Chitosan-Based Peptidopolysaccharides as Cationic Antimicrobial Agents and Antibacterial Coatings

Article abstract of DOI:10.1021/acs.biomac.8b00270

The rapid spread of multidrug-resistant bacteria has called for effective antimicrobial agents which work on a more direct mechanism of killing. Cationic peptidopolysaccharides are developed in the present work to mimic the peptidoglycan structure of bacteria and to enhance the membrane-compromising bactericidal efficacy. Antimicrobial CysHHC10 peptide was grafted to the C-2 (amino) or C-6 (hydroxyl) position of chitosan backbone via thiol-maleimide "click" conjugation, utilizing the maleimidohexanoic linkers. The peptidopolysaccharide with primary amino backbone intact (CSOHHC) exhibited higher bactericidal activity toward Gram-positive and Gram-negative bacteria, in comparison to that with amino backbone grafted with the peptide (CSNHHC). Both peptidopolysaccharides also exhibited lower hemolytic activity and cytotoxicity than free CysHHC10 peptide due to the moderation effect contributed by the chitosan backbone. For targeting the Gram-positive bacteria in particular, the CSOHHC expressed 4- and 2-fold increases in hemo- and cytoselectivity, respectively, as compared to the CysHHC10 peptide. In an extended application, peptidopolysaccharide antibacterial coatings were formed via layer-by-layer assembly with tannic acid. The peptidopolysaccharide coatings readily killed the adhered bacteria upon contact while being cytocompatible by maintaining more than 60% viability for the adhered fibroblasts. Therefore, the peptidoglycan-mimetic peptidopolysaccharides are potential candidates for anti-infective drugs in biomedical applications.

Full text of DOI:10.1021/acs.biomac.8b00270

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Chitosan-based Peptidopolysaccharides as Cationic
Antimicrobial Agents and Antibacterial Coatings
Dicky Pranantyo, Li Qun Xu, En-Tang Kang, and Mary B Chan-Park
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00270 • Publication Date (Web): 19 Apr 2018
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Chitosan-based Peptidopolysaccharides as Cationic Antimicrobial Agents and
Antibacterial Coatings
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Dicky Pranantyo1, Li Qun Xu1, En-Tang Kang1*, Mary B. Chan-Park2*
1 Department of Chemical & Biomolecular Engineering
National University of Singapore
4 Engineering Drive 4, Kent Ridge
Singapore 117585
2 Centre of Antimicrobial Bioengineering
School of Chemical and Biomedical Engineering
Nanyang Technological University
Singapore 637459
* Corresponding Authors
Eꢀmail: cheket@nus.edu.sg (E.T.K)
mbechan@ntu.edu.sg (M.B.C.P)
* ORCID
EnꢀTang Kang: 0000ꢀ0003ꢀ0599ꢀ7834
Mary B. ChanꢀPark: 0000ꢀ0003ꢀ3761ꢀ7517
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Abstract
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The rapid spread of multidrug resistant bacteria has called for effective antimicrobial
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agents which work on
a
more direct mechanism of killing. Cationic
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peptidopolysaccharides are developed in the present work to mimic the peptidoglycan
structure of bacteria and to enhance the membraneꢀcompromising bactericidal
efficacy. Antimicrobial CysHHC10 peptide was grafted to the Cꢀ2 (amino) or Cꢀ6
(hydroxyl) position of chitosan backbone via thiolꢀmaleimide ‘click’ conjugation,
utilizing the maleimidohexanoic linkers. The peptidopolysaccharide with primary
amino backbone intact (CSOHHC) exhibited higher bactericidal activity towards
Gramꢀpositive and Gramꢀnegative bacteria, in comparison to that with amino
backbone grafted with the peptide (CSNHHC). Both peptidopolysaccharides also
exhibited lower hemolytic activity and cytotoxicity than free CysHHC10 peptide due
to the moderation effect contributed by the chitosan backbone. For targeting the
Gramꢀpositive bacteria in particular, the CSOHHC expressed fourꢀ and twoꢀfold
increases in hemoꢀ and cytoselectivity, respectively, as compared to the CysHHC10
peptide. In an extended application, peptidopolysaccharide antibacterial coatings were
formed via layerꢀbyꢀlayer assembly with tannic acid. The peptidopolysaccharide
coatings readily killed the adhered bacteria upon contact while being cytocompatible
by maintaining more than 60% viability for the adhered fibroblasts. Therefore, the
peptidoglycanꢀmimetic peptidopolysaccharides are potential candidates for antiꢀ
infective drugs in biomedical applications.
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Keywords: peptidopolysaccharide, chitosan, peptidoglycanꢀmimetic, antimicrobial
peptide, antibacterial coating.
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1. Introduction
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In the era where the widespread exploitation of antibiotics has been exercised
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haphazardly, there is an overwhelming demand for the development of novel
antimicrobial compounds in order to match the rapid growth of drugꢀresistant traits in
clinically significant pathogens.1 An alternative approach to reduce the propagation of
drugꢀresistant evolvement is by employing cationic compounds to directly target the
cytoplasmic membrane of bacteria. A library of cationic polymethacrylates,2,3
polycarbonates,4ꢀ6 polyamides,7,8 polypyridines,9 polynorbornenes,10,11 and
polypeptoids12,13 have been developed to combat pathogenic bacteria. As an intrinsic
part of the innate immune system in many organisms, cationic peptides have recently
emerged as antimicrobial compounds with high killing potency towards a broad
spectrum of bacteria due to the combined electrostatic effects and amphiphilic
functions. Despite the minimum risk for inducing the resistant trait in bacteria, the
applicability of antimicrobial peptides (AMPs) is limited by poor pharmacokinetics
and proteolytic stability, as well as high toxicity towards blood and mammalian
cells.14 In addition to being disruptive towards plasma membrane of bacteria, cationic
compounds also interrupt cells membrane of other organisms. Molecular design and
engineering approaches have been carried out to prepare the next generation of AMPs
that exhibit moderate toxicity and retain the membrane targeting ability. Conjugation
of poly(ethylene glycol) (PEG) to the Nꢀterminal of synthetic CaLL peptide
comprising fragments of LLꢀ37 and cecropin A peptides retained 50% microbial
inhibitory concentration, which is 3ꢀfold higher than free CaLL, while improving lung
tissue compatibility following airway delivery.15 Conjugation of cationic KSLW
decapeptide to PEGylated phospholipid micelles also retained antimicrobial activity
while inhibiting lipopolysaccharideꢀinduced severe vascular inflammatory responses
in human umbilical vein endothelial cells, as compared to the free peptide.16
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Hyperbranched polyglycerols decorated with pendant aurein 2.2 peptide and its RW
mutant, 77c peptide, demonstrated better cell and blood compatibilities, albeit lower
antimicrobial activity, than the free peptides.17,18
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Bacterial cell walls are different from those other organisms due to the presence of
peptidoglycan, which is located immediately outside their cytoplasmic membrane. In
Gramꢀpositive bacteria, the peptidoglycan layer is substantially thicker than that in
Gramꢀnegative bacteria. This peptidoglycan consists of a polysaccharide backbone
with pendent alternating residues of Nꢀacetylmuramic acid and Nꢀacetylglucosamine
in equal amounts. Inspired by this structure, there is a growing interest to combine
peptides with polysaccharide in order to recreate a peptidoglycan mimetic for
targeting bacterial cell walls. A large number of polysaccharides are biocompatible in
nature and therefore can also contribute to reducing the toxicity of cationic AMPs.19
In particular, chitosan is a class of linear mucopolysaccharide comprising of
Dꢀ
glucosamine and Nꢀacetylꢀ ꢀglucosamine derived from deacetylation of natural chitin,
D
which is widely known for its hemocompatible, histocompatible, and biodegradable
properties.20 Molecular conjugation of chitosan with αꢀ or εꢀpolylysines produced
cationic peptidopolysaccharides which mimicked the peptidoglycan structures,
resulting in higher selectivity towards bacteria as compared to mammalian cells.21,22
Chitosan conjugated with AMP and gelatinaseꢀcleavable peptide was reported to
transform into a fibrous nanostructure upon reaching the bacterial infection site,
exposing the AMP residues for multivalent cooperative electrostatic interactions with
bacterial membranes.23 Nisin was grafted to quaternary ammonium chitosan through
an enzyme catalysed reaction to enhance its limited antimicrobial activity.24
Conjugation of chitosan with anoplin, a decapeptide isolated from the venom of
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solitary wasp, was also reported to enhance the antibacterial activity and
hemocompatibility of the AMP.25 However, almost all conjugations with peptides
were carried out at the Cꢀ2 (amino) position of chitosan, and the structural
configuration of these conjugates has one fundamental difference from the target
peptidoglycan, which has pendent amino acid residues located on the Cꢀ3 (hydroxyl)
position of its polysaccharide backbone.
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In this study, maleimideꢀfunctionalized linkers were introduced to the Cꢀ2 or Cꢀ6
(hydroxyl) positions of chitosan backbone, which subsequently served as conjugation
sites for cysteineꢀterminated HHC10 (KRWWKWIRW) AMP via thiolꢀmaleimide
‘click’ chemistry (Scheme 1). The antimicrobial activity of HHC10 peptide has been
confirmed in in vitro and in vivo tests,26,27 and the ‘clickability’ of its cysteineꢀ
terminated form has been reported previously.28 The resulting peptidopolysaccharide
with the backbone amino groups intact was hypothesized to provide higher
protonation capacity and waterꢀsolubility than that with the backbone amino groups
grafted. Maleimide functionalization on the Cꢀ2 position of chitosan was carried out
by maintaining continuous protection of the backbone primary amines in the
protonating medium. Under this condition, there was a finite probability that the
maleimide functionalization could also occur on the Cꢀ3 position of chitosan,29 and
subsequently led to a configuration more closely resembling the peptidoglycan
structure. In addition, the intact amino groups of chitosan backbone could contribute
additional electrostatic affinity to target the negatively charged surfaces of bacterial
membrane. The antimicrobial activity towards Gramꢀpositive and Gramꢀnegative
bacteria, hemocompatibility, and cytotoxicity of the two peptidopolysaccharides with
different configurations were compared. To extend their potential applications,
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antibacterial coatings of the peptidopolysaccharides were constructed via layerꢀbyꢀ
layer assembly mediated by polyphenolic tannic acid. Bacteria adhesion assays were
performed on the peptidopolysaccharide coatings to evaluate their antibacterial
efficacy and cytocompatibility, the two important criteria for biomaterials application.
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2. Experimental Section
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2.1. Materials
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Chitosan (CS, Mn 50–190 kD), 6ꢀmaleimidohexanoic acid (MIAc, 90%), oxalyl
chloride (99%), Nꢀ(3ꢀdimethylaminopropyl)ꢀN’ꢀethylcarbodiimide (EDC, 97%), Nꢀ
hydroxysuccinimide (NHS, 98%), methanesulfonic acid (99%), tannic acid (TA, ACS
reagent), calcium chloride (93%), and methylthiazolyldiphenylꢀtetrazolium bromide
(MTT, 98%) were purchased from SigmaꢀAldrich Chem. Co. (St. Louis, MO).
CysHHC10 (HꢀCKRWWKWIRWꢀNH2, 97%) peptide was purchased from China
Peptides Co. Ltd. (Shanghai, China). Stainless steel (SS) foils (AISI type 304, 0.05
mm thick) were purchased from Goodfellow Ltd. (Cambridge, UK).
Dichloromethane, dimethylformamide, dimethyl sulfoxide, and acetone were of
analytical grade, while phosphateꢀbuffered saline (PBS) was of ultrapure grade.
Escherichia coli (E. coli, ATCC 25922), Pseudomonas aeruginosa (P. aeruginosa,
ATCC 15692), Staphylococcus aureus (S. aureus, ATCC 25923), Staphylococcus
epidermidis (S. epidermidis, ATCC 12228), and standard 3T3 mouse fibroblast cells
were purchased from American Type Culture Collection (ATCC, Manassas, VA).
Fresh whole blood of Wistar Hannover rat was purchased from InVivos Pte Ltd.
(Singapore). The LIVE/DEAD BacLight Bacterial Viability Kit (containing SYTO 9
and propidium iodide), phalloidin, and 4',6ꢀdiamidinoꢀ2ꢀphenylindole (DAPI) were
purchased from Thermo Fisher (Waltham, MA).
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2.2. Conjugation of antimicrobial peptide to the Cꢀ6 (hydroxyl) position of chitosan
6ꢀMaleimidohexanoyl chloride (MICl) was prepared according to procedures reported
in the literature with some modification.30,31 MIAc (1056 mg, 5 mmol) was dissolved
in 15 mL of dichloromethane, and 20 µL (1 drop) of dimethylformamide was added.
The solution was cooled down to 0 °C in an ice bath and maintained under vigorous
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stirring. The flask was vented with calcium chloride packing. Oxalyl chloride (762
mg, 6 mmol) was added dropwise to the solution for 15 min. After the addition, the
reaction was allowed to proceed at room temperature for 3 h. The solvent was
removed using rotary evaporator and dried under reduced pressure to obtain MICl
(dark yellow solid, yield 95%).
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CS (645 mg, 4 mmol saccharide unit) was dissolved in 20 mL of methanesulfonic acid
under vigorous stirring. MICl (459 mg, 2 mmol) was dissolved in 4 mL
methanesulfonic acid, and was added dropwise to the CS solution. The reaction was
allowed to proceed overnight at room temperature. The solution was precipitated and
washed in excess acetone, dialyzed against deionized water for 3 days (molecular
weight cutꢀoff, MWCO 3.5 kDa), and lyophilized to obtain maleimidoꢀOꢀ
functionalized chitosan (CSOMI, brownish fluffy solid, yield 76%).
CSOMI (103 mg, 0.2 mmol maleimido moieties) was dissolved in 1 mL of PBS at pH
7.4. The solution was stirred and degassed with purified argon for 30 min. CysHHC10
(309 mg, 0.2 mmol) was added to the solution and the vial was quickly sealed. The
solution was stirred at room temperature for 24 h. After reaction, the solution was
dialyzed against deionized water for 3 days (MWCO 3.5 kDa) and lyophilized to
obtain peptideꢀfunctionalized chitosan (CSOHHC, paleꢀyellow fluffy solid, yield
57%).
2.3. Conjugation of antimicrobial peptide to the Cꢀ2 (amino) position of chitosan
CS (1289 mg, 8 mmol saccharide unit) was dissolved in 50 mL of aqueous HCl
solution 0.3
M. MIAc (845 mg, 4 mmol), EDC (745 mg, 4.8 mmol), and NHS (552
mg, 4.8 mmol) were dissolved in 20 mL of dimethyl sulfoxide, stirred for 30 min, and
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added dropwise to the CS solution under vigorous stirring. The reaction was allowed
to proceed at room temperature for 24 h. The mixture was dialyzed against deionized
water for 3 days (MWCO 3.5 kDa) to obtain aqueous solution of maleimidoꢀNꢀ
functionalized chitosan (CSNMI). The concentration of CSNMI solution was
determined after lyophilizing small amount of the solution (yield 65%).
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Aqueous CSNMI solution (52 mg CSNMI, 0.1 mmol maleimido moieties) was mixed
with 2 mL of PBS at pH 7.4. The solution was stirred and degassed with purified
argon for 30 min. CysHHC10 (155 mg, 0.1 mmol) was added to the solution and the
vial was quickly sealed. The solution was stirred at room temperature for 24 h. After
reaction, the solution was dialyzed against deionized water for 3 days (MWCO 3.5
kDa) and lyophilized to obtain peptideꢀfunctionalized chitosan (CSNHHC, white
cottonꢀlike solid, yield 55%).
2.4. Deposition of peptidopolysaccharides on SS substrates by TAꢀmediated layerꢀ
byꢀlayer (LbL) assembly
The SS foils were cut into 1 cm × 1 cm coupons, cleaned with piranha solution,
washed and dried according to the previous procedure.32 TA solution (2 mg/mL) was
prepared by dissolving TA in deionized water at pH 8.5 (adjusted with NaOH 2
M).
Peptideꢀgrafted CS solutions (2 mg/mL) were prepared by dissolving CSNHHC or
CSOHHC in deionized water. The clean SS coupons were immersed alternatingly in
the TA solution and peptideꢀgrafted CS solution for 1 h each. The SS functionalized
with TA (SSꢀTA) and SS functionalized with nꢀbilayers of TA and peptideꢀgrafted CS
(SSꢀCSNHHCn or SSꢀCSOHHCn) substrates were immersed in deionized water for 1
h to remove any looselyꢀadhered compound and gently blownꢀdried with purified
argon.
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2.5. Characterization
Chemical structures of the compounds in deuterated water, dimethylsulfoxide, or
chloroform were characterized by 1H NMR spectroscopy on a Bruker ARX 500 MHz
spectrometer. Sizeꢀexclusion gel permeation chromatography (GPC) was performed
on a Waters GPC system equipped with a Waters 1515 isocratic HPLC pump, a
Waters 717 plus autosampler injector, a Waters 2414 refractive index detector, and an
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Agilent PL aquagelꢀOH mixedꢀH 8 µm column, using aqueous sodium sulfate 0.05
M
eluent at 35oC and a flow rate of 1 mL/min. Zeta potential of the compounds in PBS
at pH 7.4 and 22oC was measured on a Malvern zetasizer. Surface compositions of the
modified SS substrates were determined by Xꢀray photoelectron spectroscopy (XPS)
on a Kratos AXIS Ultra DLD spectrometer, equipped with a monochromatized Al Kα
Xꢀray source of 1486.71 eV photons with a constant dwell time of 100 ms, a pass
energy of 40 eV, and a photoelectron takeꢀoff angle (α) of 90° with respect to the
sample surface. Thickness of the bilayer coatings were determined using a VASE J.A.
Woollam variable angle spectroscopic ellipsometer. Crossꢀsectional images of
modified SS substrates were observed using a JEOL JSMꢀ6700 fieldꢀemission
scanning electron microscopy (FESEM). Bacteria and cell adhesions on the substrates
were observed under a Nikon ECLIPSE TiꢀU fluorescence microscope.
2.6. Antimicrobial assays of the peptidopolysaccharides and peptidopolysaccharideꢀ
functionalized SS substrates
Bacterial cells were cultured to a midꢀlog phase in the respective growth media at
37°C according to ATCC protocols, and diluted to 2 × 105 colony forming units
(CFU)/mL in MuellerꢀHinton broth (MHB). Stock solutions of sample compounds
were prepared in MHB at a concentration of 2048 ꢀg/mL, serially diluted by 2ꢀfold,
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and 100 ꢀL of each dilution was placed in a 96ꢀwell plate (Greiner Bioꢀone,
Germany). Then, 100 ꢀL of bacterial suspension was added to each compound
solution. The plate was incubated at 37°C overnight and observed by naked eye.
Bacterial growth made the suspension appear cloudy, while the suspension with no
bacterial growth remained clear.33 The lowest concentration of the compound that
inhibited the growth of bacteria was recorded as minimum inhibitory concentration
(MIC). Subsequently, 100 ꢀL of the suspension in the wells with no visible growth
was spread on a MuellerꢀHinton agar and incubated at 37°C overnight to observe the
viability of the bacteria. The lowest concentration of the compound that killed 99.9%
of the initially inoculated bacteria was recorded as minimum bactericidal
concentration (MBC).
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Midꢀlog phase bacterial cultures were washed and resuspended in PBS at a
concentration of 107 cells/mL. The pristine and functionalized SS substrates were
placed in a 24ꢀwell plate and immersed in 1 mL of the bacterial suspension at 37 °C
for 4 h. After washing thrice with PBS to remove the looselyꢀadhered bacteria, each
sample substrate was stained with LIVE/DEAD BacLight solution for 15 min and
washed once with ultrapure water. The live (appearing green) and dead (appearing
red) bacterial cells adhered on the sample surfaces were observed on a fluorescence
microscope under a green filter (excitation/emission: 470 nm / 525 nm) and a red
filter (excitation/emission: 535 nm / 645 nm).
2.7. Determination of hemolytic activity
Stock solutions of sample compounds were prepared in PBS at a concentration of
16384 ꢀg/mL, serially diluted by 2ꢀfold, and 100 ꢀL of each dilution was placed in a
96ꢀwell plate. Fresh whole rat blood was dispersed in PBS at a concentration of 8%
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(v/v), and 100 ꢀL of the blood suspension was added to each compound solution. The
blood suspension was added to blank PBS as negative control, and to PBS containing
0.1% (v/v) Triton Xꢀ100 as positive control. The plate was incubated at 37°C for 1 h
to allow the interaction between the compounds and the blood cells. Each mixture was
centrifuged at 1500 rpm for 10 min, and the optical absorbance of the supernatant was
measured at 560 nm wavelength under a UV microplate reader (Multiskan GO,
Thermo Scientific). The percentage of hemoglobin release was calculated as [(AS –
AN) / (AP – AN)] × 100%, where AS, AN, and AP are the optical absorbance of the
supernatant from the incubated sample, negative control, and positive control,
respectively.
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2.7. Cytotoxicity assay
Metabolic MTT assay was carried out to determine the cytotoxicity of the sample
compounds. 3T3 fibroblast cells were cultured and resuspended in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1
mM
Lꢀglutamine, and 100 IU/mL penicillin. Then, 100 µL of the suspension
containing 5000 cells were placed in each well of a 96ꢀwell plate. The plate was
incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 24 h.
Sample solutions were prepared by dissolving the respective compounds in the
supplemented DMEM at 2ꢀfold serial dilutions. The medium in each well was
replaced with 100 µL of the sample solutions, and the plate was incubated at 37°C for
24 h. Nontoxic control experiment was carried out using the supplemented DMEM
without any compound. The medium in each well was then replaced with 100 µL of
MTT solution (0.5 mg/mL of MTT concentration in supplemented DMEM). After
additional incubation at 37°C for 4 h, the supernatant was aspirated and 100 µL of
dimethyl sulfoxide was added to dissolve the internalized formazan crystal. After 15
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min, the optical absorbance was measured at 600 nm wavelength using a UV
microplate reader and expressed as a percentage relative to the absorbance of the
nontoxic control.
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Cytotoxicity of the modified substrates was determined using cells adhesion assay.
The 3T3 fibroblasts were resuspended in supplemented DMEM at a concentration of
2 × 105 cells/mL. The pristine and modified SS substrates were placed in a 24ꢀwell
plate and covered with 1 mL of cells suspension. The substrates were incubated at
37°C for 24 h and washed thrice with PBS. The adhered cells were fixated using 1 mL
of PBS containing 4% (v/v) paraformaldehyde for 15 min and washed thrice with
PBS. The fixated cells were treated with PBS containing 0.1% (v/v) Triton Xꢀ100 for
5 min and washed thrice with PBS. The treated cells were stained using 0.5 mL of
PBS containing 1.5 µL of phalloidin and 1 µL of DAPI for 15 min, and washed thrice
with PBS. The stained cells on the substrates were observed on a fluorescence
microscope under a green filter (excitation/emission: 470 nm / 525 nm) and a blue
filter (excitation/emission: 350 nm / 460 nm). The amount of adhered 3T3 fibroblast
cells was counted using National Institute of Health (NIH) ImageJ software.
Statistical analysis of the results over 3 fluorescence micrographs was performed
under oneꢀway analysis of variance (ANOVA) using a Tukey’s post hoc test.
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3.1. Preparation of the chitosanꢀbased peptidopolysaccharides (Scheme 1)
The Oꢀacylation of chitosan with acyl chloride to introduce maleimide functions was
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carried out by maintaining salt formation of the primary amino groups with a strong
protonating solvent. In this amineꢀprotected environment, the substitution site with
acyl group occurred mainly at the Cꢀ6 position and less at the Cꢀ3 position.29 To
ensure major substitution at the Cꢀ6 position, the acyl chloride was set as the limiting
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reactant in the molarꢀfeed composition. The H NMR spectrum of the maleimidoꢀOꢀ
functionalized chitosan (CSOMI, Figure 1) is predominated by the chemical shifts of
glucosamine unit protons at 3.1, 3.5–4.1, and 4.8 ppm.34 New chemical shifts at 1.3,
1.5, 2.0, and 3.3 ppm arise from the alkane protons of hexanoyl linker, indicating
successful acetylation of chitosan.35 The appearance of chemical shift at 6.8 ppm also
signifies the presence of ‘clickable’ maleimido group on CSOMI. The conjugation of
cationic peptide with chitosan was carried out via thiolꢀmaleimide ‘click’ reaction
between the cysteine unit of CysHHC10 and the maleimido groups of CSOMI. This
reaction occurred spontaneously without catalytic assistance, thus avoiding any metal
or organic residues which may interfere with the biological functions. Below pH 7.4,
the maleimido group might also react with the nucleophilic amines of peptide,
ensuring complete conjugation of the maleimide functions, albeit resulting in
uncertainty of the final structure. The chemical shift characteristics of CSOMI and
1
CysHHC10 appear concomitantly in the H NMR spectrum of the HHC10ꢀOꢀ
functionalized chitosan (CSOHHC), suggesting the presence of pendent peptide
moieties grafted to the chitosan backbone (Figure 1).
The introduction of maleimide functions to the Cꢀ2 position of chitosan was carried
out by Nꢀamidation with succinimideꢀactivated 6ꢀmaleimidohexanoic acid (MIAc) to
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obtain the maleimidoꢀNꢀfunctionalized chitosan (CSNMI) solution. Notably, the solid
form of CSNMI was water insoluble, probably due to the lack of protonation ability of
the secondary amines after amide formation. After dialysis, the aqueous CSNMI
solution was subjected to thiolꢀmaleimide ‘click’ reaction with CysHHC10 peptide to
obtain the HHC10ꢀNꢀfunctionalized chitosan (CSNHHC). The chemical shift
characteristics of CS, maleimidohexanoic linker, and CysHHC10 appear
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concomitantly in the H NMR spectrum of the CSNHHC peptidopolysaccharide
(Figure 1), confirming the successful conjugation of pendent peptide moieties to the
chitosan backbone. The solid form of CSNHHC was water soluble due to the
protonation of the abundant primary amino groups in the grafted peptide.
In the 2D 1H–13C heteronuclear multiple bond correlation (HMBC) spectra of CSOMI
and CSNMI (Figure S1 and S2, Supporting Information), the signal crowding at f1
55–98 ppm were dominated by the correlations among chitosan signals, while most of
the correlations among the hexanoic linker signals are present at f1 22–38 ppm. In the
CSOMI spectrum, the signal at f1 174 ppm and f2 2 ppm arose from the correlation
between ester carbon and the adjacent proton of hexanoic linker, indicating the
successful Oꢀacylation. In the CSNMI spectrum, the signal at f1 184 ppm and f2 2 ppm
emerged due to the correlation between amide carbon and the adjacent proton of
hexanoic linker, indicating successful Nꢀamidation. Because amide carbon exhibits
higher shift than ester carbon, this evidence confirms the difference in molecular
configuration between CSOMI and CSNMI. It is assumed that the Oꢀacylation
occurred mainly at the Cꢀ6 position and less at the Cꢀ3 position due to steric
orientation, although the exact percentage cannot be concluded.29
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In the 1H NMR spectra of the peptidopolysaccharides (Figure 1), the chemical shift at
3.5 ppm is attributable to the methine proton in the Cꢀ5 position of the chitosan units,
whereas that at 0.75 and 0.63 ppm are attributable to the methyl protons of the
CysHHC10 units. Based on the integration of the peak areas of these chemical shifts,
the mass fractions of the peptide unit in the CSNHHC and CSOHHC
peptidopolysaccharides are calculated to be 45.2% and 47.6%, respectively (Table 1).
The degree of substitution (DS) of CSOMI was slightly higher than that of CSNMI.
Chitosan unit provides two sites (Cꢀ6 and Cꢀ3 positions) for Oꢀacylation, as compared
to only one site (Cꢀ2 position) for Nꢀamidation. Upon the peptide conjugations, the
DS for conjugated peptide decreased only slightly from the conjugated maleimide,
implying high efficiency of the thiolꢀmaleimide ‘click’ reaction. The zeta potential (ζ)
values of the chitosan and maleimidoꢀfunctionalized chitosans did not show
appreciable difference, but increased significantly upon conjugation with the cationic
peptide, implying the superior electrophoretic mobility of peptidopolysaccharides due
to the presence of positive charges on the molecules.
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From the sizeꢀexclusion gel permeation chromatography (GPC), the numberꢀaverage
molecular weight (Mn) and polydispersity (Mw/Mn) of the chitosanꢀbased compounds
increased progressively with the conjugations of maleimide linkers and CysHHC10
peptide (Figure S3), in agreement with the increasing size and complexity of the
macromolecules. According to the Mn data, the pristine chitosan consists of 119 repeat
units. Previously, Mn determination of chitosan compounds has been reported using
PL aquagelꢀOH mixed (300 mm) column, aqueous acetic acid/sodium acetate eluent,
and nonꢀcharged poly(ethylene oxide)/poly(ethylene glycol) standards of 1970–
1345000 g/mol.36 In the present work, similar type of column and standards were
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used, and aqueous sodium sulfate eluent was employed to minimize the interaction
between the charged polymers and the column. The use of nonꢀcharged standards and
vinylꢀbased stationary phase to characterize charged polymers imposed a limitation in
the Mn determination. It should be noted that GPC data show estimates of relative Mn
and might not reflect the actual values, because correct determination of Mn for these
polymers was challenging due to structural variation accompanied by differences in
hydrodynamic size, shape, and refractive index increment (dn/dc ratio).
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3.2. Preparation of the peptidopolysaccharide coatings via polyphenolꢀmediated
chemistry (Scheme 1)
The antibacterial coatings can be constructed via layerꢀbyꢀlayer (LbL) assembly of
tannic acid (TA) and peptidopolysaccharides. The polyphenolic groups of TA readily
chelate the metal centre of stainless steel (SS) substrates by the formation of tridentate
coordination complexes.37 In comparison of the XPS wideꢀscan spectra (Figures 2a
and 2b) of the SS and TAꢀanchored SS surface (SSꢀTA), the increase in intensity of C
1s coreꢀlevel signal in the latter is consistent with the presence of an organic TA layer
on the SS substrate. The disappearance of characteristic signals of SS metal viz., Cr
2p3/2, Fe 2p3/2 and Ni 2p3/2 coreꢀlevel components with respective binding energies
(BEs) at 577, 711 and 860 eV, suggests that the thickness of TA layer had exceeded
the ~8 nm probing depth in organic matrices of the XPS technique.38 The abundant
trihydroxyphenyl sites on the SSꢀTA surfaces can readily bind with the amino
moieties of the peptidopolysaccharides through Michael addition or Schiff base
reaction.39 A new N 1s coreꢀlevel signal has emerged in the SSꢀCSNHHCn and SSꢀ
CSOHHCn surfaces (Figure 2c–f), confirming the incorporation of
peptidopolysaccharides in the multilayer coatings. The intensity of N 1s signal also
increases with the increase in number of the peptidopolysaccharide layer. From the
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crossꢀsectional view of fieldꢀemission scanning electron microscopy (FESEM) images
(Figure 3a–c), the coating thicknesses of the bilayers on SSꢀCSNHHC10 and SSꢀ
CSOHHC10 surfaces have reached 270 and 400 nm, respectively. The coating
thickness also increases approximately linearly with the number of bilayers (Figure
3d).
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3.3. Antimicrobial activities of the chitosanꢀbased peptidopolysaccharides
The antimicrobial properties of the peptidopolysaccharides towards Gramꢀnegative
(Escherichia coli and Pseudomonas aeruginosa) and Gramꢀpositive (Staphylococcus
aureus and epidermidis) bacteria were investigated based on their minimum inhibitory
concentration (MIC) using the broth dilution method. Polymers that do not contain
AMP, namely CS and CSOMI, did not show any activity towards all the bacteria
challenged up to the highest concentration tested (Table 2). On the contrary, the native
CysHHC10 peptide exhibited strong antimicrobial activity in the MIC range of 8 to 64
µg/mL towards all bacteria. In comparison to that of native AMP, the MIC values of
CSNHHC peptidopolysaccharides increased by severalꢀfold towards all bacteria. The
increase in MIC values was due to the inclusion of chitosan moiety, implying that the
antimicrobial effect was mainly contributed by the AMP content in the
peptidopolysaccharide molecules. It has also been reported that nonꢀbactericidal CS
backbone contributed to overall mass of nisinꢀconjugated CS, resulting in lower
antimicrobial potency towards E. coli and S. aureus as compared to free nisin.24
Interestingly, compared to the native AMP, CSOHHC showed similar MIC values
towards Gramꢀpositive bacteria, and only twoꢀfold increase of MIC values towards
Gramꢀnegative bacteria. On the basis of peptide mass fraction, CSOHHC exhibited
MIC values lower than that of the native AMP, namely at 15 and 4 µg/mL (in
comparison to the corresponding values of 32 and 8 µg/mL) towards S. aureus and
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epidermidis, respectively. In general, the outermost surface of Gramꢀpositive bacteria
is encased with a thick peptidoglycan layer, whereas the cell wall of Gramꢀnegative
bacteria mainly consists of lipid bilayer. Due to the resemblance of CSOHHC with the
peptidoglycan structure, it is postulated that the peptidopolysaccharide exhibited
affinity and infiltration capability towards the cell wall of Gramꢀpositive bacteria,
leading to the improved inhibitory of growth. The antimicrobial activity of CSOHHC
was found to be higher than that of CSNHHC, presumably due to the free amino
group on the peptidopolysaccharide backbone which increased the protonation
capacity and water solubility. It was also reported that primary amine could exist in an
equilibrium form between free amine (no charge) and protonated amine (positively
charged), and its antibacterial effect decreased upon introduction of more alkyl arms
or rigid structure.40 The CSOHHC retained a higher DS than CSNHHC, and thus also
exhibited higher antimicrobial activity due to higher peptide content. The influence of
DS on activity has been reported and reviewed.41 It has also been reported that the
arginineꢀfunctionalized chitosan with higher DS was more effective in permeabilizing
the cell membrane of E. coli and P. fluorescens than the same derivative with lower
DS.42 The parameter of killing efficacy is expressed as minimum bactericidal
concentration (MBC, Table S1), which were consistently recorded twice the MIC
values as an indication to the potency of peptidopolysaccharides to eradicate
pathogens.
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3.5. Hemotoxicity and cytotoxicity of the chitosanꢀbased peptidopolysaccharides
Hemotoxicity and cytotoxicity of the peptidopolysaccharides were assayed with rat
whole blood and mouse 3T3 fibroblast cells, respectively, as the model mammalian
standards. The hemolytic parameter was determined by measuring the lowest
concentration of a compound which incites lysis towards 50% of the red blood cells
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(HC50). The half maximal inhibitory concentration (IC50) of a compound was
determined by measuring the metabolic methylthiazolyldiphenylꢀtetrazolium bromide
(MTT) residue to express its cytotoxicity. The compounds with no AMP content,
namely CS and CSOMI, did not show any hemolytic activity up to the highest
concentration tested at 16384 µg/mL (Table 2). Similarly, they did not show any
indication of cytotoxicity up to the highest concentration tested at 2048 µg/mL. The
CysHHC10 peptide exhibited strong hemoꢀ and cytotoxicity with HC50 and IC50
values of 1024 and 84 µg/mL, respectively. Upon conjugation to chitosan backbone,
the HC50 and IC50 values of the CSNHHC and CSOHHC peptidopolysaccharides
increased several folds in comparison to that of the CysHHC10 peptide. In addition, at
any effective concentration, the cell viability of fibroblasts incubated with the
peptidopolysaccharides was higher than that incubated with CysHHC10 (Figure 4).
These results indicate that the biocompatible nature of the chitosan backbone can be
molecularly engineered to moderate the toxicity of AMP.
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Therapeutic indices of the antimicrobial compounds were expressed by measuring the
quantitative ratio between biocompatibility and efficacy to obtain the hemoselectivity
(HC50/MIC) and cytoselectivity (IC50/MIC) values (Table 2). The hemoselectivity of
CysHHC10 is higher than other AMPs, for example indolicidin,43 melittin,44 magainin
and protegrin45 with HC50/MIC (towards S. aureus) values of 8, 8, 5 and 3,
respectively. In general, the CSNHHC and CSOHHC peptidopolysaccharides
exhibited HC50/MIC values higher than that of the CysHHC10, implying that
conjugation with the chitosan backbone have increased the hemoselectivity of the
native AMP. In comparison to the CysHHC10 peptide, the CSOHHC
peptidopolysaccharides exhibited fourꢀ and twoꢀfold increases in HC50/MIC and
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IC50/MIC values, respectively, when calculated using MIC towards both
Staphylococci. The structural mimetic of peptidoglycan in CSOHHC presumably has
contributed to its selective targeting of the Gramꢀpositive bacterial membranes,
leading to the improved therapeutic indices.
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3.6. Antibacterial activity and cytocompatibility of the peptidopolysaccharideꢀ
functionalized surfaces
Antibacterial coatings consisting of the chitosanꢀbased peptidopolysaccharides and
tannic acid (TA) were constructed on stainless steel (SS) surface via layerꢀbyꢀlayer
assembly. After being immersed in the bacterial suspension (107 cells/mL) for 4 h, the
pristine SS and TAꢀfunctionalized SS (SSꢀTA) surfaces were colonized by live
(appearing green) E. coli and S. aureus, indicating their susceptibility towards
bacterial adhesion (Figure 5). On the contrary, most of the bacteria adhered on the
TAꢀpeptidopolysaccharide functionalized SS (SSꢀCSNHHC10 and SSꢀCSOHHC10)
surfaces were compromised (appearing red), confirming the antibacterial properties of
the peptidopolysaccharide coatings. The cationic peptidopolysaccharide layers
presumably have instigated membrane permeability and destabilization upon contact,
leading to the death of Gramꢀnegative and ꢀpositive bacteria. Surface
cytocompatibility is also of an equally important criterion in biomaterial applications.
Mouse fibroblasts (2 × 105 cells/mL) were incubated with the pristine and
functionalized SS surfaces, followed by the nucleic and cytoplasmic staining with
4',6ꢀdiamidinoꢀ2ꢀphenylindole (DAPI) and phalloidin. A large number of viable cells
were able to adhere and proliferate on the nontoxic SS surface, while a smaller
number of viable cells adhered on the peptidopolysaccharideꢀfunctionalized SS
surfaces (Figure 6). The viability of adhered cells on the SSꢀCSNHHC10 and SSꢀ
CSOHHC10 surfaces were 66 and 62%, respectively, relative to that of the SS surface.
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These results demonstrated potential applications of the peptidopolysaccharides as
antibacterial coatings with moderate cytocompatible properties.
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Peptidopolysaccharides with peptidoglycanꢀmimetic structures were prepared by
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grafting CysHHC10 peptide to the Cꢀ2 (amino) or Cꢀ6 (hydroxyl) position of the
chitosan backbone via thiolꢀmaleimide ‘click’ conjugation, utilizing the
maleimidohexanoic linkers. The peptidopolysaccharide with primary amino backbone
intact (CSOHHC) showed higher bactericidal activity as compared to that with the
amino backbone grafted with the peptide (CSNHHC). The CSOHHC
peptidopolysaccharide increased the hemoꢀ and cytoselectivity of the free peptide
towards Staphylococci by fourꢀ and twoꢀfold, respectively, consistent with its
peptidoglycanꢀmimetic structure for targeting the Gramꢀpositive bacteria. This
improved therapeutic index indicates the successful molecular engineering approach
to moderating the toxicity of AMP without losing its activity. Antibacterial coatings
consisting of peptidopolysaccharide and tannic acid layers could be constructed via
layerꢀbyꢀlayer assembly, to give rise to peptidopolysaccharidesꢀbased antimicrobial
surfaces. In the static adhesion assay, E. coli and S. aureus adhered on the
peptidopolysaccharide coatings were killed upon contact. On the other hand, the
adhered 3T3 cells retained more than 60% viability on the peptidopolysaccharide
coatings. The antibacterial efficacy and cytocompatibility of the present
peptidopolysaccharides and coatings thus readily suggest their applicability in
biomedical and healthcare fields.
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Associated Content
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The Supporting Information is available free of charge on the ACS Publications
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website.
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MBC values, 1H–13C HMBC spectra, GPC chromatograms of the chitosan derivatives
and peptidopolysaccharides, XPS spectra and surface elemental stoichiometries of the
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peptidopolysaccharide coatings, detailed H NMR spectra of all compounds, and
determinations of DS, yields, and surface coatings density.
Acknowledgement
This work was funded and supported by a Singapore Ministry of Education Tier 3
Grant (MOE2013ꢀT3ꢀ1ꢀ002).
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Captions for Scheme, Tables and Figures
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Scheme 1. Preparation of the peptidopolysaccharides by conjugation of AMP to the
Cꢀ2 (amine) or Cꢀ6 (hydroxyl) positions of chitosan, and peptidopolysaccharide
functionalization of SS substrates via LbL assembly mediated by tannic acid.
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Table 1. Molecular weight, zeta potential, and composition of the polysaccharides
and peptidopolysaccharide conjugates.
Table 2. Antimicrobial activity, hemolytic effect, cytotoxicity and therapeutic index
of the polysaccharides and peptidopolysaccharide conjugates.
Figure 1. 1H NMR spectra of the polysaccharides and peptidopolysaccharide
conjugates.
Figure 2. XPS wideꢀscan spectra of the (a) pristine SS, (b) TAꢀmodified SS, and (c–f)
SS surfaces coated with 5ꢀ and 10ꢀbilayers of TA and peptidopolysaccharides.
Figure 3. Crossꢀsectional FESEM images of the (a) pristine SS, (b) SSꢀCSNHC10, (c)
SSꢀCSOHHC10 surfaces, and (d) coating thickness of the TAꢀpeptidopolysaccharide
bilayers on SS surfaces measured using ellipsometry. Scale bar is 100 nm. Error bar
denotes standard deviation obtained from three replicates.
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