Polymer

Chemistry
View Article Online
PAPER View Journal | View Issue

Methacrylate-ended polypeptides and

Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.

Cite this: Polym. Chem., 2017, 8,

polypeptoids for antimicrobial and antifouling
6386 coatings†
Qiang Gao,a Peng Li, *b Hongyang Zhao,c Yashao Chen,d Liu Jiangd and
Peter X. Ma*a,e,f,g,h

Methacrylate-ended polypeptides/polypeptoids were successfully synthesized via ring-opening

Received 3rd September 2017,
Accepted 25th September 2017
DOI: 10.1039/c7py01495c
rsc.li/polymers
polymerization (ROP) of N-carboxyanhydrides (NCA). These oligomers were further initiated under ultra-
violet (UV) irradiation by a polydopamine ( pDA) layer which is attachable to the surface of virtually all
materials to generate a polymer brush coating. This brush-like polymer coating comprising cationic anti-
microbial polypeptides (MePpep) and antifouling polysarcosine (MePsar) exhibited effective antimicrobial
activity against four pathogens (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and
Candida albicans), as well as antifouling activity in the resistance to protein and platelet adhesion, and
thus prevented biofilm formation for up to 7 days. An in vitro cytotoxicity study showed that this coating is
biocompatible with mouse fibroblast (L929) cells. More importantly, this coating exhibited significant
anti-infectivity in vivo. This dual-functional polymer brush coating can be immobilized on the surface of
multiple categories of materials through the mussel-inspired pDA coating, and thus should be widely
applicable for combating infection in many classes of bio-medical materials.

1. Introduction human beings.1 According to the estimation in a review com-

The worldwide overuse of antibiotics has become a recent
cause for concern due to its link to the emergence of resistant
microbial strains. “Superbugs” that show resistance to most
current antibiotics are a prospective unimaginable threat to all
a less likely to trigger resistance, have attracted more attention
Center of Biomedical and Engineering and Regenerative Medicine, Frontier Institute
of Science and Technology, Xi’an Jiaotong University, Xi’an 710054, China.
E-mail: mapx@umich.edu; Fax: +(86)29-83395131; Tel: +(86)29-83395361
b
Key Laboratory of Flexible Electronics (KLOFE) and Institute of Advanced Materials
(IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials
(SICAM), Nanjing Tech University, Nanjing 211816, China.
E-mail: iampli@njtech.edu.cn; Fax: +(86) 25-83587982; Tel: +(86) 25-83587982
c
Center of Applied Chemical Research, Frontier Institute of Science and Technology,
Xi’an Jiaotong University, Xi’an 710054, China
d
Key Laboratory of Applied Surface and Colloid Chemistry, School of Chemical and
Chemical Engineering, Shaanxi Normal University, Xi’an 710119, China
e
Department of Biomedical Engineering, University of Michigan, Ann Arbor,
Michigan 48109, USA
f
Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor,
Michigan 48109, USA
g
Macromolecular Science and Engineering Center, University of Michigan, Ann Arbor,
Michigan 48109, USA
h
Department of Materials Science and Engineering, University of Michigan,
Ann Arbor, Michigan 48109, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7py01495c
missioned by the UK Government, 10 million people could die
each year from drug-resistant bacteria by 2050.2 Also, the
development of new antibiotics is far surpassed by the rate of
development of bacterial resistance. To update the arsenal
against bacteria, scientists have taken different approaches.
Instead of traditional antibiotics, natural antimicrobial pep-
tides (AMPs, also known as host defense peptides), which are
recently.3 However, AMPs exist in very low concentrations natu-
rally, and the production of these species is expensive. Hence,
a number of chemical synthetic AMPs and their mimics have
been developed.4–13 Recently, the ring-opening polymerization
(ROP) of α-/N-substituted amino acid N-carboxyanhydride
(NCA) monomers for the synthesis of polypeptides and poly-
peptoids has gained prominence.14,15 Although this approach
cannot generate precise amino acid sequences, it allows the
facile and low-cost synthesis of random or block co-polypep-
tides/polypeptoids with potential applications, such as in anti-
microbials, tissue engineering, drug delivery, coatings, etc.16–22
Microbial colonization and growth on the surface of
implantable materials and medical devices (e.g., catheters,
indwelling needles, hip replacements, etc.) is a serious
problem that imperils human health, especially when caused
by drug-resistant “superbugs”.23 Surface-associated bacterial
communities embedded in the extracellular polymeric matrix,

6386 | Polym. Chem., 2017, 8, 6386–6397 This journal is © The Royal Society of Chemistry 2017

Polymer Chemistry
i.e., so-called biofilms, further increase the resistance towards
conventional drugs.24 Recent efforts have been dedicated to
preparing AMP coatings in order to inhibit bacterial attach-
ment and thereby prevent subsequent biofilm formation.25,26
AMPs have been immobilized on surfaces by surface graft
polymerization,27,28 click chemistry,29 layer-by-layer assem-
bly,30 etc. However, most of the current methods involve multi-
step treatments or post-synthesis.31,32 The post-synthesis of
AMPs usually requires rigorous chemistry conditions and may
Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.
also change the biological properties of the AMPs.
Furthermore, these surface modifications generally do not
fulfill the requirements of different categories of substances.
Recently, research groups have expended more effort to
develop a simple and universal method of modifying the
surface of biomaterials. Mussel-inspired materials for bio-
medical applications (e.g., coatings, hydrogels and drug car-
riers) are being extensively studied.33–35 Zhou et al. found that
a mussel-inspired pDA coating which was attachable to vir-
tually all inorganic and organic surfaces, could generate free
radicals under irradiation and initiate the surface grafting
reaction of vinyl monomers.36
Although immobilized antimicrobial molecules such as
AMPs and their synthetic mimics were capable of killing the
bacteria on contact, these coatings were often stuck with
fouling issues.37 Non-specific adhesion of protein, dead cells
and debris on the surface blocks the coating from contact with
bacteria, resulting in the loss of its efficacy. Thus the use of
just antimicrobial molecules was not enough for an effective and
long-lasting anti-infective coating.38 Coatings which retain not
only antimicrobial activity but also antifouling or cell-releasing
features have been reported lately.39–45 In our recent work,
block copolymers containing bactericidal and cell/protein-
repellent segments were synthesized.46–48 Polyethylene glycol
(PEG) was used as the antifouling segment. However, some
Scheme 1 Synthesis of methacrylate-ended polymers (MePs) and pDA-assisted grafting of MePs. (A) Synthesis of methacrylate-ended polypeptide
(MePpep) and polysarcosine (MePsar). (B) Schematic illustration of thin-coating deposition of pDA (Step 1) and surface grafting of MeP polymer
brushes through UV irradiation (Step 2).
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
studies indicated that PEG can be easily oxidatively degraded,
and its degradation products may have unexpected toxicity.49,50
Polysarcosine (Psar), a hydrophilic homo-polypeptoid, has
exhibited excellent antifouling activity comparable with PEG.51
Furthermore, Psar is originating from the natural and non-
toxic amino acid, which is more safe and reliable for bio-
medical usage. Thus Psar is an excellent alternative to PEG. In
addition, the synthesis of block copolymers is also tedious.
Hence, our main target herein is to design facile synthesized
functional biomolecules for the development of anti-infective
coatings that are attachable to multiple categories of surfaces
via a simple method under ambient conditions without using
harsh chemicals or conditions.
In order to avoid the post-synthesis of biomolecules, metha-
crylate-ended peptides, as well as peptoids, were synthesized
via NCA chemistry (Scheme 1A). These methacrylate-ended
polymers (MePs) were ready-made for further polymerization
to form a functional surface coating via facile mussel-inspired
surface modification (Scheme 1B). 2-Aminoethyl methacrylate
hydrochloride (AEMA) was used to initiate the methacrylate-
ended antimicrobial polypeptides (MePpep) and antifouling
polypeptoids (MePsar). Cationic antimicrobial MePpep, which
was synthesized by random copolymerization of lysine (Lys)
and phenylalanine (Phe) residues, had an excellent minimal
inhibitory concentration (MIC) (25 μg mL−1, 4.9 µM) toward
Gram-positive bacteria (S. aureus), Gram-negative bacteria
(E. coli and P. aeruginosa), and fungus (C. albicans) (ESI
Table S1†). MePsar with excellent protein/cell repellent pro-
perties and biocompatibility was synthesized by the polymeriz-
ation of sarcosine-NCA monomers.52 The methacrylate groups
at the end of these biomolecules undergo initiation by free
radicals generated on the pDA film under UV, and then
polymerized into surface polymer brushes. Two brush coat-
ings, poly(Ppep) and poly(Ppep/Psar), were successfully pre-
Polym. Chem., 2017, 8, 6386–6397 | 6387

Paper
pared, as confirmed by various characterization techniques.
Comparatively, the poly(Ppep/Psar) coating not only exhibited
exceptional antimicrobial activity with an inhibition rate of
above 94.6% for all four clinically significant pathogens, but also
greatly reduced non-specific protein adsorption and platelet
adhesion in vitro. Additionally, this polymer brush coating was
non-toxic to mammalian cells in vitro, and retained its excel-
lent biological properties in a rat infection model, successfully
preventing implant-related infection by S. aureus. Besides its
Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.
use as an anti-infective coating, we anticipate that this category
of methacrylate-ended polypeptide/polypeptoid oligomers will
also be applicable in other areas, such as tissue engineering,
drug delivery, etc.
2. Experimental section
2.1. Materials
H-Lys(z)-OH (99%), L-phenylalanine (99%), dopamine hydro-
chloride (DA, 98%), and 2-aminoethyl methacrylate hydro-
chloride (AEMA, 90%) were obtained from Sigma-Aldrich
(USA). Anhydrous N,N-dimethylformamide (DMF, >99.8%), tri-
phosgene (98%), hydrogen bromide (33 wt% solution in acetic
acid), anhydrous sodium sulfate (99%), bovine serum albumin
(BSA, Mn = 66 000 g mol−1, 98%), and lysozyme (Mn = 14 400
g mol−1, 98%) were purchased from J&K Scientific Ltd, China.
Anhydrous tetrahydrofuran (THF), ethyl ether, and n-hexane
were obtained by using a solvent-drying system (PureSolv
MD-5, Innovative Technology). Staphylococcus aureus (ATCC
29253), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa
(ATCC 27853), and Candida albicans (ATCC 10231) were
obtained from the American Type Culture Collection (ATCC).
The AlamarBlue® cell viability assay reagent, LIVE/DEAD®
BacLight™ bacterial viability kit, and Micro BCA™ protein
assay kit were purchased from Thermo Fisher Scientific Inc.
Phosphate buffered saline (PBS) was obtained from Sigma-
Aldrich and sterilized in an autoclave at 121 °C before use.
Deionized water used in the experiments was purified by using
a Millipore water purification system with a minimum resis-
tivity of 18.2 MΩ cm. Poly(dimethylsiloxane) (PDMS) was pre-
pared by mixing a silicone elastomer and a curing agent in a
weight ratio of 10 : 1 (Sylgard 184, Dow Corning, MI, USA) and
curing in 90 mm Petri dishes for polymerization at 80 °C
for 12 h.
2.2. Synthesis of NCA monomers
Lys(z)-NCA, Phe-NCA, and Sar-NCA were synthesized as pre-
viously reported in the literature and confirmed by 1H-NMR
(ESI Fig. S1†).48,53 All synthesized NCA monomers were herme-
tically stored at −80 °C before use.
2.3. Synthesis of methacrylate-ended polypeptides (MePpep)
Lys(z)-NCA (2.08 g, 12.5 mmol) and Phe-NCA (1.30 g,
12.5 mmol) were dissolved in 35 mL of anhydrous DMF under
a nitrogen atmosphere in a glovebox. The initiator, AEMA
(166 mg, 1 mmol), was dissolved in another 5 mL of anhy-
6388 | Polym. Chem., 2017, 8, 6386–6397
View Article Online
Polymer Chemistry
drous DMF and subsequently added to the reaction mixture.
The reaction solution was stirred at 60 °C for 72 h. After com-
pletion of the reaction, the methacrylate-ended polypeptide
precursor (Lys(CBz)12.5-ran-Phe12.5) was precipitated in 300 mL
of cold ether and centrifuged (3200g, 5 min), and then dried
under reduced pressure (white solid, 3.11 g, 92%). 1H-NMR
(400 MHz, d6-DMSO): δH 1.22–1.77 (m, –CH2CH2CH2–), 1.86 (s,
–CH3–), 2.94–3.22 (m, –CH2–), 4.21–4.48 (m, –CH–), 4.99
(C6H5–CH2–), 5.66 (s, vCHH), 6.05 (s, vCHH), 7.21–7.33 (m,
–C6H5–) ppm (ESI Fig. S2†).
Deprotection of the benzyl-protected polypeptide precursor
(MePpep(z), 200 mg) was performed by the addition of HBr
solution (300 µL, 33% in acetic acid), followed by continuous
agitation at 0 °C for 1 h. The deprotected polypeptide (Lys
(NH3+)12.5-ran-phe12.5) was precipitated by the addition of cold
ether (10 mL) and centrifuged (3200g, 5 min), and then col-
lected solid precipitate was washed with ether thrice (10 mL).
Finally, the polypeptide was dialyzed (MWCO 3500) against de-
ionized water for 2 days and subsequently freeze-dried (fluffy
white solid, 175 mg, 87.5%). 1H-NMR (400 MHz, d6-DMSO):
δH 1.35–1.65 (m, 70H, –CH2CH2CH2–), 1.86 (s, 3H, –CH3–),
2.95–3.16 (m, 48H, –CH2–), 4.21–4.56 (m, 24H, –CH–), 5.70 (s,
1H, vCHH), 6.06 (s, 1H, vCHH), 7.27 (m, 60H, –C6H5–) ppm.
Mw = 5080 g mol−1 (ESI Fig. S2†).
2.4. Synthesis of methacrylate-ended polysarcosine (MePsar)
Sar-NCA (1.56 g, 50 mmol) was dissolved in 16 mL of
anhydrous DMF. AEMA (166 mg, 1 mmol) was dissolved in
another 5 mL of anhydrous DMF and subsequently added to
the reaction mixture. The reaction solution was stirred at
60 °C for 72 h. Directly after completion of the reaction,
the polymer was precipitated in 200 mL of cold ether,
centrifuged (3200g, 5 min), and then isolated polysarcosine
was dried under reduced pressure, dissolved in water, and
subsequently freeze-dried (light yellow solid, 1.42 g, 91%).
1
H-NMR (400 MHz, d6-DMSO): δH 1.86 (s, 3H, –CH3), 2.75–3.25
(m, 145H, N–CH3), 4.14–4.44 (m, 90H, –CH2–), 5.67 (s, 1H,
vCHH), 6.07 (s, 1H, vCHH) ppm. Mw = 3365 g mol−1
(ESI Fig. S3†).
2.5. Measurement of minimal inhibitory concentration (MIC)
The antimicrobial activity of the synthesized MePs against
S. aureus (Gram positive), E. coli and P. aeruginosa (Gram
negative), and C. albicans (fungi) was investigated by using a
broth micro-dilution MIC assay. Briefly, 100 μL of MeP solu-
tion in Mueller-Hinton broth (MHB, for bacteria) or Yeast-Malt
broth (YMB, for fungi) at different concentrations (3.1, 6.3,
12.5, 25, 50, 100, 200, and 400 µg mL−1) was filled into the
wells of a 96-well culture plate by a 2-fold dilution method.
Another 100 µL of 106 CFU mL−1 bacterial suspension was
then carefully added to each well. The plate was incubated at
37 °C for 24 h (28 °C for C. albicans), and the turbidity was
read by a microplate reader (SpectraMax Paradigm, Molecular
Devices) at 600 nm. The MIC was determined to be the poly-
peptide concentration at which no bacterial growth could be
observed.
This journal is © The Royal Society of Chemistry 2017

Polymer Chemistry
2.6. Photo-polymerization of polymer brush coatings of MePs
Pristine PDMS films were cut into 1 × 1 cm squares and sub-
sequently ultrasonically cleaned in hexane for 1 h, then rinsed
thoroughly with a copious amount of acetone, and finally
vacuum dried overnight. pDA coating was carried out by
immersing the substrate in a buffer solution (10 mM Tris-HCl,
pH 8.5) containing 2 mg mL−1 of DA, followed by shaking (200
rpm) at room temperature for 2 h with exposure to air. All
Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.
samples were then rinsed with deionized water and dried
under a stream of nitrogen. The pDA-coated substrates were
immersed in 2 mL of MePpep solution (25 wt%, 4.92 µM) or
MePpep/MePsar mixed solution (12.5/8.3, wt%, 2.46/2.46 µM)
respectively, then placed in a quartz reaction vessel and
degassed with nitrogen for 30 min. The reaction vessel was
subjected to UV irradiation (365 nm, 10 mW cm−2) under
nitrogen protection for 1 h in a photochemical reactor (XPA-5,
Xujiang Machine-electronic Plant, China). The coated sub-
strates were then rinsed with deionized water and dried under
a stream of nitrogen.
2.7. In vitro antimicrobial test
The in vitro bactericidal potency of the coatings was deter-
mined by following a contact protocol. Firstly, the bacteria
(S. aureus, E. coli, and P. aeruginosa) were pre-cultured in MHB
at 37 °C (YMB for C. albicans at 28 °C) and subcultured until
their optical density at 600 nm reached 0.5–0.7. The bacteria
were then harvested by centrifugation (1400g, 5 min), washed
with PBS thrice, and resuspended in PBS at a final concen-
tration of ∼108 CFU mL−1. A 10 μL-aliquot of the inoculum
suspension was pipetted and spread onto the uncoated
(control), poly(Ppep)- and poly(Ppep/Psar)-coated PDMS sur-
faces (1 × 1 cm2). After a contact time of 1 h, all samples were
placed in 1 mL of PBS and vigorously washed and sonicated
for 10 min; the surviving bacteria were plated with 10-fold
serial dilutions and counted. The antimicrobial rate was calcu-
lated as: kill% = [(CFUControl − CFUCoatings)/(CFUControl )] × 100.
All experiments were carried out in triplicate for each
formulation. LIVE/DEAD staining was also performed to
investigate the viability of the bacteria upon contact with these
coatings.
2.8. Field-emission scanning electron microscopy (FE-SEM)
of biofilms
The bacterial attachment and biofilm formation by S. aureus
and E. coli on the surfaces were observed by FE-SEM.54 Fresh
bacteria at the logarithmic stage of growth were collected and
resuspended (to OD600 = 0.01) in MHB. The uncoated, poly
(Ppep)-, and poly(Ppep/Psar)-coated PDMS films (1 × 1 cm2)
were individually immersed in 2 mL of the bacterial suspen-
sion and incubated at 37 °C for 7 days to allow biofilm growth.
The broth was changed every day. At day 1 and 7, the samples
were washed thrice with sterile PBS to remove the unattached
bacteria, then fixed with 2.5% glutaraldehyde for 2 h, de-
hydrated through a series of graded ethanol solutions (10%,
30%, 50%, 70%, 90%, and 100% each for 15 min), and then
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
dried under a stream of nitrogen. All samples were pre-coated
with gold and observed using FE-SEM.
2.9. Protein adsorption assay
The coating was directly prepared on the bottom surface of the
wells in a 12-well plate according to the method described in
section 2.6; the protein adsorption properties were determined
by the Micro BCA assay. Briefly, the uncoated and polymer
brush-coated surfaces were equilibrated in PBS ( pH 7.4) for
12 h; 2 mL of BSA and lysozyme protein solution (5 mg mL−1
in PBS) was added to the wells and incubated at 37 °C for 24 h.
After this protein adsorption period, each well was washed
with PBS and distilled water twice, respectively. A 1 mL aliquot
of 2.0 wt% aqueous sodium dodecyl sulfate (SDS) solution was
then added to each well, followed by shaking for 2 h and sub-
sequent sonication for 1 h in an ice bath to detach the surface-
absorbed protein. A 100 μL sample of the protein solution was
collected from each well and transferred to a new 96-well plate,
supplemented with 100 μL of BCA reagent and incubated at
60 °C for 1 h. The protein concentration was measured based
on the absorbance at 560 nm by using a microplate reader
(SpectraMax Paradigm, Molecular Devices). Here, the efficiency
of the protein resistance was determined by applying the
equation: protein resistance (%) = [(O.D.TCPS − O.D.coatings)/
O.D.TCPS] × 100.
2.10. Platelet adhesion study
Platelet-rich plasma (PRP) was obtained by centrifuging
citrated whole blood (424g, 15 min).55 PRP (50 μL) was intro-
duced onto the uncoated, poly(Ppep)-, and poly(Ppep/Psar)-
coated (1 × 1 cm2) samples. After incubation at 37 °C for 2 h,
all samples were washed thrice with PBS. The adherent plate-
lets were then fixed with 2.5% glutaraldehyde at 4 °C for 2 h,
dehydrated through a series of graded ethanol solutions (10%,
30%, 50%, 70%, 90%, and 100%, each for 15 min), and dried
under a stream of nitrogen. All samples were pre-coated with
gold and observed using FE-SEM.
2.11. In vitro biocompatibility of poly(Ppep/Psar) coating
All samples were cut into disks with a diameter of 5 mm and
sterilized with 70% ethanol prior to use. L929 cells were
seeded on a tissue culture polystyrene (TCPS) plate at a density
of 6000 cells per cm2 and cultured for 4 h to allow attachment
of the cells. The samples were then gently placed onto the
well, allowing the surface coating to make contact with the
cells. The cells were cultured for 4 days, and the medium was
changed every two days. The cell viability was determined by
AlamarBlue and LIVE/DEAD assays at day 1 and 4.56
2.12. In vivo evaluation of infections
All procedures were carried out according to the Regulation on
the Administration of Laboratory Animals of China (2017 revi-
sion) and approved by the Animal Ethical Committee of the
Xi’an Jiaotong University. Female Sprague-Dawley rats
(8 weeks, ∼200 g) were purchased from the Laboratory Animal
Unit of the College of Medicine, Xi’an Jiaotong University and
Polym. Chem., 2017, 8, 6386–6397 | 6389

Paper
allowed to acclimatize for 7 days in the lab. A rat subcutaneous
S. aureus infection model was generated using a pre-seeding
protocol. The uncoated PDMS (control) and poly(Ppep/Psar)-
coated PDMS films (0.2 × 0.6 cm) were firstly inoculated with
10 µL of S. aureus suspension (108 CFU mL−1 in PBS) and then
subcutaneously implanted into the back of the rats. Post-
operatively, all rats were monitored daily. All of the implants
were retrieved after 5 days, then individually placed in 1 mL of
sterile PBS and sonicated for 5 min to detach the adhered bac-
Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.
terial cells. The bacteria in the resulting suspensions were
counted after serial dilutions and plating. For histological
observations, the tissues around the implants were sectioned
to a thickness of 1–2 mm with a sterilized pair of scissors and
immediately fixed in 4% neutral formalin at 4 °C for 4 days,
and then paraffin embedded. The paraffin embedded tissues
were sectioned and stained with hematoxylin–eosin.
2.13. Characterization
1 pathogenic microbes. Cationic MePpep had a dramatically
H-NMR spectra were recorded on a Bruker Avance 400 MHz
spectrometer. Fourier transform infrared (FTIR) spectra were
collected on a Nexus 670 FTIR ESP spectrometer (Nicolet
Instrument Corp., Waltham, USA) at a resolution of 4 cm−1
using 32 scans. FE-SEM analysis was performed on a Hitachi
S-3000N instrument. The surface morphology and roughness
of the samples were analyzed by atomic force microscopy
(AFM). AFM measurements were performed with a MultiMode
STM microscope controlled by the NanoScope III form the
Digital Instruments system, operating in tapping mode at a fre-
quency of 1 Hz. The surface chemical composition was ana-
lyzed by X-ray photoelectron spectroscopy (XPS) using an Axis
UltraDLD spectrometer (Kratos Analytical Ltd, UK) with a mono-
chromatic Al-Kα X-ray source (1486.71 eV photons). The static
contact angle of the surface was measured at room tempera-
ture by the sessile drop method using a 2 μL water droplet in a
Ramé-Hart telescopic goniometer.
2.14. Statistical analysis
The results are expressed as the mean ± standard deviation.
All experimental data were analyzed using the Student’s t test;
p < 0.05 was considered to be statistically significant.
3. Results and discussion
3.1. Synthesis, characterization, and biological activities of
MePs
Advancements in the ROP-NCA method have offered a facile
route for the synthesis of various polypeptides or polypeptoids.
Ammonium salts are relatively stable and easy to handle and
purify and can initiate the ROP of NCA monomers.57 Here,
methacrylate-ended antimicrobial polypeptides (MePpep) and
antifouling polypeptoids (MePsar) were successfully syn-
thesized using AEMA as the initiator and characterized by
1
H-NMR and FTIR analyses (ESI Fig. S1–S4†). 1H-NMR analysis
confirmed the polymerization and provided further infor-
mation. The 1H-NMR signals of the methacrylate group of the
6390 | Polym. Chem., 2017, 8, 6386–6397
View Article Online
Polymer Chemistry
protected MePpep appeared at 5.66 and 6.05 ppm, and the
signal of the Cbz groups appeared at 4.99 ppm and dis-
appeared after the deprotection process (ESI Fig. S2†). For
MePsar, two obvious and assignable signals were apparent at
2.75–3.15 ppm (m, Ha) and 3.85–4.45 ppm (m, Hb) and the
degree of polymerization was determined to be about 45 (ESI
Fig. S3†). The FTIR results further confirmed the successful
polymerization. The two characteristic stretching vibrations of
the NCA monomers at 1850 cm−1 and 1790 cm−1 disappeared
after initiation with AEMA (Fig. S4†).
In order to achieve potent antimicrobial activity, the Lys/
Phe ratio was set as 1 : 1 and the molar feed ratio of the
monomer/initiator ([MLys + MPhe]/[IAEMA]) was 25, according to
the optimized composition described in previous studies.16,48
The NMR results confirmed that the degree of polymerization
of MePpep was about 24 and the content of hydrophilic
groups (Lys) was about 50% (Fig. S2C†). The antimicrobial
activity of the synthesized MePs was determined with four
lower MIC of 25 μg mL−1 (4.9 µM) for the four clinically signifi-
cant pathogens: Gram-positive (S. aureus), Gram-negative
(E. coli and P. aeruginosa), and fungi (C. albicans). MePsar,
which lacks the cationic and hydrophobic groups, did not
exhibit significant inhibition of the four pathogens at concen-
trations of up to 1000 μg mL−1 (297 μM).
3.2. Polymer brush coatings of MePs
Methacrylate-ended molecules are capable of polymerizing in
the presence of free radicals. The mussel-inspired pDA film is
attachable to virtually all organic, inorganic and metallic sur-
faces, and is capable of generating free radicals (e.g., CO•
and C•) under UV irradiation to initiate the graft polymeriz-
ation of vinyl monomers.36 Inspired by the universality of the
photo-initiated polymerization using the pDA coating, we
decided to use a similar strategy to graft the MePs onto the
surface of pDA pre-coated materials to generate antimicrobial
and antifouling coatings. To produce a polymer brush coating,
pDA-coated PDMS was immersed in an aqueous solution com-
prising a mixture of MePpep and MePsar and irradiated with
UV light in a nitrogen environment. As shown in Fig. 1A, the
Fig. 1 (A) ATR-FTIR spectra of the uncoated PDMS (i), pDA (ii) and poly
(Ppep/Psar) (iii) coated PDMS surfaces. (B) Contact angle measurement.
This journal is © The Royal Society of Chemistry 2017

Polymer Chemistry
attenuated total reflection (ATR)-FTIR spectra of the uncoated
PDMS, pDA-coated PDMS, and poly(Ppep/Psar)-coated
PDMS surfaces presented three groups of well-defined
peaks, indicating the successful formation of the coating.
The peaks around 3400 cm−1 were ascribed to the aromatic
–NHx and –OH stretching vibrations, and the intense, broad
peaks at 3000–2750 cm−1 in the spectrum of the poly(Ppep/
Psar) coating were assigned to the CH2 stretching vibrations
of Group I. The peaks at 1720 cm−1, 1640 cm−1, and
Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.
1530 cm−1 are the representative peaks of the CvO, amide
I, and amide II bonds of the polypeptides (Group II). 48,58
The peaks at 1260 cm−1 (–Si–CH3), 1000 cm−1 (Si–O–Si), and
780 cm−1 (–Si(CH3)2) are attributed to the groups of the
uncoated PDMS. The intensity of Group III decreased sig-
nificantly due to the presence of the poly(Ppep/Psar)
coating covering the PDMS substrate.59 Grafting with the
MePs also altered the wettability of the material surface
(Fig. 1B). MePpep comprises 50% hydrophilic lysine resi-
dues; the contact angle of uncoated PDMS was reduced
from 102 ± 1° to 50 ± 1° after coating with the MePpep
brush. The introduction of MePsar, which is composed of
100% hydrophilic sarcosine residues, further decreased the
contact angle to 44 ± 4°.
Fig. 2 AFM images (i), topography (nm) ( profile corresponding to the white line in panel i) (ii), and high resolution N 1s XPS spectra (iii) of the
(A) uncoated, (B) pDA-coated, (C) poly(Ppep)-coated, and (D) poly(Ppep/Psar)-coated PDMS surfaces. (E) Schematic illustration of patterned
poly(Ppep/Psar) brush coating (i); AFM images (ii), topography (nm) ( profile corresponding to the white line in panel ii) (iii) of the patterned
poly(Ppep/Psar) brush coating.
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
The presence of the polymer brush coating was also deter-
mined by AFM and XPS characterization. Fig. 2A-i and ii show
that the uncoated PDMS surface was flat, with a roughness
(root mean square, RMS) of ∼0.6 nm. The nitrogen peak was
almost undetectable under XPS (Fig. 2A-iii). Fig. 2B-i and ii
show a homogeneous pDA coating with an increased rough-
ness of 58.4 nm. Two new peaks were observed at 399.2 and
401.6 eV (Fig. 2B-iii), which are attributed to the major second-
ary amine and minor amine groups in the pDA coating.60 As
shown in Fig. 2C-i and ii, the poly(Ppep) coating exhibited a
roughness of 35.4 nm, which was a bit lower than the pDA
coating. However, the intensity of the peak (401.8 eV) which
represented amine groups increased significantly owing to the
abundant lysine residues existing in the poly(Ppep) coating
(Fig. 2C-iii).61 The poly(Ppep/Psar) coating showed a roughness
of 44.1 nm (Fig. 2D-i and ii). The incorporation of Psar
reduced the concentration of the lysine residues on the
surface, thus leading to a decreased intensity of the amine
peak at 402.1 eV for the poly(Ppep/Psar) coating (Fig. 2D-iii).
To further verify that the surface-attached pDA film can initiate
polymerization of the MePs, we fabricated a patterned poly
(Ppep/Psar) coating by using the photomask technique; the
formed coating and its thickness could be easily observed with
Polym. Chem., 2017, 8, 6386–6397 | 6391

Paper
AFM (Fig. 2E). Fig. 2E-ii and iii show successful coating with
the patterned poly(Ppep/Psar) brushes; the average thickness
was 310 nm. These results indicated that MeP brushes were
successfully grafted onto the surface of the pDA-coated
materials under UV irradiation.
3.3. Antimicrobial activities of the coatings
After the successful preparation of the MeP polymer brush
Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.
coatings, their contact-active bactericidal ability was evaluated
with four pathogenic microbes (S. aureus, E. coli, P. aeruginosa,
and C. albicans). The antimicrobial activity of the two brush
coatings, poly(Ppep) and poly(Ppep/Psar), grafted on PDMS
was assessed by confrontation with four pathogens through a
surface-contact method; the uncoated PDMS was used as a
control.62 As shown in Fig. 3A, the poly(Ppep) coating, totally
composed of cationic polypeptides, exhibited higher than
99.6% reduction against all four pathogens. The poly(Ppep/
Psar) coating also exhibited robust bactericidal ability against
the four pathogens, and exhibited 97.6%, 99.7%, 94.6%, and
95.6% reduction of S. aureus, E. coli, P. aeruginosa, and
C. albicans, respectively. The bactericidal activity for E. coli was
the same for both coatings, whereas the poly(Ppep/Psar)
coating exhibited slightly lower reduction for the other 3 bac-
teria and fungus.
Fig. 3 Contact bactericidal activity of MeP coatings. (A) Antimicrobial
activity of the poly(Ppep) and poly(Ppep/Psar) coatings against various
pathogens (each data point represents the mean ± standard deviation
for three separately prepared samples). (B) LIVE/DEAD bacterial viability
assay of E. coli on the uncoated (i), poly(Ppep) (ii), and poly(Ppep/Psar)
(iii) brush coatings after 1 h incubation at 37 °C and washing (scale bar =
20 µm).
6392 | Polym. Chem., 2017, 8, 6386–6397
View Article Online
Polymer Chemistry
One important aspect to consider is that although efficient
contact-killing coatings have been developed, they often fail to
maintain their bactericidal activity as they can be easily
masked by an absorbed conditioning film of organic com-
pounds such as proteins or remnants of dead cells. Thus, the
adherence of dead cells to the surface after the contact-killing
assay was observed using the LIVE/DEAD assay (Fig. 3B). The
uncoated, poly(Ppep)-, and poly(Ppep/Psar)-coated PDMS sur-
faces were respectively brought into contact with E. coli for 1 h,
then rinsed with PBS to remove the un-adhered bacteria or
components, and stained with a mixture of SYTO-9 and propi-
dium iodide (PI). SYTO-9 enters all bacterial cells and stains
them green, whereas PI is only able to permeate the damaged
membrane and reduce the fluorescence of SYTO-9, staining
the cells red. As shown in Fig. 3B-i, an abundance of live E. coli
cells (stained green) adhered to the uncoated surface. In con-
trast, no live cells were present on the surface of the poly
(Ppep) coating due to its strong bactericidal activity, whereas
numerous dead cells (stained red) adhered to the surface
(Fig. 3B-ii). This result indicated that the bacterial cell mem-
brane was damaged by our poly(Ppep) coating, thus allowed
the penetration of PI. Previously, Zhou et al. reported that
similar synthesized polypeptides exhibit their antimicrobial
activity by disrupting the bacterial membrane in solution.16
The cell membrane of bacteria contains abundant negative
charged lipids.63 Once the bacteria came into contact with
poly(Ppep) brushes, the positively charged lysine residues and
hydrophobic phenylalanine segments could interact with the
lipid bilayers strongly, thus disrupting the cell membrane and
leading them to death. The strong interaction between the
poly(Ppep) coating and the bacteria also results in the adher-
ence of the dead cells. The accumulation of dead cells on the
surface may block contact of the coating with other pathogens,
thus leading to the failure of the coating in long-term appli-
cations.37 To prevent the fouling of the surface, MePsar was
copolymerized with MePpep to form a dual-functional anti-
microbial and antifouling coating. Polysarcosine is a very
hydrophilic peptoid with excellent cell/protein-resistant pro-
perties.51 As shown in Fig. 3B-iii, no live or dead cells were
found on the surface of the poly(Ppep/Psar) coating. Therefore,
the antimicrobial cationic polypeptides killed the live cells and
antifouling polysarcosine prevented adhesion of the dead
cells.
3.4. Biofilm-resistant activity of the coatings
After adhesion on the surface of the biomaterials, bacteria can
form a biofilm. Bacteria in this form are embedded in a poly-
meric matrix, which makes them much harder to kill than
planktonic individuals. Often, surgical removal of the biofilm-
infected implants may even fail to solve this problem because
the residual bacteria in the body cause recurring infections.64
Thus, the biofilm-resistance is a key factor for antimicrobial
coatings to prevent biomaterial-associated infections. The
uncoated and poly(MeP)-coated PDMS were subjected to
biofilm growth for 7 days and then analyzed by FE-SEM and
LIVE/DEAD staining. As shown in Fig. 4A, numerous S. aureus
This journal is © The Royal Society of Chemistry 2017

Polymer Chemistry
Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.
Fig. 4 Formation of the biofilm by S. aureus (left) and E. coli (right) on the uncoated, poly(Ppep) and poly(Ppep/Psar) coatings, observed with
(A) FE-SEM (scale bar = 10 μm), and (B) LIVE/DEAD bacterial viability assay (scale bar = 20 µm).
or E. coli cells were adhered to the surface of the pristine PDMS
(control) at day 1 and 7. The poly(Ppep) coating, which has
potent bactericidal activity, showed a reduction in the adhered
bacteria compared with the uncoated PDMS. However, the poly
(Ppep) coating was unable to prevent biofilm formation by
S. aureus and E. coli. More bacteria adhered to the surface of
poly(Ppep)-coated PDMS from day 1 to day 7. This may be due
to fouling of the surface by dead cells and other biomolecules
such as proteins. Comparatively, the poly(Ppep/Psar) coating
was capable of preventing biofilm formation by S. aureus and
E. coli up to day 7; no bacteria adhered on the surface. This
result may be due to the existence of numerous poly(Psar)
chains in the surface polymer brushes, which prevents fouling
of the surface. LIVE/DEAD bacterial staining was performed to
further study the biofilm-resistance of the coatings. As shown in
Fig. 4B, a bacterial biofilm was formed on the surface of
uncoated PDMS after 7 days of incubation. The bacteria adher-
ing to the poly(Ppep) coating were also alive at day 7, which may
be attributed to the remnants of adhered dead cells that
blocked the coating, thus causing it to lose its antimicrobial
activity. No live or dead bacteria adhered to the poly(Ppep/Psar)
coating, which indicated that this coating has excellent biofilm-
resistance. Therefore, although the poly(Ppep/Psar) coating has
slightly poorer contact bactericidal potency than the poly(Ppep)
coating, the biofilm-resistance of the poly(Ppep/Psar) coating is
better than that of the poly(Ppep) coating.
3.5. Antifouling activities
For implantable biomaterials, the fouling of their surfaces with
proteins and platelets may promote biofilm formation and may
also result in thrombosis and inflammation.65 Apart from the
antimicrobial activity, the antifouling activity is also a very impor-
tant factor for the successful coating of implantable materials.
Thus, the protein-resistant ability of the two polymer brush coat-
ings was evaluated by the Micro BCA protein assay (Fig. 5A–C).
BSA and lysozyme were used as model proteins for adsorption
on the surfaces. After adsorption for 24 h, the surface-attached
protein was chelated with the BCA reagent and formed a violet-
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
colored complex (Fig. 5A). The protein adsorption was quantitat-
ively determined by the absorbance at 560 nm (Fig. 5B). Taking
the pristine PDMS as a control, the protein absorption was
greatly reduced for both the poly(Ppep) and poly(Ppep/Psar) coat-
ings. The adsorption of protein on the poly(Ppep) coating was
reduced by 54.7% (BSA) and 64.8% (lysozyme) respectively com-
pared with that on the pristine PDMS. For the poly(Ppep/Psar)
coating, which has more hydrophilic antifouling Psar chains, the
adsorption of the protein was further reduced by 83.4% for BSA
and 87.8% for lysozyme (Fig. 5C).
The platelet adhesion test was performed by placing the
surface in contact with platelet-rich plasma (PRP) for 2 h, fol-
lowed by FE-SEM imaging. As shown in Fig. 5D-i, the platelet
adhesion was extensive, most of which were highly active and
spread out with many typical pseudopods littering the surface
of pristine PDMS. A few platelets adhered to the surface of
poly(Ppep), but with a small degree of deformation and a
small amount of pseudopods (Fig. 5D-ii). No platelets adhered
to the surface of poly(Ppep/Psar) (Fig. 5D-iii). Therefore, the
presence of the poly(Psar) component greatly improved the
antifouling activity of the polymer brush coating to enhance
the resistance to protein and platelet adhesion. In summary,
the poly(Ppep/Psar) brush coating itself exhibits antimicrobial
and anti-biofilm ability as well as excellent resistance to
protein/platelet adhesion. These advanced features of the poly
(Ppep/Psar) coating are expected to support long-lasting and
effective anti-infective effects in the body.
3.6. In vitro biocompatibility of surface coating of MePs
Prior to animal testing, the in vitro biocompatibility of the poly
(Ppep/Psar) coating was evaluated by placing the coating in
contact with L929 cells for up to 4 days. A tissue culture poly-
styrene dish (TCPS) was used as a control. AlamarBlue and LIVE/
DEAD cell viability assays showed that the L929 cells proliferated
well in contact with the poly(Ppep/Psar) coating (Fig. 6). The
AlamarBlue assay measures the mitochondrial activity and cell
viability and proliferation. Fig. 6A shows that the absorbance of
AlamarBlue increased with the culture time, indicating that the
Polym. Chem., 2017, 8, 6386–6397 | 6393
 View Article Online

Paper Polymer Chemistry

Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.

Fig. 5 Antifouling ability of polymer brush coatings. (A) BCA reagent (i, blank control) chelated with protein (BSA) from the surface of uncoated
PDMS (ii), poly(Ppep) (iii) and poly(Ppep/Psar) (iv) coatings formed a violet-colored complex. (B) The protein (BSA and lysozyme) adsorption quanti-
tatively determined from the absorbance at 560 nm. (C) Reduction in the protein adsorption of poly(Ppep) and poly(Ppep/Psar) coatings versus that
of the pristine PDMS surface (error bars represent mean ± standard deviation of mean for n = 5, **p < 0.01). (D) Platelet adhesion on the uncoated
PDMS (i), poly(Ppep) (ii), and poly(Ppep/Psar) (iii) coatings observed with FE-SEM (scale bar = 20 µm (lower), 5 µm (upper)).

an animal subcutaneous infection model. S. aureus, one of the

Fig. 6 In vitro biocompatibility study of L929 cells after 1 and 4 days of
culture with poly(Ppep/Psar) coating. Cell viability determined with (A)
AlamarBlue assay (error bars represent mean ± standard deviation of
mean for n = 5); (B) LIVE/DEAD assay (scale bar = 100 µm).
cells proliferated in the presence of the coating. In Fig. 6B, the
cells were mostly stained green, indicating that they remained
alive in contact with the coating, suggesting that the poly(Ppep/
Psar) coating is in vitro biocompatible.
3.7. In vivo anti-infective activity of surface coating of MePs
The anti-infective activity of this poly(Ppep/Psar) brush-coated
PDMS (a commonly used catheter material) was evaluated in
key pathogenic microbes in infections associated with bio-
medical implants, was used as model bacteria to induce infec-
tion using a pre-seeding protocol.66 Incisions (1 cm) were care-
fully cut on two sides of the back of each of eight rats, and
uncoated and poly(Ppep/Psar)-coated implants (size: 0.2 ×
0.6 cm) inoculated with 10 µL (∼106 CFU) of fresh S. aureus
bacteria were placed into the incisions (Fig. 7A-i). After 5 days,
the conditions of the wounds were observed. Fig. 7A-ii shows a
serious infection in the incisions with the uncoated implants,
and there were obvious pyogenic fluids. However, the incisions
with the poly(Ppep/Psar)-coated implants looked different
from that of the control groups, where no obvious sign of
infection was observed (Fig. 7A-iii). All implants were retrieved
and washed with PBS under ultrasonication to determine the
number of viable bacteria on the implants. As shown in
Fig. 7A-iv and v, there were numerous S. aureus growing on the
uncoated implants, but few bacteria were retrieved from the
poly(Ppep/Psar)-coated implants. The bacterial count for the
eight uncoated control implants ranged from 2.6 × 105 to 6.2 × 107
CFU per implant. In contrast, two out of the eight implants
with the poly(Ppep/Psar) coating had no visible bacteria, and
the other six implants had fewer than 320 CFU per implant.
The bacterial count for the poly(Ppep/Psar)-coated implants

6394 | Polym. Chem., 2017, 8, 6386–6397 This journal is © The Royal Society of Chemistry 2017
 View Article Online

Polymer Chemistry Paper

Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.

Fig. 7 In vivo anti-infective activity of the poly(Ppep/Psar) coating. (A) Schematic of the rat subcutaneous infection model: photographs of surgical
wound with implants seeded with S. aureus at day 1 (i) and day 5 (ii and iii), bacterial cultures from uncoated (iv) and poly(Ppep/Psar)-coated
(v) implants. (B) Numbers of viable S. aureus cells recovered from the uncoated and poly(Ppep/Psar)-coated implants (error bars represent mean ±
standard deviation of mean for n = 8, **p < 0.01). (C) Histological study of the infected tissues around the uncoated (i and ii) and poly(Ppep/Psar)-
coated (iii and iv) implants.

was significantly lower than that of the control group ( p <
0.01), with a log reduction of 5.2. Additionally, histological
analysis was used to evaluate the anti-infective activity of the
poly(Ppep/Psar) coating. Inflammatory cells (blue color, as
indicated with the arrows) in the tissue around the implants
represent the severity of the inflammation and infection. As
shown in Fig. 7C, more inflammatory cells were observed in
the tissue around the uncoated implants (i an ii) than in the
case of the poly(Ppep/Psar)-coated implants (iii and iv). Thus,
the poly(Ppep/Psar) coating greatly reduced the bacterial count
and the inflammation in this rat subcutaneous infection
model.
malian cells in vitro, and retained its excellent biological pro-
perties in a rat infection model by successfully preventing
implant-related infections caused by S. aureus. This polymer
brush coating can be easily UV-grafted onto the surface of
medical materials and exhibits antimicrobial and antifouling
capabilities that can potentially combat biomaterial-associated
infections.
Conflicts of interest
There are no conflicts to declare.

4. Conclusions
A dual-functional poly(Ppep/Psar) brush coating was developed
as a viable strategy to deal with biomaterial-associated infec-
tions. The rationally synthesized MePs not only possess excel-
lent antimicrobial or antifouling groups, but also have metha-
crylate groups capable of facile grafting onto the pDA-coated
surface under UV irradiation. Two brush coatings, poly(Ppep)
and poly(Ppep/Psar), were successfully prepared as confirmed
by ATR-FTIR, water contact angle, AFM, and XPS characteriz-
ation. Comparatively, the poly(Ppep/Psar) coating showed
excellent antimicrobial activity with an inhibition rate above
94.6% for all four clinically significant Gram-positive/-negative
bacteria and fungi, and also prevented biofilm formation for
up to 7 days. The poly(Ppep/Psar) coating greatly reduced non-
specific protein adsorption and platelet adhesion in vitro.
Additionally, this polymer brush coating is non-toxic to mam-
Acknowledgements
This work was funded and supported by the Natural Science
Foundation of China (No. 51403173), the Natural Science Basic
Research Plan in Shaanxi Province of China (No. 2015JQ5139),
and the Fundamental Research Funds for the Central
Universities of China. The authors thank Prof. Peng Yang
(Shaanxi Normal University, China) for their kind assistance
with AFM experiments.
References
1 E. D. Brown and G. D. Wright, Nature, 2016, 529, 336–343.
2 J. O’Neill, Tackling Drug-Resistant Infections Globally: final
report and recommendations, UK Department of Health,
Review on Antimicrobial Resistance, London, 2016.
3 M. Zasloff, Nature, 2002, 415, 389–395.

This journal is © The Royal Society of Chemistry 2017 Polym. Chem., 2017, 8, 6386–6397 | 6395

Paper
4 Z. Y. Ong, N. Wiradharma and Y. Y. Yang, Adv. Drug
Delivery Rev., 2014, 78, 28–45.
5 P. Li, X. Li, R. Saravanan, C. M. Li and S. S. J. Leong, RSC
Adv., 2012, 2, 4031–4044.
6 X. B. Qi, C. C. Zhou, P. Li, W. X. Xu, Y. Cao, H. Ling,
W. N. Chen, C. M. Li, R. Xu, M. Lamrani, Y. G. Mu,
S. S. J. Leong, M. W. Chang and M. B. Chan-Park, Biochem.
Biophys. Res. Commun., 2010, 398, 594–600.
7 R. H. Liu, X. Y. Chen, Z. Hayouka, S. Chakraborty,
Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.
S. P. Falk, B. Weisblum, K. S. Masters and S. H. Gellman,
J. Am. Chem. Soc., 2013, 135, 5270–5273.
8 R. H. Liu, X. Y. Chen, S. Chakraborty, J. J. Lemke,
Z. Hayouka, C. Chow, R. A. Welch, B. Weisblum,
K. S. Masters and S. H. Gellman, J. Am. Chem. Soc., 2014,
136, 4410–4418.
9 R. H. Liu, X. Y. Chen, S. P. Falk, B. P. Mowery,
A. J. Karlsson, B. Weisblum, S. P. Palecek, K. S. Masters and
S. H. Gellman, J. Am. Chem. Soc., 2014, 136, 4333–4342.
10 R. H. Liu, J. M. Suarez, B. Weisblum, S. H. Gellman and
S. M. McBride, J. Am. Chem. Soc., 2014, 136, 14498–14504.
11 R. H. Liu, X. Y. Chen, S. P. Falk, K. S. Masters, B. Weisblum
and S. H. Gellman, J. Am. Chem. Soc., 2015, 137, 2183–
2186.
12 A. Muñoz-Bonilla and M. Fernández-García, Prog. Polym.
Sci., 2012, 37, 281–339.
13 A. Muñoz-Bonilla and M. Fernández-García, Eur. Polym. J.,
2015, 65, 46–62.
14 T. J. Deming, Chem. Rev., 2016, 116, 786–808.
15 N. Gangloff, J. Ulbricht, T. Lorson, H. Schlaad and
R. Luxenhofer, Chem. Rev., 2016, 116, 1753–1802.
16 C. C. Zhou, X. B. Qi, P. Li, W. N. Chen, L. Mouad,
M. W. Chang, S. S. J. Leong and M. B. Chan-Park,
Biomacromolecules, 2010, 11, 60–67.
17 M. H. Xiong, Z. Y. Han, Z. Y. Song, J. Yu, H. Z. Ying,
L. C. Yin and J. J. Cheng, Angew. Chem., Int. Ed., 2017, 56,
10826–10829.
18 H. Lu, J. Wang, Z. Y. Song, L. C. Yin, Y. F. Zhang,
H. Y. Tang, C. L. Tu, Y. Lin and J. J. Cheng, Chem.
Commun., 2014, 50, 139–155.
19 D. Pranantyo, L. Q. Xu, Z. Hou, E. T. Kang and M. B. Chan-
Park, Polym. Chem., 2017, 8, 3364–3373.
20 P. Li, C. C. Zhou, S. Rayatpisheh, K. Ye, Y. F. Poon,
P. T. Hammond, H. W. Duan and M. B. Chan-Park, Adv.
Mater., 2012, 24, 4130–4137.
21 C. Tian, J. Ling and Y. Q. Shen, Chin. J. Polym. Sci., 2015,
33, 1186–1195.
22 Y. Shen, Z. B. Li and H. A. Klok, Chin. J. Polym. Sci., 2015,
33, 931–946.
23 H. C. Flemming, J. Wingender, U. Szewzyk, P. Steinberg,
S. A. Rice and S. Kjelleberg, Nat. Rev. Microbiol., 2016, 14,
563–575.
24 D. Davies, Nat. Rev. Drug Discovery, 2003, 2, 114–122.
25 M. Riool, A. de Breij, L. de Boer, P. H. S. Kwakman,
R. A. Cordfunke, O. Cohen, N. Malanovic, N. Emanuel,
K. Lohner, J. W. Drijfhout, P. H. Nibbering and S. A. J. Zaat,
Adv. Funct. Mater., 2017, 27, 1606623.
6396 | Polym. Chem., 2017, 8, 6386–6397
View Article Online
Polymer Chemistry
26 Q. Yu, Z. Q. Wu and H. Chen, Acta Biomater., 2015, 16, 1–
13.
27 K. Yu, J. C. Y. Lo, M. Yan, X. Yang, D. E. Brooks,
R. E. W. Hancock, D. Lange and J. N. Kizhakkedathu,
Biomaterials, 2017, 116, 69–81.
28 C. C. Zhou, P. Li, X. B. Qi, A. R. M. Sharif, Y. F. Poon,
Y. Cao, M. W. Chang, S. S. J. Leong and M. B. Chan-Park,
Biomaterials, 2011, 32, 2704–2712.
29 R. T. C. Cleophas, M. Riool, H. C. Quarles van Ufford,
S. A. J. Zaat, J. A. W. Kruijtzer and R. M. J. Liskamp, ACS
Macro Lett., 2014, 3, 477–480.
30 G. Xu, D. Pranantyo, B. Zhang, L. Xu, K. G. Neoh and
E. T. Kang, RSC Adv., 2016, 6, 14809–14818.
31 G. Gao, D. Lange, K. Hilpert, J. Kindrachuk, Y. Zou,
J. T. J. Cheng, M. Kazemzadeh-Narbat, K. Yu, R. Wang,
S. K. Straus, D. E. Brooks, B. H. Chew, R. E. W. Hancock
and J. N. Kizhakkedathu, Biomaterials, 2011, 32, 3899–
3909.
32 C. Yang, X. Ding, R. J. Ono, H. Lee, L. Y. Hsu, Y. W. Tong,
J. Hedrick and Y. Y. Yang, Adv. Mater., 2014, 26, 7346–7351.
33 G. Pan, S. Sun, W. Zhang, R. Zhao, W. Cui, F. He, L. Huang,
S. H. Lee, K. J. Shea, Q. Shi and H. Yang, J. Am. Chem. Soc.,
2016, 138, 15078–15086.
34 H. Cheng, K. Yue, M. Kazemzadeh-Narbat, Y. Liu,
A. Khalilpour, B. Li, Y. S. Zhang, N. Annabi and
A. Khademhosseini, ACS Appl. Mater. Interfaces, 2017, 9,
11428–11439.
35 H. M. Yuan, B. R. Yu, L. H. Fan, M. Wang, Y. W. Zhu,
X. K. Ding and F. J. Xu, Polym. Chem., 2016, 7, 5709–5718.
36 W. B. Sheng, B. Li, X. L. Wang, B. Dai, B. Yu, X. Jia and
F. Zhou, Chem. Sci., 2015, 6, 2068–2073.
37 W. Hartleb, J. S. Saar, P. Zou and K. Lienkamp, Macromol.
Chem. Phys., 2016, 217, 225–231.
38 E. K. Riga, M. Vöhringer, V. T. Widyaya and K. Lienkamp,
Macromol. Rapid Commun., 2017, DOI: 10.1002/marc.
201700216.
39 T. Wei, Q. Yu, W. J. Zhan and H. Chen, Adv. Healthcare
Mater., 2016, 5, 449–456.
40 T. Wei, W. J. Zhan, L. M. Cao, C. M. Hu, Y. C. Qu, Q. Yu
and H. Chen, ACS Appl. Mater. Interfaces, 2016, 8, 30048–
30057.
41 T. Wei, W. J. Zhan, Q. Yu and H. Chen, ACS Appl. Mater.
Interfaces, 2017, 9, 25767–25774.
42 S. S. Yuan, Y. G. Li, S. F. Luan, H. C. Shi, S. J. Yan and
J. H. Yin, J. Mater. Chem. B, 2016, 4, 1081–1089.
43 S. J. Yan, H. C. Shi, L. J. Song, X. H. Wang, L. Liu,
S. F. Luan, Y. M. Yang and J. H. Yin, ACS Appl. Mater.
Interfaces, 2016, 8, 24471–24481.
44 B. L. Wang, Q. W. Xu, Z. Ye, H. H. Liu, Q. K. Lin, K. H. Nan,
Y. Z. Li, Y. Wang, L. Qi and H. Chen, ACS Appl. Mater.
Interfaces, 2016, 8, 27207–27217.
45 B. L. Wang, Z. Ye, Q. W. Xu, H. H. Liu, Q. K. Lin, H. Chen
and K. Nan, Biomater. Sci., 2016, 4, 1731–1741.
46 Z. L. Zhi, Y. J. Su, Y. W. Xi, L. Tian, M. Xu, Q. Wang,
S. Padidan, P. Li and W. Huang, ACS Appl. Mater. Interfaces,
2017, 9, 10383–10397.
This journal is © The Royal Society of Chemistry 2017

Polymer Chemistry
47 Y. J. Su, Z. L. Zhi, Q. Gao, M. H. Xie, M. Yu, B. Lei, P. Li and
P. X. Ma, Adv. Healthcare Mater., 2017, 6, 1601173.
48 Q. Gao, M. Yu, Y. J. Su, M. H. Xie, X. Zhao, P. Li and
P. X. Ma, Acta Biomater., 2017, 51, 112–124.
49 R. H. Wang, T. Hughes, S. Beck, S. Vakil, S. Li, P. Pantano
and R. K. Draper, Nanotoxicology, 2013, 7, 1272–1281.
50 V. Gaberc-Porekar, I. Zore, B. Podobnik and V. Menart,
Curr. Opin. Drug Discovery Dev., 2008, 11, 242–250.
51 K. H. A. Lau, C. L. Ren, T. S. Sileika, S. H. Park, I. Szleifer
Published on 26 September 2017. Downloaded by Iowa State University on 1/24/2019 7:45:54 AM.
and P. B. Messersmith, Langmuir, 2012, 28, 16099–16107.
52 S. T. Xuan, S. Gupta, X. Li, M. Bleuel, G. J. Schneider and
D. H. Zhang, Biomacromolecules, 2017, 18, 951–964.
53 A. Birke, D. Huesmann, A. Kelsch, M. Weilbacher, J. Xie,
M. Bros, T. Bopp, C. Becker, K. Landfester and M. Barz,
Biomacromolecules, 2014, 15, 548–557.
54 Z. X. Voo, M. Khan, K. Narayanan, D. Seah, J. L. Hedrick
and Y. Y. Yang, Macromolecules, 2015, 48, 1055–1064.
55 J. Gu, Y. J. Su, P. Liu, P. Li and P. Yang, ACS Appl. Mater.
Interfaces, 2017, 9, 198–210.
56 Y. J. Su, L. Tian, M. Yu, Q. Gao, D. Wang, Y. W. Xi, P. Yang,
B. Lei, P. X. Ma and P. Li, Polym. Chem., 2017, 8, 3788–3800.
57 C. D. Vacogne and H. Schlaad, Chem. Commun., 2015, 51,
15645–15648.
58 S. H. Wibowo, A. Sulistio, E. H. H. Wong, A. Blencowe and
G. G. Qiao, Adv. Funct. Mater., 2015, 25, 3147–3156.
This journal is © The Royal Society of Chemistry 2017
View Article Online
Paper
59 B. Dhananjay and K. M. Chantal, Microelectron. Eng., 2006,
83, 1277–1279.
60 F. Bernsmann, A. Ponche, C. Ringwald, J. Hemmerle,
J. Raya, B. Bechinger, J. C. Voegel, P. Schaaf and V. Ball,
J. Phys. Chem. C, 2009, 113, 8234–8242.
61 R. T. C. Cleophas, J. Sjollema, H. J. Busscher,
J. A. W. Kruijtzer and R. M. J. Liskam, Biomacromolecules,
2014, 15, 3390–3395.
62 P. Li, Y. F. Poon, W. F. Li, H. Y. Zhu, S. H. Yeap, Y. Cao,
X. B. Qi, C. C. Zhou, M. Lamrani, R. W. Beuerman,
E. T. Kang, Y. G. Mu, C. M. Li, M. W. Chang, S. S. J. Leong
and M. B. Chan-Park, Nat. Mater., 2011, 10, 149–156.
63 G. J. Gabriel, A. Som, A. E. Madkour, T. Eren and G. N. Tew,
Mater. Sci. Eng., R, 2007, 57, 28–64.
64 G. G. Anderson, J. J. Palermo, J. D. Schilling, R. Roth,
J. Heuser and S. J. Hultgren, Science, 2003, 301, 105–107.
65 D. C. Leslie, A. Waterhouse, J. B. Berthet, T. M. Valentin,
A. L. Watters, A. Jain, P. Kim, B. D. Hatton, A. Nedder,
K. Donovan, E. H. Super, C. Howell, C. P. Johnson, T. L. Vu,
D. E. Bolgen, S. Rifai, A. R. Hansen, M. Aizenberg,
M. Super, J. Aizenberg and D. E. Ingber, Nat. Biotechnol.,
2014, 32, 1134–1140.
66 M. Riool, L. de Boer, V. Jaspers, C. M. van der Loos,
W. J. B. van Wamel, G. Wu, P. H. S. Kwakman and
S. A. J. Zaat, Acta Biomater., 2014, 10, 5202–5212.
Polym. Chem., 2017, 8, 6386–6397 | 6397