09/18/executive-order-combating-antibiotic-resistant-
bacteria (accessed November 2015).
(2) Kock, R.; Becker, K.; Cookson, B.; van Gemert-Pijnen, J.;
̈
Harbarth, S.; Kluytmans, J.; Mielke, M.; Peters, G.; Skov, R. L.;
Struelens, M. J.; Tacconelli, E.; Navarro Torne, A.; Witte, W.;
Friedrich, A. W. Euro Surveil. 2010, 15, 19688.
(3) Nichols, D.; Cahoon, N.; Trakhtenberg, E. M.; Pham, L.; Mehta,
A.; Belanger, A.; Kanigan, T.; Lewis, K.; Epstein, S. S. Appl. Environ.
Microbiol. 2010, 76, 2445−2450.
(4) Ling, L. L.; Schneider, T.; Peoples, A. J.; Spoering, A. L.; Engels,
I.; Conlon, B. P.; Mueller, A.; Schaberle, T. F.; Hughes, D. E.; Epstein,
̈
S.; Jones, M.; Lazarides, L.; Steadman, V. A.; Cohen, D. R.; Felix, C.
R.; Fetterman, K. A.; Millett, W. P.; Nitti, A. G.; Zullo, A. M.; Chen,
C.; Lewis, K. Nature 2015, 517, 455−459.
(5) Wenzel, M.; Chiriac, A. I.; Otto, A.; Zweytick, D.; May, C.;
Schumacher, C.; Gust, R.; Albada, H. B.; Penkova, M.; Kramer, U.;
̈
Erdmann, R.; Metzler-Nolte, N.; Straus, S. K.; Bremer, E.; Becher, D.;
Brotz-Oesterhelt, H.; Sahl, H. − G.; Bandow, J. E. Proc. Natl. Acad. Sci.
U. S. A. 2014, 111, E1409−E1418.
(6) Mohanram, H.; Bhattacharjya, S. Antimicrob. Agents Chemother.
2014, 58, 1987−1996.
ASSOCIATED CONTENT
* Supporting Information
■
S
(7) Deming, T. J. Nature 1997, 390, 386−389.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.5b01766.
(8) Nederberg, F.; Connor, E. F.; Moller, M.; Glauser, T.; Hedrick, J.
̈
L. Angew. Chem., Int. Ed. 2001, 40, 2712−2715.
(9) (a) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2005, 58,
379−410. (b) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem.
2009, 62, 1402−1472.
NMR, GPC, and DLS characterizations as well as
biocompatibility studies (PDF)
(10) Cobo, I.; Li, M.; Sumerlin, B. S.; Perrier, S. Nat. Mater. 2015, 14,
143−159.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: edgar.wong@unsw.edu.au.
*E-mail: mbechan@ntu.edu.sg.
(11) Nguyen, T. − K.; Selvanayagam, R.; Ho, K. K. K.; Chen, R.;
Kutty, S. K.; Rice, S. A.; Kumar, N.; Barraud, N.; Duong, H. T. T.;
Boyer, C. Chem. Sci. 2016, 7, 1016−1027.
■
(12) Byrne, M.; Victory, D.; Hibbitts, A.; Lanigan, M.; Heise, A.;
Cryan, S. − A. Biomater. Sci. 2013, 1, 1223−1234.
(13) Lam, S. J.; Sulistio, A.; Ladewig, K.; Wong, E. H. H.; Blencowe,
A.; Qiao, G. G. Aust. J. Chem. 2014, 67, 592−597.
(14) Dong, Y.; Qin, Y.; Dubaa, M.; Killion, J.; Gao, Y.; Zhao, T.;
Zhou, D.; Duscher, D.; Geever, L.; Gurtner, G. C.; Wang, W. Polym.
Chem. 2015, 6, 6182−6192.
Present Address
⊥E.H.H.W.: Centre for Advanced Macromolecular Design and
Australian Centre for Nanomedicine, School of Chemical
Engineering, The University of New South Wales, Sydney,
NSW 2052, Australia.
1177
DOI: 10.1021/acs.biomac.5b01766
Biomacromolecules 2016, 17, 1170−1178

Biomacromolecules
Article
(15) Undin, J.; Illanes, T.; Finne-Wistrand, A.; Albertsson, A. − C.
Polym. Chem. 2012, 3, 1260−1266.
(42) Rotem, S.; Radzishevsky, I.; Mor, A. Antimicrob. Agents
Chemother. 2006, 50, 2666−2672.
(43) Ruden, S.; Hilpert, K.; Berditsch, M.; Wadhwani, P.; Ulrich, A.
S. Antimicrob. Agents Chemother. 2009, 53, 3538−3540.
(16) Kesik, M.; Akbulut, H.; Soylemez, S.; Cevher, S
̧
. C.; Hızalan, G.;
̈
Udum, Y. A.; Endo, T.; Yamada, S.; Çırpan, A.; Yagcı, Y.; Toppare, L.
̆
Polym. Chem. 2014, 5, 6295−6306.
(17) Takara, M.; Toyoshima, M.; Seto, H.; Hoshino, Y.; Miura, Y.
Polym. Chem. 2014, 5, 931−939.
(18) Engler, A. C.; Shukla, A.; Puranam, S.; Buss, H. G.; Jreige, N.;
Hammond, P. T. Biomacromolecules 2011, 12, 1666−1674.
(19) Nederberg, F.; Zhang, Y.; Tan, J. P. K.; Xu, K.; Wang, H.; Yang,
C.; Gao, S.; Guo, X. D.; Fukushima, K.; Li, L.; Hedrick, J. L.; Yang, Y.
− Y. Nat. Chem. 2011, 3, 409−414.
(20) Fu, T. − H.; Li, Y.; Thaker, H. D.; Scott, R. W.; Tew, G. N. ACS
Med. Chem. Lett. 2013, 4, 841−845.
(21) Al-Ahmad, A.; Laird, D.; Zou, P.; Tomakidi, P.; Steinberg, T.;
Lienkamp, K. PLoS One 2013, 8, e73812.
(22) (a) Zhou, C.; Qi, X.; Li, P.; Chen, W. N.; Mouad, L.; Chang, M.
W.; Leong, S. S. J.; Chan-Park, M. B. Biomacromolecules 2010, 11, 60−
67. (b) Li, P.; Poon, Y. F.; Li, W.; Zhu, H. − Y.; Yeap, S. H.; Cao, Y.;
Qi, X.; Zhou, C.; Lamrani, M.; Beuerman, R. W.; Kang, E. − T.; Mu,
Y.; Li, C. M.; Chang, M. W.; Leong, S. S. J.; Chan-Park, M. B. Nat.
Mater. 2011, 10, 149−156. (c) Li, P.; Zhou, C.; Rayatpisheh, S.; Ye, K.;
Poon, Y. F.; Hammond, P. T.; Duan, H.; Chan-Park, M. B. Adv. Mater.
2012, 24, 4130−4137.
(23) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004−2021.
(24) Liang, L.; Astruc, D. Coord. Chem. Rev. 2011, 255, 2933−2945.
(25) Barner-Kowollik, C.; Du Prez, F. E.; Espeel, P.; Hawker, C. J.;
Junkers, T.; Schlaad, H.; Van Camp, W. Angew. Chem., Int. Ed. 2011,
50, 60−62.
(26) Leriche, G.; Budin, G.; Brino, L.; Wagner, A. Eur. J. Org. Chem.
2010, 2010, 4360−4364.
(27) Yang, Y.; Song, X.; Yuan, L.; Li, M.; Liu, J.; Ji, R.; Zhao, H. J.
Polym. Sci., Part A: Polym. Chem. 2012, 50, 329−337.
(28) Wong, E. H. H.; Lam, S. J.; Nam, E.; Qiao, G. G. ACS Macro
Lett. 2014, 3, 524−528.
(29) McKenzie, T. G.; Fu, Q.; Wong, E. H. H.; Dunstan, D. E.; Qiao,
G. G. Macromolecules 2015, 48, 3864−3872.
(30) McKenzie, T. G.; Wong, E. H. H.; Fu, Q.; Sulistio, A.; Dunstan,
D. E.; Qiao, G. G. ACS Macro Lett. 2015, 4, 1012−1016.
(31) Zhao, C.; Patel, K.; Aichinger, L. M.; Liu, Z.; Hu, R.; Chen, H.;
Li, X.; Li, L.; Zhang, G.; Chang, Y.; Zheng, J. RSC Adv. 2013, 3,
19991−20000.
(32) Vorobii, M.; de los Santos Pereira, A.; Pop-Georgievski, O.;
Kostina, N. Y.; Rodriguez-Emmenegger, C.; Percec, V. Polym. Chem.
2015, 6, 4210−4220.
(33) Huesmann, D.; Birke, A.; Klinker, K.; Turk, S.; Rader, H. J.;
̈
̈
Barz, M. Macromolecules 2014, 47, 928−936.
(34) (a) Blencowe, A.; Tan, J. F.; Goh, T. K.; Qiao, G. G. Polymer
2009, 50, 5−32. (b) Wong, E. H. H.; Blencowe, A.; Qiao, G. G. Polym.
Chem. 2013, 4, 4562−4565. (c) McKenzie, T. G.; Wong, E. H. H.; Fu,
Q.; Lam, S. J.; Dunstan, D. E.; Qiao, G. G. Macromolecules 2014, 47,
7869−7877.
(35) Gao, H.; Matyjaszewski, K. Prog. Polym. Sci. 2009, 34, 317−350.
(36) Wei, X.; Moad, G.; Muir, B. W.; Rizzardo, E.; Rosselgong, J.;
Yang, W.; Thang, S. H. Macromol. Rapid Commun. 2014, 35, 840−845.
(37) Kuroda, K.; Caputo, G. A.; DeGrado, W. F. Chem. - Eur. J. 2009,
15, 1123−1133.
(38) Saha, K.; Moyano, D. F.; Rotello, V. M. Mater. Horiz. 2014, 1,
102−105.
(39) Jindal, H. M.; Le, C. F.; Yusof, M. Y. M.; Velayuthan, R. D.; Lee,
V. S.; Zain, S. M.; Isa, D. M.; Sekaran, S. D. PLoS One 2015, 10,
e0128532.
(40) Ramamoorthy, A.; Thennarasu, S.; Tan, A.; Gottipati, K.;
Sreekumar, S.; Heyl, D. L.; An, F. Y. P.; Shelburne, C. E. Biochemistry
2006, 45, 6529−6540.
(41) Zhu, W. L.; Nan, Y. H.; Hahm, K. − S.; Shin, S. Y. J. Biochem.
Mol. Biol. 2007, 40, 1090−1094.
1178
DOI: 10.1021/acs.biomac.5b01766
Biomacromolecules 2016, 17, 1170−1178