Synthesis of Antibacterial Glycosylated Polycaprolactones Bearing Imidazoliums with Reduced Hemolytic Activity

Article abstract of DOI:10.1021/acs.biomac.8b01577

Most synthetic antimicrobial polymers are not biodegradable, thus limiting their potential for large-scale applications in personal care disinfection and environmental contaminations. Poly(?-caprolactone) (PCL) is known to be both biodegradable and biocom

Full text of DOI:10.1021/acs.biomac.8b01577

Article
Cite This: Biomacromolecules XXXX, XXX, XXX−XXX
pubs.acs.org/Biomac
Synthesis of Antibacterial Glycosylated Polycaprolactones Bearing
Imidazoliums with Reduced Hemolytic Activity
†
‡
‡
†
,‡,§
and Xue-Wei Liu*,†,§
Yuan Xu, Kaixi Zhang, Sheethal Reghu, Yichao Lin, Mary B. Chan-Park,*
†Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University,
1 Nanyang Link, Singapore 637371, Republic of Singapore
2
‡
School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459,
Republic of Singapore
§
Centre for Antimicrobial Bioengineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Republic of
Singapore
*
S Supporting Information
ABSTRACT: Most synthetic antimicrobial polymers are not
biodegradable, thus limiting their potential for large-scale
applications in personal care disinfection and environmental
contaminations. Poly(ε-caprolactone) (PCL) is known to be
both biodegradable and biocompatible, thus representing an
ideal candidate biopolymer for antimicrobial applications.
Here we successfully grafted alkylimidazolium (Im) onto PCL
to mimic the cationic properties of antimicrobial peptides.
The poly(ε-caprolactone)-graft-butylimidazolium had only
moderate MICs (32 μg/mL), reasonably good red blood cell selectivity (36) and relatively good fibroblast compatibility
(
81% cell viability at 100 μg/mL), indicating that combining the hydrophobic PCL backbone with the most hydrophilic
butylimidazolium gives a good balance of MIC and cytotoxicity. On the other hand, the PCL-graft-hexylimidazolium and
octylimidazolium demonstrated better MICs (4−32 μg/mL), but considerably worse cytotoxicity. We postulated that the
-
worse hydrophilicity of hexylimidazolium and octylimidazolium was responsible for their higher cytotoxicity and sought to
moderate their cytotoxicity with different sugar compositions and lengths. Through our screening, we identified a candidate
polymer, P(C6Im)0.35CL-co-P(Man)0.65CL, that demonstrated both superior MIC and very low cytotoxicity. We further
demonstrated that our biopolymer hit had superior antimicrobial kinetics compared to the antibiotic vancomycin. This work
paves the way forward for the use of biodegradable polyesters as the backbone scaffold for biocompatible antibacterial agents, by
clicking with different types and ratios of alkylimidazolium and carbohydrate moieties.
INTRODUCTION
biodegradable polymers and their roles as scaffolds for
■
biodegradable antibacterial polymers have been rarely
Antimicrobial peptides (AMPs) generally possess amphiphilic
properties, arising from both hydrophilic and hydrophobic side
chains, which are responsible for their activities. Synthetic
13,17,26,27
investigated.
Moreover, antibacterial polycarbonates
1
have been well studied, which presented a good example of
degradable antibacterial polymers. In comparison with
polycarbonates, PCL (poly(ε-caprolactone)) has more efficient
polymers which mimic the amphiphilic structures of AMPs,
have attracted great attention as a new class of disinfectants
because of their ease of synthesis and low-cost of production in
28−30
degradation behavior,
and little has been studied using
2
−4
PCL as the backbone for antibacterial polymers. Besides, some
reported polyesters have the ability of self-degradation despite
large quantities.
The antimicrobial polymers, inspired by
the cationic amino acids residues in AMPs, generally have
cationic groups to facilitate their adsorption to negatively
charged bacterial membranes. For example, DeGrado and
co-workers synthesized a biomimetic poly(arylamide) with
amphiphilic structures bearing a primary amine, which showed
broad antibacterial activity. Also, many other polymers have
been synthesized and tested for activities against different
3
1,32
their relatively high hemolytic activity.
Intriguingly,
5
,6
cationic coumarin polyesters and polyurethanes were reported
to exhibit selective activity against Gram-negative bacteria
which is very surprising compared with other cationic
33,34
7
,8
polymers.
The backbone of the cationic antibacterial
polymers is known to profoundly affect the resultant biological
properties. Therefore, these interesting and attractive results
had inspired us to design new antibacterial polymers based on
9
−12
pathogens, for example, polynorbornenes,
21,22
polycarbon-
and polyacry-
1
3−17
18−20
ates,
poly(β-lactam),
polypeptides
2
3−25
late derivatives.
However, most synthetic antimicrobial
polymers consist of carbon-based backbones that are not
biodegradable, thus limiting their potential in clinical
applications. Aliphatic polyesters constitute a class of
Received: October 31, 2018
Revised: December 22, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.biomac.8b01577
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Figure 1. Design and synthesis of new antibacterial agent with biodegradable poly(ε-caprolactone) (PCL). (a) Synthesis of PCLs: (i) NBS,
NH OAc, Et O, rt, 30 min; (ii) m-CPBA, DCM, rt, 24 h; (iii) BnOH, Sn(Oct) , 80 °C, 48 h; (iv) NaN , DMF, rt, overnight. (b) Synthesis of
4
2
2
3
P(C6Im)0.64-co-P(Glc)0.36CL in a one-pot reaction: (i) CuI, Et N, DMF, 40 °C, overnight (Glc = glucose). (c) Calculation of the ratio of grafted
3
1
imidazolium and carbohydrate by the integrals in H NMR, Ratio(
= I /(I − I ), which should be 0.64 and 0.36 for P(C6Im)0.64-co-
C6Im/Glc)
d
c+f
d
P(Glc)0.36CL (predicted ratio is 70:30). (d) Characteristic signals in resulting PCLs: signals d and e are from imidazolium ring while c and f are
from the formed triazole rings.
polycaprolactones. As a well-known biodegradable polyester,
PCL has attracted considerable attention for the past decades,
but the major drawback is the lack of side functional groups
which can be solved by introducing functional groups to the α-
to increase the hydrophilicity. Noteworthily, the effect of
glycounits was studied on antimicrobial polyacrylates system-
53
atically but with only one sugar.
Here, we report the synthesis of a new class of antibacterial
compounds based on the biodegradable PCL backbone.
Imidazolium was grafted to mimic the cationic properties of
antimicrobial peptides whereas the alkyl chain on the
imidazoliums and carbohydrates were used to balance the
hydrophobicity/hydrophilicity of the resultant polymer. The
synthesis of the polymers could be successfully achieved
through ring-opening polymerization and subsequent click
reaction between the azido PCL and alkyne-imidazoliums (Im)
and alkyne-carbohydrates in a one-pot grafting reaction
(Figure 1). Subsequently, the PCL-Im-carbohydrates grafts
were tested for activity against both Gram-positive and Gram-
negative bacteria, including up to 9 MRSA strains. In addition,
to evaluate selectivity against bacteria, these polymers were
also tested for toxicities against red blood cells and 3T3 cells.
35,36
position of the carbonyl.
Interestingly, functionalized ε-
caprolactone has been prepared. It has been successfully
polymerized and modified by click reactions, thus implying it is
a great candidate substrate for producing biodegradable
3
5,37,38
antimicrobial polyesters.
Furthermore, imidazolium
salts have gained considerable attention in antimicrobial
applications owing to their relative hydrophilicity, permanent
39−41
charges and stability.
Furthermore, like natural AMPs, most cationic polymers are
42
hemolytic or cytotoxic to human cells, limiting their further
applications. Thus, studies have been widely pursued to
manipulate the antimicrobial activity and biocompatibility of
the synthetic polymers through various factors such as charge
9
,43
12,44
45,46
density,
molecular weight,
type of active moiety
2
3,24,47
and alkyl chain length.
In particular, the balance between
EXPERIMENTAL SECTION
■
hydrophilicity and hydrophobicity of the entire polymer is
most crucial to the selectivity. This can be done by adding alkyl
chains to increase hydrophobicity or adding biocompatible
Materials. Chemicals and solvents are purchased from Alfa-Aesar,
Sigma-Aldrich and VWR and used without further purification unless
otherwise noted. Benzyl alcohol (Alfa-Aesar, 99%) was stirred with
sodium at room temperature and distilled under nitrogen and stored
48,49
50−53
compounds such as poly(ethylene glycol)
and sugars
B
DOI: 10.1021/acs.biomac.8b01577
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in Schlenk flask in desiccator until further use. Anhydrous toluene
tin(II) 2-ethylhexanoate (Sn(Oct) , 648 mg, 1.6 mmol) were added
2
(
over sodium/benzophenone), tetrahydrofuran (THF, over sodium/
sequentially. The mixture in the Schlenk flask was subject to three
freeze−pump−thaw cycles, sealed under nitrogen and stirred at 80 °C
for 48 h before quenched with highly diluted HCl in methanol. The
resulted mixture was diluted with DCM and precipitated with hexane,
centrifuged and dried under vacuum, giving the poly(α-azido-ε-
benzophenone) and dichloromethane (DCM, over calcium hydride)
were freshly distilled under nitrogen atmosphere before use. All the
other anhydrous solvents were purchased from Sigma-Aldrich and
used as received. Deuterated solvents are obtained from Cambridge
Isotope Laboratories and used as received. Thin layer chromatog-
raphy (TLC) with Merck TLC silica gel 60 F254 plate was used to
monitor reaction. UV, potassium permanganate and iodine staining
was used to visualize compounds on TLC plates. Regenerated
cellulose dialysis tubing (MWCO 3500 Da) was purchased from
Fisher Scientific. Deionized water was obtained from a Merck
Millipore Integral 3 water purification system. For biological studies,
NIH 3T3 fibroblasts were purchased from Millipore, Singapore. All
broth and agar were purchased from Becton Dickinson Company
1
caprolactone) (PBrCL) in 83% yield (7.2 g). H NMR (300 MHz,
CDCl ): δ 7.37 (m, Ar−H from Bn−), 5.21 (s, PhCH −), 4.53−3.88
3
2
(m, −OCH − and −CH(Br)−), 3.66 (t, −CH OH), 2.24−1.91 (m,
2
2
1
3
−CH(Br)CH −), 1.85−1.37 (m, −CH(Br)CH CH CH −).
NMR (75 MHz, CDCl ): δ 169.7, 65.5, 45.7, 34.3, 27.8, 23.8.
C
2
2
2
2
3
Mn,NMR = 4.8 kDa, Mn,GPC = 4.4 kDa, PDI = 1.29. To a solution of
PBrCL (7.5 g, 39 mmol of repeating unit) in DMF (120 mL) was
added sodium azide (NaN , 5.1 g, 78 mmol), and the mixture was
3
stirred at room temperature overnight before concentrated under
vacuum. The concentrated solution was diluted with toluene followed
by centrifugation to remove the insoluble solid. The supernatant was
concentrated and dried in vacuo afterward to afford the final desired
(
Franklin Lakes, US) and used as received. Bacteria strains used (
Escherichia coli ATCC8739, Staphylococcus aureus ATCC29213,
Pseudomonas aeruginosa PAO1, Bacillus subtilis, Enterococcus faecalis
ATCC 29212, Enterococcus faecium ATCC 19434 and drug-resistant
Staphylococcus aureus MRSA BAA40, MRSA USA300) were
purchased from ATCC. Drug-resistant Staphylococcus aureus MRSA
1
PN CL (5.6 g, 93%). H NMR (400 MHz, CDCl ): δ 7.38 (m, Ar−H
3
3
from Bn−), 5.22 (s, PhCH −), 4.40−4.11 (m, −OCH −), 3.97−3.78
2
2
(m, −CH(N )−), 3.67 (t, −CH OH), 1.94−1.68 (m, −CH(Br)
3
2
1
3
1
−7 were clinical strains isolated from local hospital (TTSH).
CH CH CH −), 1.62−1.44 (m, −CH(Br)CH CH CH −). C NMR
2
2
2
2
2
2
1
13
Instruments for Characterization. H and C and 2D NMR
(100 MHz, CDCl ): δ 170.4, 65.4, 61.9, 30.9, 28.1, 22.3. M
= 4.0
n,NMR
3
spectra were recorded on Bruker Avance 300, Bruker Avance 400,
Bruker AVIII 400 and JEOL JNM-ECA 400 spectrometers using
deuterated solvents as reference. Mass spectra were obtained using an
Agilent 6230 TOF LC/MS with an electrospray (ESI) source with
purine and HP-0921 as an internal calibrates. Organic phase gel
permeation chromatography (GPC) was carried out on a Shimadzu
liquid chromatography system equipped with a Shimadzu refractive
index detector (RID-10A) and two Agilent Polargel columns
operating at 40 °C using DMF (with 1 wt % LiBr) or THF as the
eluent at a flow rate of 1 mL/min using polystyrene kit as standard.
Aqueous phase GPC was carried out on an Agilent liquid
chromatography system equipped with an Agilent refractive index
detector with two Shodex OHpak columns operating at 40 °C using
kDa, Mn,GPC = 4.6 kDa, PDI = 1.30.
General Procedure for Modification of PN CL with Grafting
3
Compounds. The modification of PN CL was modified from
3
37
literature procedure. All the modification reactions were conducted
using a similar procedure; therefore, the procedure of modification
with 1-propargyl-3-butyl-imidazolium bromide is taken as an example
herein. To the solution of PN CL (129 mg, 1.0 equiv. azide group)
3
and 1-propargyl-3-butyl-imidazolium bromide (212 mg, 1.05 equiv)
in DMF (5 mL), anhydrous triethylamine (8.4 mg, 0.1 equiv) was
added and the mixture was subject to two freeze−pump−thaw cycles
before the addition of CuI (15.8 mg, 0.1 equiv). The mixture was
subject to another two freeze−pump−thaw cycles before sealed under
argon and stirred at 40 °C overnight. The resulted mixture was diluted
with water and transferred into dialysis tubing directly followed by
dialyzed against highly diluted EDTA solution (0.25 mg/L) for 2 days
and deionized water for another 1 day. Puffy solid could be obtained
after lyophilization.
Degradability Examination. The degradation ability of polymers
was studied using the selected sample as a model. Polymer was
incubated in phosphate-buffered-saline (PBS, PH = 7.4) at 37 °C.
Aliquots were taken out at certain time intervals and monitored
directly by GPC.
0.05 M NaCl solution as the eluent at a flow rate of 0.5 mL/min using
pullulan kit as standard to determine M , M and polydispersity index
n
w
(
PDI = M /M ).
n w
Synthesis of α-Bromo-ε-caprolactone (α-BrCL). The α-
bromo-ε-caprolactone (α-BrCL) was synthesized according to the
54
literature procedure started from cyclohexanone. Briefly, cyclo-
hexanone (14.7 g, 0.15 mol) was dissolved in dry diethyl ether (150
mL) followed by the addition of N-bromosuccinimide (NBS, 28.0 g,
0
0
.158 mol). The solution was stirred and ammonium acetate (1.16 g,
.015 mol) was added portion-wise. After stirring for 0.5 h at room
Minimum Inhibitory Concentration (MIC) Determination.
temperature, the solid was filtered off and the filtrate was washed with
water, dried over Na SO , concentrated and subject to flash column
Minimum inhibition concentrations (MICs) were measured following
55
standard broth dilution method with minor modification. Bacterial
cells were grown overnight in Mueller−Hinton broth (MHB, Difco,
Becton, Dickinson and Company) at 37 °C. The bacteria were 1:100
2
4
chromatography, giving pale yellow liquid α-bromo-cyclohexanone
17.8 g, 67%). To the solution of α-bromo-cyclohexanone (10.0 g, 56
(
5
mmol) in anhydrous dichloromethane (DCM, 200 mL), 3-
chloroperoxybenzoic acid (m-CPBA, 25.7 g, 85 mmol, ca. 70% with
water) was added and the solution was stirred at room temperature
for 24 h. The mixture was cooled to −20 °C and filtered. The filtrate
was washed successively with Na S O and saturated NaHCO
subcultured in MHB to a midexponential phase and diluted to 5 × 10
−
1
CFU·mL in fresh MHB. Stock solutions of PCLs were prepared in
−
1
the MHB medium at a concentration of 1024 μg·mL . The solutions
were 2-fold serially diluted in MHB medium, and 50 μL of each
dilution was placed in each well of 96-well microplates (Nunc,
ThermoScientific) followed by the addition of 50 μL of the bacterial
suspension. The plate was mixed in a shaker incubator for 10 min
before incubated at 37 °C for 18 h, and the absorbance at 600 nm was
measured with a microplate reader (TECAN, infinite F200). A
positive control without polymer and a negative control without
bacteria were included. MIC was determined as the lowest
concentration of the compound that inhibited the growth of bacteria
by more than 90%. All tests were done in three independent tests with
duplicate per test.
2
2
3
3
solution thoroughly until no acid could be detected by TLC. The
organic layer was dried over Na SO , concentrated and subject to
2
4
flash column chromatography, affording the desired monomer α-
1
bromo-ε-caprolactone in 41% yield (4.4 g). H NMR (400 MHz,
CDCl ): δ 4.85 (dd, J = 6.2, 3.6 Hz, 1H), 4.77−4.63 (m, 1H), 4.37−
3
4
(
2
.20 (m, 1H), 2.20−2.11 (m, 2H), 2.11−1.93 (m, 2H), 1.92−1.76
m, 2H). 13C NMR (125 MHz, CDCl ): δ 169.8, 69.7, 48.2, 31.8,
3
7
9
+
9.1, 25.2. MS (ESI) m/z calcd. for C H BrO [M + H] 192.99,
6 10 2
8
1
+
found 192.96; calcd. for C H BrO2 [M + H] 194.98, found
6
10
1
94.96.
Hemolysis Studies. Fresh human blood was collected from a
health donor (IRB-2015-03-040) and used within the same day.
Human blood was drawn directly into K2-EDTA-coated Vacutainer
tubes to prevent coagulation of blood and stored at 4 °C for 30 min. 1
mL of blood was mixed with 9 mL of PBS and centrifuged at 1,000
rpm for 5 min. Supernatant was discarded, and red blood cells
Synthesis of Poly(α-azido-ε-caprolactone) (PN CL). The
3
poly(α-azido-ε-caprolactone) (PN CL) was synthesized according
to the reported procedure with some modifications.
solution of α-bromo-ε-caprolactone (8.7 g, 45 mmol) in anhydrous
toluene (20 mL), benzyl alcohol (BnOH, 162 mg, 1.5 mmol) and
3
3
7,38
To the
C
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Figure 2. Confirmation of the characteristic protons from the main chain of PCL with 2D NMR spectra. (a) Confirmation of C from the main
γ
chain. (b) Confirmation of H from the main chain (c,d). Confirmation that the H is isolated from other signals in imidazolium bearing PCLs.
γ
γ
(
RBCs) were collected. The RBCs were washed with PBS three times
seeded into a 96-well tissue culture plate and incubated at 37 °C in a
and resuspended to a final concentration of 5% (v/v) in PBS. A 2-fold
dilution series of polymer in PBS solution was prepared, 50 μL red
blood cell suspension was mixed with 50 μL polymer solution in each
well and incubated for 1 h at 37 °C in an inoculation shaker with
continuous shaking at 150 rpm. The 96-well plates were centrifuged at
humidified incubator with 5% CO for 24 h. Polymer in culture
2
medium solutions at 100 μg/mL and 200 μg/mL were added into the
96-well plate seeded with cells and incubated at 37 °C in a humidified
incubator with 5% CO incubator for 24 h. Cells incubated with only
2
DMEM were used as positive nontoxic controls. Afterward, the
culture medium containing polymer was removed, and each well was
1
,000 rpm for 10 min. After centrifugation, 80 μL centrifuge
−
1
washed with PBS prior to the addition of MTT solution (1 mg·mL
supernatant samples were transferred to a new 96-well plate and
diluted with 80 μL PBS, and hemolytic activity was calculated by
measuring absorbance at 540 nm using a 96-well plate spectropho-
tometer (Benchmark Plus, BIO-RAD). PBS buffer (pH 7.4) was used
as a negative hemolysis control, and Triton X-100 (0.1% v/v in PBS)
was used as a positive control. The percentage of hemoglobin release
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. All data
were obtained from the mean value of three replicates.
in DMEM). After another 4 h of incubation, the MTT solution was
aspirated and 100 μL dimethyl sulfoxide (DMSO) was added into
each well, and the plate was shaken at 150 rpm for 10 min, after which
the absorbance of each well was measured at 570 nm using a
microplate reader spectrophotometer (BIO-RAD, Benchmark Plus).
The cell viability results were expressed as percentages relative to the
absorbance obtained in the control experiment.
Average abs of treated cells
Average abs of controls
%Cell viability =
× 100%
Cytotoxicity Assays. The mammalian cell biocompatibility study
was tested toward 3T3 fibroblast cell using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) in colorimetric assay.
Bacteria Killing Kinetics. Time kill study was conducted by
incubating bacteria with different concentrations of polymer/anti-
−1
biotics and determining CFU·mL at various time points. Bacterial
cells were grown overnight in Mueller−Hinton broth medium at 37
°C. After subculturing to a midexponential phase, bacteria were
3
T3 cells (ATCC) were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum
FBS) and 1% antibiotics (penicillin/streptomycin). The cells in
5
−1
(
diluted to 5 × 10 CFU·mL in fresh MHB. Polymer or antibiotic
were added to 1000 μL bacteria in MHB suspension in Eppendorf
tubes to achieve a final polymer/antibiotic concentration of 4 × MIC,
2 × MIC, 1 × MIC and 0.5 × MIC respectively. Bacteria in MHB
suspension without addition of polymer were used as positive control.
tissue culture flask were cultured at 37 °C in a humidified incubator
with 5% CO until 80% confluence was reached. 3T3 cells were
2
harvested from the confluence flask by trypsinization. Cell number
4
was determined using a hemocytometer and 10 cells/well were
D
DOI: 10.1021/acs.biomac.8b01577
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Table 1. Antibacterial, Hemolytic Activity and Mammalian Cell Biocompatibility of Resulting PCLs
Cell viability (%),
3T3
b
MIC (μg/mL)
Selectivity
a
S. aureus
29213
MRSA
BAA40
E. coli
8739
P. aeruginosa
HC50 (μg/mL),
HC /
100 μg/
mL
200 μg/
mL
50
MIC
Entry
P1
P2
P3
P4
P(3)5
P(3)6
P(3)7
P(3)8
P(2)9
Sample
PAO1
RBC
P(C4Im)CL
P(C6Im)CL
P(C8Im)CL
32
4
8
32
8
8
16
128
4
32
4
8
16
8
16
32
256
8
32
8
16
64
32
32
16
512
16
16
128
32
32
128
32
64
256
>512
64
1158
687
332
24
149
346
386
1361
139
337
1497
1796
>12500
1380
>12500
36
172
42
1
19
43
24
11
35
42
47
56
>195
43
81
20
18
4
27
6
9
4
6
5
7
95
6
6
35
45
72
51
82
P(C10Im)CL
P(C8Im)0.63CL-co-P(Glc)0.37CL
P(C8Im)0.48CL-co-P(Glc)0.52CL
P(C8Im)0.37CL-co-P(Glc)0.63CL
P(C8Im)0.2CL-co-P(Glc)0.8CL
P(C6Im)0.76CL-co-P(Glc)0.24CL
31
32
48
98
33
38
80
84
92
85
90
P(2)10 P(C6Im)0.64CL-co-P(Glc)0.36CL
P(2)11 P(C6Im)0.5CL-co-P(Glc)0.5CL
P(2)12 P(C6Im)0.38CL-co-P(Glc)0.62CL
P(2)13 P(C6Im)0.29CL-co-P(Glc)0.71CL
P(2)14 P(C6Im)0.33CL-co-P(Gal)0.67CL
8
8
64
16
32
64
32
64
32
32
128
64
64
64
256
512
>512
256
512
128
256
64
P(2)15 P(C6Im)0.35CL-co-
128
>195
P(Man)0.65CL
P(2)16 P(C6Im)0.33CL-co-
64
64
64
512
5782
90
96
99
93
96
P(GlcNAc)0.67CL
P17
P(Glc)CL
>512
>512
>512
>512
>12500
−
a
b
Values obtained from the plotted hemolysis curve. Calculated based on the MIC values of S. aureus 29213.
The bacteria suspensions with polymers were incubated in an
inoculation shaker at 37 °C with continuous shaking. Aliquots were
taken at different time intervals (0, 0.25, 0.5, 1, 2, 4, 6, and 24 h) and
been found to be active against bacteria with moderate
21,42,56
toxicities.
Thus, various functionalized polycaprolactone
derivatives with moderate molecular weight of about 4 to 5
kDa were targeted by controlling the feeding ratio and a single
peak was observed in GPC for each polymer, indicating a well-
controlled polymerization. For example, a PBrCL of 4.8 kDa
(DP of 25) was obtained by controlling the feeding ratio of
benzyl alcohol and monomer at 1:30. Considering the 83%
yield, we can conclude that the targeted molecular weight was
successfully obtained in a controlled manner (Supporting
Information, Figure S1 and S2). Subsequently, the bromo
substituents on PBrCL were converted into azido groups by
1
0-fold serial diluted in PBS for plating. The diluted aliquots were
plated on LB agar and incubated at 37 °C and CFU of each sample
was determined after 20 h. The killing model for the comparison with
polymer degradation was carried out in PBS with same conditions
except no medium. Two independent experiments with duplicate for
each test were performed for each polymer/pathogen combination,
−1
and the resulting average values are plotted on CFU·mL against
time.
RESULTS AND DICUSSION
■
reaction with sodium azide (NaN ) in DMF at room
3
Synthesis of Poly(azido-ε-caprolactone) (PN CL) with
3
temperature. The resulting poly(α-azido-ε-caprolactone)
Clickable Side Groups. The functionalizable monomer of α-
bromo-ε-caprolactone was synthesized using previously
1
(
PN CL) was characterized with H NMR and the results
3
54
indicated the complete conversion of bromo to azido groups
reported procedure and functionalizable poly(ε-caprolac-
(
Supporting Information, Figure S1).
Conjugation of Poly(α-azido-ε-caprolactone) with
tone) were synthesized by reported procedure with some
37,38
modifications (Figure 1).
Cyclohexanone was converted
Alkyne-Imidazoliums and Alkyne-Carbohydrates via
CuAAC Click Chemistry. The postmodification of the
into α-bromocyclohexanone by bromination with N-bromo-
succinimide (NBS) followed by Baeyer−Villiger oxidation with
obtained PN CL was conducted using CuAAC click reaction
3
-chloroperoxybenzoic acid (m-CPBA) as the oxidant,
3
37
via a modified literature procedure. Briefly, the click reaction
was carried out utilizing the CuI/triethylamine combination in
anhydrous and degassed DMF at a slightly elevated temper-
ature of 40 °C because of poor solubility of cationic
compounds and carbohydrates at room temperature. Initially,
as a first trial, a functionalized PCL modified with 1-propargyl-
3-hexyl-imidazolium bromide (C6Im) and prop-2-ynyl-D-
glucopyranoside (Glc) was synthesized successfully in a one-
producing the monomer α-bromo-ε-caprolactone (α-BrCL)
in moderate yield (41%). With the monomer in hand, the
poly(α-bromo-ε-caprolactone) (PBrCL) was prepared by ring-
opening polymerization using benzyl alcohol (BnOH) as
initiator and tin(II) 2-ethylhexanoate (Sn(Oct) ) as catalyst.
2
The antibacterial activities and toxicity of cationic polymers
have been found to be influenced by molecular weights.
Polymers with moderate molecular weights have generally
E
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Article
Table 2. Antibacterial Activity of Selected Samples against other Gram-Positive Strains Including Resistant Strains
a
MIC (μg/mL)
Entry
B. subtilis 6633 E. faecalis 29212 E. faecium 19434 MRSA USA300 MRSA 1 MRSA 2 MRSA 3 MRSA 4 MRSA 5 MRSA 6 MRSA 7
P(3)6
P(3)7
8
32
8
16
64
16
32
32
16
32
64
64
256
64
8
32
16
32
64
32
32
32
8
32
32
32
128
32
8
16
16
16
64
32
64
32
8
32
16
16
64
32
64
32
16
64
8
16
16
16
64
32
32
32
16
64
32
32
128
32
16
32
32
64
128
64
8
32
32
32
128
64
P(2)11
P(2)12
P(2)13
P(2)14
P(2)15
P(2)16
128
128
256
128
256
128
128
128
64
64
64
64
64
64
64
64
a
B. subtilis, E. faecalis, E. faecium, MRSA BAA40, MRSA USA300 were purchased from ATCC. MRSA 1−7 were clinical strains isolated from local
hospital (TTSH).
positive bacteria (Table 1, P1−P4). Among these PCLs,
polymers possessing imidazolium with intermediate length (6
or 8) carbon tails have better antibacterial activities than those
possessing shorter and longer carbon chains (P1 and P4).
With the exception of P1, the other imidazolium function-
alized PCLs had relatively high toxicity toward mammalian
cells. The hemolytic activities of P1 to P3 were reasonable,
with a selectivity index of 171 and 41 for P2 and P3,
respectively. P4 is too hemolytic, likely because the side chain
with 10 carbons is too long.
signals on the main chain of PCL are isolated from the others,
which could be confirmed by 2D NMR and applied in
calculating the click efficiency as well as molecular weight
To determine whether increasing the hydrophilicity of the
polymers can improve the toxicity profile of the polymers,
carbohydrates were introduced into two series with 8 and 6
alkyl side chains (i.e., P5 to P8 and P9 to P13, respectively)
and their effects were systematically studied with different
molar ratios and various carbohydrates.
52
(
Figure 2, full spectra see Supporting Information). The
HMBC of P(Glc)CL indicates H coupled with the adjacent
β
carbonyl C and C , proving H is on the adjacent carbon C of
α
γ
β
β
carbonyl group. With the information from HMQC of
P(Glc)CL, H has coupled with only C , proving it is the
Comparing P3 with their glycosylated derivatives (P5 to
P8), the MIC values against Gram-negative strains increased
significantly with increasing ratios of carbohydrate in the
polymers whereas the MIC values went up less significantly for
Gram-positive bacteria. It is probably due to the imperme-
γ
γ
only proton connected with carbon C . Furthermore, the H
γ
γ
only coupled with the C in the HMQC of PCLs bearing
γ
imidazolium as well, indicating H is the isolated proton signal
γ
in the main chain. The molecular weight of the polymers was
ability of the outer membranes of Gram-negative bacteria to
1
59,60
calculated from both H NMR and GPC, and the results are
large hydrophilic molecules (Table 1, P5−P8).
However,
summarized in Table S1. (The GPC traces were unimodal
the C8-Im derivatives’ hemolysis and cytotoxicity did not
decrease until the sugar content is rather large (i.e., with P8).
On the other hand, for similar ratios of carbohydrate and
imidazolium, PCLs bearing C6-Im has higher MICs than those
possessing C8-Im despite their lower hemolysis and lower 3T3
toxicity (Table 1, P6 vs P11 and P7 vs P12). As with P3
derivatives (i.e., P5−P8), the MIC increased much less
significantly with Gram-positive bacteria when the ratio of
glycosylated substitution was increased. However, their toxicity
to 3T3 cells, as well as their hemolytic toxicity, was significantly
reduced. For example, P13 has fairly balanced profiles of
bactericidal activities against Gram-positive bacterial and good
selectivity indices for 3T3 and red blood cells. Additionally,
more Gram-positive strains including B. subtilis, Enterococcus
faecalis, Enterococcus faecium and MRSA USA300, together
with a panel of other clinical MRSA strains from local
hospitals, were used to evaluate selected compounds.
Comparable results (Table 2) to those in Table 1 were
obtained, indicating they (including P11 which has good 3T3
and red blood cell compatibility, together with MICs) are
potentially bactericidal toward deadly MRSA strains. Thus, we
can conclude, the alkyl side chain length, and content of
imidazolium in these PCL-backbone polymers are crucial to
the antibacterial activity. Interestingly, only the shorter C6-Im
can achieve a more balanced MIC and selectivity values
(
Figure S2).) Moreover, the actual content of resulting
polymers is very close to the designed feeding ratio of grafting
agents (Table S2).
Antibacterial Activities and Toxicity of the Resulting
Poly(ε-caprolactone). The antibacterial efficacy of the
resulting PCLs was first evaluated by employing both Gram-
negative (Escherichia coli (E. coli) and Pseudomonas aeruginosa (
P. aeruginosa)) and Gram-positive bacteria (Staphylococcus
aureus (S. aureus) and methicillin-resistant Staphylococcus
aureus (MRSA)). The minimum inhibitory concentration
(
MIC), or the lowest concentration of compound needed to
prevent visible growth of bacteria, of various polymer
derivatives were measured against the different bacterial
strains. We measured hemolytic toxicity as the concentration
causing 50% hemolysis of human red blood cells (HC ). We
measured the selectivity of the functionalized PCLs as the ratio
between HC and MIC values (Herein, MIC values against S.
5
0
5
0
aureus was used). Toxicity toward a typical mammalian cell
3T3 fibroblasts) at 100 and 200 μg/mL (several times the
(
MICs) was also measured.
As imidazolium salts were reported as good antibacterial
5
7,58
agents,
imidazolium salts with variable alkyl tail length
were grafted onto the PCLs. The resultant PCL derivatives
have good efficacy against both Gram-negative and Gram-
F
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Figure 3. Dose-dependent hemolytic activity of resultant PCLs. (a) PCLs bearing different ratios of glucose. (b) PCLs bearing different
carbohydrates. Glc = glucose, Gal = galactose, Man = mannose, GlcNAc = glucosamine.
Figure 4. Killing kinetics against (a) MRSA BAA40: (i) P(2)15, (ii) vancomycin. (b) MRSA USA300: (i) P(2)15, (ii) vancomycin.
possibly because the PCL backbone is relatively hydrophobic.
Also, with the decreasing ratios of imidazolium in the
intermediate range (Table 1, P5−P7, and P9−P12), the
MIC values increased moderately for Gram-positive bacteria
but dramatically for Gram-negative bacteria. Overall, the
hemolysis of the PCL-Im derivatives are generally quite good
3b), and C6-Im with glucose, mannose and glucosamine had
markedly improved 3T3 compatibility.
With regard to selectivity against bacteria over red blood
cells, P13 and P15 showed the best results (>195), followed by
P2 (>172) and P16 (>90). However, considering both the
hemolytic selectivity and cytotoxicity, P15 (P(C6Im)0.35CL-co-
P(Man)0.65CL) was postulated to have the most satisfactory
balance in this polymer system.
(
with selectivity ratios of at least 40 even for P3).
Furthermore, to study the effects of different carbohydrates,
various sugars (glucose, galactose, mannose, glucosamine)
were attached in comparable molar ratios. No significant
difference was observed in MIC values (Table 1, P13−P16).
However, it is noteworthy that PCLs with C6-Im bearing
glucose and mannose are relatively less hemolytic probably
attributed to their difference in polarity (Table 1 and Figure
With this promising compound in hand, the killing kinetics
of the compound was studied and compared against
vancomycin, which is the last resort antibiotics against
MRSA infection. By exposing MRSA BBA40 to a series of
concentrations of synthesized polymer and vancomycin, rapid
bactericidal effects were observed at 1 × MIC concentration of
G
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Article
our polymer whereas vancomycin could not kill the bacteria
within 6 h even at 4 × MIC concentration (Figure 4a).
Another MRSA strain tested (USA300) has revealed the same
trend although some regrowth could be observed at lower
concentration probably due to slight degradation of anti-
bacterial agents (Figure 4b). Besides, to examine the
degradability of our polymers, a model study was carried out
with P15 in physiological pH condition at 37 °C. The
preliminary results indicated that the polymers are hydrolyti-
cally degradable. In addition, no noticeable degradation
occurred before inhibiting bacteria and significant loss of
activity after degradation was observed (Figure S5). This
feature endows the polymers with great advantages over
enzymatically degradable polymers, rendering such polymers
more promising in further applications.
ORCID
Mary B. Chan-Park: 0000-0003-3761-7517
Xue-Wei Liu: 0000-0002-8327-6664
Funding
Nanyang Technological University (RG14/16 and RG132/14)
and the Ministry of Education, Singapore (MOE 2013-T3-1-
002).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded and supported by Nanyang Techno-
logical University (RG14/16 and RG132/14) and the Ministry
of Education, Singapore (MOE 2013-T3-1-002).
Compared with bacteriostatic antibiotics, cationic polymers
are preferred in many situations against resistant bacteria,
biofilm bacteria or persistent bacteria where clinical studies
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Supporting Information
The Supporting Information is available free of charge on the
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AUTHOR INFORMATION
Corresponding Authors
Email: xuewei@ntu.edu.sg (Xue-Wei Liu).
Email: mbechan@ntu.edu.sg (Mary B. Chan-Park).
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