Development of nonfouling polypeptides with uniform alternating charges by polycondensation of the covalently bonded dimer of glutamic acid and lysine

Article abstract of DOI:10.1039/c3tb21333a

In this work, nonfouling polypeptides with homogenous alternating charges were synthesized by polycondensation of the covalently bonded dimer of glutamic acid (E) and lysine (K) (EK dimer) with benzyloxycarbonyl (Z)-protected side chains. This facile method successfully solved the uniformity problem of nonfouling peptides caused by the copolymerization of two different monomers and enabled the incorporation of various terminal functional groups for future applications. The molecular weights (MWs) of the nonfouling peptides can be easily controlled by the ratio of the terminal group, lipoic acid, to the EK dimer. The nonfouling peptides can form self-assembling monolayers (SAMs) on a gold surface through two terminal thiol groups, which were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and ellipsometry (ELL). The resistance to nonspecific protein adsorption, cell attachment and bacterial adhesion of these nonfouling peptide SAMs and the in vitro cytotoxicity and haemolytic activity of these peptides were also evaluated. The results show that the lowest relative protein adsorptions of antibody (anti-IgG) and fibrinogen (Fg) on the SAMs are 5.1 ± 1.6% and 7.3 ± 1.8%, respectively, determined by enzyme-linked immunosorbent assay (ELISA), where the protein adsorption on a tissue culture polystyrene (TCPS) surface was set to 100%. Almost no obvious cell attachment and bacterial adhesion were observed, and no cytotoxicity and no haemolytic activity in vitro were detected. With the advantages of biocompatibility, biodegradability and the abundance of moieties for ligand immobilization, these nonfouling peptides developed by the facile method can be used in a wide range of biomedical applications. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3tb21333a

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Materials Chemistry B
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PAPER
Development of nonfouling polypeptides with
uniform alternating charges by polycondensation
of the covalently bonded dimer of glutamic acid
and lysine
Cite this: J. Mater. Chem. B, 2014, 2,
577
Qinghua Yang,ac Longgang Wang,a Weifeng Lin,a Guanglong Ma,a Jiang Yuanb
ab
*
and Shengfu Chen
In this work, nonfouling polypeptides with homogenous alternating charges were synthesized by
polycondensation of the covalently bonded dimer of glutamic acid (E) and lysine (K) (EK dimer) with
benzyloxycarbonyl (Z)-protected side chains. This facile method successfully solved the uniformity
problem of nonfouling peptides caused by the copolymerization of two different monomers and
enabled the incorporation of various terminal functional groups for future applications. The molecular
weights (MWs) of the nonfouling peptides can be easily controlled by the ratio of the terminal group,
lipoic acid, to the EK dimer. The nonfouling peptides can form self-assembling monolayers (SAMs) on a
gold surface through two terminal thiol groups, which were characterized by attenuated total reflection
Fourier transform infrared (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and ellipsometry (ELL).
The resistance to nonspecific protein adsorption, cell attachment and bacterial adhesion of these
nonfouling peptide SAMs and the in vitro cytotoxicity and haemolytic activity of these peptides were also
evaluated. The results show that the lowest relative protein adsorptions of antibody (anti-IgG) and
fibrinogen (Fg) on the SAMs are 5.1 ꢀ 1.6% and 7.3 ꢀ 1.8%, respectively, determined by enzyme-linked
immunosorbent assay (ELISA), where the protein adsorption on a tissue culture polystyrene (TCPS)
surface was set to 100%. Almost no obvious cell attachment and bacterial adhesion were observed, and
no cytotoxicity and no haemolytic activity in vitro were detected. With the advantages of
biocompatibility, biodegradability and the abundance of moieties for ligand immobilization, these
nonfouling peptides developed by the facile method can be used in a wide range of biomedical applications.
Received 26th September 2013
Accepted 4th November 2013
DOI: 10.1039/c3tb21333a
www.rsc.org/MaterialsB
and weak interactions with cell membranes.4,14–17 However, they
are not in the most ideal format for bio-applications due to a
Introduction
lack of biodegradability through natural metabolism. They
might disrupt normal cell metabolism and eventually cause
host or untargeted cell death in gene or nano medicine therapy
since their inherent lack of non-biodegradability will lead to the
accumulation of the polymers in cells. Therefore, it is necessary
to develop biodegradable alternatives to the classical poly-
zwitterionic nonfouling materials.
For this purpose, biodegradable and biocompatible natural
amino acid-based nonfouling peptides, composed of homoge-
nous alternating negative glutamic acid (E) and positive lysine
(K) residues, were proposed and investigated since the peptide
could possess similar charge distribution features to the
zwtterionic polymer, uniformity and neutrality.12 Recently, the
Jiang group18 also demonstrated that an alternating EK oligo-
peptide with a four-proline linker and a cysteine residue can
form a self-assembling ordered structure on a gold surface. The
results clearly show that these peptide coated surfaces effi-
ciently resist nonspecic protein adsorption. Moreover, there
With the accelerated investigation of the nonfouling mecha-
nisms in recent years, polyzwitterionic materials, such as
poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC),1–3
poly(sulfobetaine) (pSB),4,5 and poly(carboxybetaine) (pCB),6–8
have been recognized as effective nonfouling materials. They
are superior to poly(ethylene glycol) (PEG) in the elds of
resistance to nonspecic protein adsorption, endurance in an
oxidating environment, multi-functionalization,6,9–13 etc. All of
these polyzwitterionic materials show good biocompability
through excellent resistance to nonspecic protein adsorption
aState Key Laboratory of Chemical Engineering, Department of Chemical and
Biological Engineering, Zhejiang University, Hangzhou 310027, PR China. E-mail:
schen@zju.edu.cn; Tel: +86 15958163615
bJiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu
Key Laboratory of Biomedical Materials, College of Chemistry and Materials Science,
Nanjing Normal University, Nanjing 210046, China
cSchool of Medical Engineering, Hefei University of Technology, Hefei 230009, PR
China
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are plenty of functional groups on the uniformly charged cells (HUVECs) were purchased from China Center for Type
peptides for immobilizing biorecognition molecules in future Culture Collection. All other chemicals were of reagent grade
biomedical applications. All results indicate that this is a new and were used without further purication.
group of promising nonfouling materials. However, to the best
of our knowledge, all high quality nonfouling peptides with
exact EK-repeating sequences have to use a peptide synthesizer,
which is not suitable for mass production. Thus, copolymeri-
Synthesis route
The synthesis route is summarized in Scheme 1.
zation of two monomers containing E and K through N-
carboxyanhydride (NCA) ring opening polymerization has been
Synthesis of Na-(d-benzyl-N-tert-butoxycarbonyl-L-glumatyl)-
N3-benzyloxycarbonyl-a-tert-butyl-L-lysine (4)
developed.19 However, it is rather difficult to obtain uniform EK
repeating sequences due to both the different reactivity of each
monomer and the difficulty in controlling the purity of the
monomer in each batch, although this problem could be
partially solved through different ratios of E to K NCA
monomers.
A typical procedure is described as follows: H-Lys(Z)-OtBu$HCl
(1, 5.87 g, 15.74 mmol) was dissolved in 50 mL anhydrous DMF
and 3 mL TEA (21.52 mmol) in 10 mL DMF was added dropwise.
Aer 30 min, the mixture was ltered. Boc-Glu(Z)–OH (3, 5.06 g,
15 mmol), EDC$HCl (5.75 g, 30 mmol) and HOBt (4.05 g,
30 mmol) were added into the ltrate and then the mixture was
reacted for 48 h at room temperature. Aer complete removal of
DMF, the residue was dissolved in dichloromethane and then
washed with 10% aqueous citric acid solution, saturated
aqueous NaHCO3 solution, water, and saturated brine, and
dried over anhydrous MgSO4. The organic solution was
concentrated by a rotary evaporator under reduced pressure,
and the residue was puried by column chromatography (silica
gel, dichloromethane/ethyl acetate ¼ 3 : 1, v/v) to obtain
In this work, we designed and synthesized a covalently
bonded EK dimer to achieve uniformity, which is the key feature
to generate nonfouling properties, of charge alternating non-
fouling peptides through polycondensation. Three nonfouling
peptides with different degrees of polymerization were
successfully prepared through controlling the ratio of the
terminal group, lipoic acid, to the EK dimer. The lipoic acid
capped EK-repeating polypeptides can form self-assembling
monolayers (SAMs) on a gold surface through two thiol groups.
The chemical and physical properties of these peptide SAMs
were systematically characterized to further verify the formation
of SAMs. The functionalities of the SAMs, including resistance
to protein adsorption, cell attachment and bacterial adhesion,
were investigated. The relationships between the resistance and
the peptide MWs were discussed. Moreover, the biocompati-
bility of these peptides, including in vitro cytotoxicity and hae-
molytic activity of these EK-repeating polypeptides were also
examined. All results suggest that these fully biodegradable
and biocompatible polypeptides will play important roles in
biomedical applications, especially for nano drug carriers.
1
compound 4 (8.40 g, 85.4%). H NMR (DMSO-d6, 400 MHz): d
1.23–1.45 22H, 1.49–1.71 2H, 1.71–1.83 1H, 1.85–1.98 1H, 2.36–
2.44 2H, 2.92–3.02 2H, 3.95–4.03 2H, 4.96–5.05 2H, 5.05–5.11
2H, 6.90–6.97 0.89H, 7.20–7.27 0.88H, 7.28–7.40 10H, 8.05–8.11
0.95H.
Experimental
Materials
N3-benzyloxycarbonyl-a-tert-butyl-L-lysine hydrochloride (H-
Lys(Z)-OtBu$HCl, 99%) and d-benzyl-N-tert-butoxycarbonyl-L-
glutamic acid (Boc-Glu(Z)–OH, 98%) were purchased from
Shanghai Hanhong Chemical Co., Ltd. RPMI 1640 medium,
trypsin 0.25% and fetal bovine serum (FBS) were purchased
from Sijiqing Biological Engineering Materials Co., Ltd.
Horseradish peroxidise (HRP)-conjugated goat anti-human IgG
(anti-IgG/HRP), horseradish peroxidase (HRP)-conjugated goat
anti-brinogen (anti-Fg/HRP) and Fg were purchased from
Beijing Bosheng Bio-technology Co., Ltd. 1-(3-Dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDC$HCl, 98.5%),
2-hydroxybenzotriazole (HOBt, 99%), O-phenylenediamine
(OPD, 98%), 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenyte-
trazoliumromide (MTT), uorescein diacetate (FDA), triuoro-
acetic acid (TFA), triethylamine (TEA), and anhydrous
dimethylformamide (DMF) were purchased from Aladdin
Reagent Co., Ltd. 33 wt% HBr/HOAc solution was purchased
from Sigma-Aldrich Co. LLC. Human umbilical vein endothelial Scheme 1 Synthesis route of the homogenous EK polypeptide.
578 | J. Mater. Chem. B, 2014, 2, 577–584
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Synthesis of Na-(d-benzyl-L-glumatyl)-N3-benzyloxycarbonyl -L-
lysine (5)
XPS measurements18
XPS was conducted on an X-probe spectrometer (VG Escalab
4 (4.15 g, 6.33 mmol) in 18 mL TFA/CH2Cl2 (v/v ¼ 2 : 1) solution Mark II) equipped with a monochromatic Mg Ka X-ray source
was added to a 50 mL round bottom ask. The mixture was (hn ¼ 1253.6 eV), a hemi-spherical analyzer, and a multichannel
stirred for 6 h at room temperature, followed by removal of the detector. The bare gold coated chips were used as controls.
solvent by a rotary evaporator under reduced pressure and the
residue was dissolved in ethyl acetate again. TEA was added to
adjust the pH to 7.0–8.0. Then the mixture was dried and
washed with water several times to obtain compound 5 (2.66 g,
84.2%). 1H NMR (DMSO-d6, 400 MHz): d 1.19–1.31 2H, 1.32–1.43
2H, 1.49–1.62 1H, 1.64–1.82 2H, 1.82–1.95 1H, 2.41–2.48 2H,
2.90–3.00 2H, 3.36–3.46 1H, 4.01–4.12 1H, 4.94–5.02 2H, 5.02–
5.13 2H, 7.23–7.29 0.86H, 7.30–7.40 10H, 8.16–8.28 1H.
Protein adsorption assay
Gold chips were immersed in the potassium dichromate solu-
tion overnight, followed by rinsing ve times with DI water and
ethanol, and drying by ltered air. Then pre-treated gold chips
were immersed in 2 mg mLꢁ1 polypeptide solution for 24 h
followed by rinsing with sterile PBS. Fg adsorption on the
polypeptide-based SAMs and TCPS control were quantied
using the anti-Fg/HRP adsorption.
Synthesis of lipoic acid capped poly(Na-(d-benzyl-L-glumatyl)-
N3-benzyloxycarbonyl-L-lysine) (6)
The samples were rst incubated with Fg (10 mg mLꢁ1) for
20 min, followed by rinsing with sterile PBS to remove all free
proteins. Then the chips were incubated with anti-Fg/HRP
(1 mg mLꢁ1) for another 20 min and rinsed ve times with sterile
PBS. The tested chips were transferred to a 24-well plate and
incubated with 1 mL o-phenylenediamine (OPD) (1 mg mLꢁ1) in
citrate phosphate buffer (0.1 M, pH 5.0) containing 0.03%
hydrogen peroxide. The enzyme activity was quenched by add-
ing an equal volume of 2 N H2SO4 aer 8 minutes of incubation.
The tangerine colour was measured by a UV spectrophotometer
at 492 nm. The antibody adsorption was directly measured from
the adsorbed anti-IgG/HRP.
The EK dimer (5) (0.2 g, 0.4 mmol), EDC$HCl (0.115 g,
0.6 mmol), HOBt (0.081 g, 0.6 mmol) and different amounts of
D,L-lipoic acid (based on the molar ratio of D,L-lipoic acid to the
EK dimer (5)) were added into 2 mL anhydrous DMF. Aer
complete removal of oxygen and humidity from the reaction
system by bubbling ltrated N2 for about 30 min, the resulting
mixture was kept sealed for reaction at room temperature for
48 h. The mixture was poured into excess ethyl ether to
precipitate a polymer (6). The polymer (6) was kept in oil for the
next reaction without purication.
Bacterial adhesion assay
Synthesis of lipoic acid capped poly (L-glumatyl-L-lysine) (7)
Both Gram positive Staphylococcus aureus (G+) and Gram nega-
tive E.coli (Gꢁ) bacteria were used to investigate the resistance to
bacterial adhesion. The Staphylococcus aureus seeds were rst
The Z moieties on polymer 6 were removed by a mixed solution
of 33 wt% HBr/HOAc and TFA (v/v ¼ 1 : 1) for about 6 h. The EK
polypeptide (7) was precipitated by ethyl ether, followed by
neutralization to pH 7.0 by saturated NaHCO3 solution, dialyzed
against deionised (DI) water and lyophilized to obtain a white
powder. Full cleavage was conrmed by the disappearance of
ꢂ
incubated in the culture medium at 37 C, 200 rpm for 24 h.
Then the Staphylococcus aureus was separated by centrifugation
and re-suspended in 30 mL of sterile PBS. Aer rinsing twice
with PBS, the bacterial density was adjusted to 1 ꢃ 106 cells
mLꢁ1 (OD670 ¼ 0.015). Prior to testing, the samples and TCPS
controls were immersed in alcohol/water solution (v/v ¼ 3 : 1)
for 5 min, followed by sterile PBS for 1 min. Then they were
incubated with 1 ꢃ 106 cells mLꢁ1 bacterial suspension for 1–
2 h, washed with sterile PBS and then stained with Live/Dead®
BackLight bacterial viability kits. The images were recorded by a
CCD camera on a Nikon Eclipse Ti series microscope with a 20ꢃ
lens. The operation procedure for E.coli was the same as that for
Staphylococcus aureu.
1
peaks at 7.20–7.40 and 4.90–5.20 in HNMR. A typical yield of
the EK polypeptide from the EK dimer is about 45.0%. 1H NMR
(D2O, 400 MHz): d 1.20–1.50 2H, 1.51–1.59 2H, 1.65–2.06 4H,
2.08–2.36 2H, 2.90–2.94 2H, 3.95–4.23 2H.
Gel permeation chromatography (GPC) analysis
The MW and MW distribution of the polypeptides were deter-
mined by Shimadzu gel permeation chromatography equipped
with a Waters Ultrahydrogel™ 120 and a Waters Ultra-
hydrogel™ linear column. The mobile phase was phosphate
buffered saline (PBS) (NaCl 150 mM, pH 7.4) with a ow rate of
0.5 mL minꢁ1 at 45 ꢂC. Poly(ethylene oxide) was used as a
standard.
Cell attachment assay
The samples and TCPS controls were transferred to a 24-well
plate and each well was loaded with 1 mL HUVECs (4 ꢃ 105 cells
mLꢁ1) suspension in RPMI 1640 culture medium plus 5% fetal
bovine serum (FBS) and incubated for 1 day at 37 ꢂC in 5% CO2.
ATR-FTIR for lm structure and ELL for lm thickness
ATR-FTIR was conducted on a Fourier transform infrared Aer the medium was removed, each well was loaded with 1 mL
spectrometer (Bruker Vector 22) and lm thickness measure- of 5 mg mLꢁ1 FDA solution and incubated for 5 min. Aer
ments were conducted on a spectroscopic ellipsometer (J. A. rinsing twice with PBS, each well was loaded with 1 mL RPMI
Woollam M-2000D). The bare gold coated chips were used as 1640 and the images were directly recorded by a CCD camera on
controls.
a Nikon Eclipse Ti series microscope with a 20ꢃ lens.
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MTT assay
A typical procedure for the MTT assay is as follows: a 5 mg mLꢁ1
MTT stock solution was diluted in RPMI 1640 medium to make
a 5 mg mLꢁ1 working solution. HUVECs were seeded in a 96-well
tissue culture plate at a density of 10 000 cells per well and were
cultured in RPMI 1640 medium with 10% FBS. Aer 1 day of
incubation, the medium was removed and the cells were washed
with RPMI 1640 medium twice. Then the cells were incubated in
100 mL of RPMI 1640 medium with polypeptides at various
concentrations at 37 ꢂC for 24 h, and the metabolic activity was
determined by using an MTT assay. Aer the medium was
removed, 100 mL MTT working solution was transferred into each
well and the samples were incubated at 37 ꢂC for 4 h. Then, the
medium was removed and 100 mL DMSO was added and incu-
bated for 10 min. The absorbance at 570 nm was recorded using a
96-well plate reader SpectraMaxM2, Molecular Devices, Sunny-
vale, CA. Cell viability was expressed as the percentage of absor-
bance of treated cells with various polypeptide concentrations
compared to the absorbance of cells that were not treated. Each
measurement had three replicate wells.
Fig. 1 Nuclear magnetic resonance (1H NMR) spectrum of the EK
polypeptide.
(5) was condensed by EDC$HCl/HOBt and D,L-lipoic acid serves
as the end-group to adjust the MW. The MWs of the synthesized
EK polypeptides are 2.8 kDa (PDI ¼ 1.13), 5.4 kDa (PDI ¼ 1.31),
and 12.0 kDa (PDI ¼ 1.90) (Table 1). The difference between the
feeding molar ratios of the EK dimer (5) to lipoic acid and
the MWs of the synthesized EK polypeptides might be caused by
the side reaction of minor impurities in the EK dimer (5). The
low PDI of the 2.8 kDa EK polypeptide is achieved by dialysis.
Haemolytic activity assay
The haemolytic activity assay was performed based on a
previous study.20 Red blood cells (RBCs) were collected by
centrifugation of whole blood in sterile PBS at 1500 rpm for
10 min. The RBCs were further washed three times using sterile
PBS. Aer the supernatant had been removed following the last
wash, the cells were resuspended in PBS to get a 2% w/v RBC
suspension. The tested samples were also prepared in sterile
PBS. 150 mL of the sample solution and 150 mL of the 2% w/v
RBC solution were added to the centrifuge tube to make a
sample nal concentration of 5 mg mLꢁ1 and incubated for 4 h
at 37 ꢂC. Then the mixture was centrifuged and the supernatant
was transferred to a 96-well plate. The relative adsorption of the
supernatants was measured on a microplate reader at 575 nm.
Complete haemolysis was attained using water as the positive
control and PBS as the negative control. The haemolytic activity
was dened as follows: haemolytic activity% ¼ [(sample
absorbance ꢁ negative control)/(positive control ꢁ negative
control)] ꢃ 100%.
The properties of SAMs characterized by ATR-FTIR, XPS and
ELL
In order to verify the formation of the polypeptide SAMs on the
gold coated surface, the polypeptide SAMs were investigated by
ATR-FTIR, XPS and ELL. From the FTIR spectra (Fig. 2), two
typical secondary amide bands, dened as band I (nC]O, 1600–
1750 cmꢁ1) and band II (dN–H, 1500–1560 cmꢁ1) can be seen.
The peaks over 3500 cmꢁ1 might mainly come from the water
molecules adsorbed on the zwitterionic polypeptide SAMs21
since water molecules could be strongly bound with the poly-
peptide through both ionic hydration and hydrogen bond
formation. This strong hydration capability leads to the excel-
lent resistance of these zwitterionic polypeptides to protein
adsorption, bacterial adhesion and cell attachment.
XPS survey spectra of the EK polypeptide coated gold
surfaces and bare gold surface were also measured. By
comparing these four survey spectra in Fig. 3, the emergence of
N 1s, representing the characteristic element peak of poly-
peptides, together with the obvious intensity increases of C 1s
and O 1s indicate the formation of the EK polypeptide SAMs.
Furthermore, detailed scans of carbon, nitrogen and oxygen
were also obtained to further investigate the relative element
Results and discussion
Synthesis of the EK polypeptide
In this work, the target EK polypeptide (7) with uniform alter-
nating charges was achieved by following the synthesis route in
1
Scheme 1. The H NMR spectrum of the EK polypeptide (7) is
shown in Fig. 1.
Commercially available H-Lys(Z)-OtBu$HCl (1) and Boc-
Glu(Z)–OH (3) were coupled using EDC$HCl/HOBt to obtain the
fully protected EK dimer (4), followed by cleavage of Boc (tert-
butoxycarbonyl) by using TFA/CH2Cl2 (v/v ¼ 2 : 1) solution to get
the EK dimer (5) with Z-protected side chains, which serves as a
basic block for the successful synthesis of the EK polypeptide.
Complete removal of the protection group Boc was conrmed
by disappearance of d 1.23–1.45 18H in 1H NMR. The EK dimer
Table 1 The feed molar ratio of the EK dimer (5) to lipoic acid, MW
(Mw) and polydispersity index (PDI) of the target EK polypeptide (7)
n(EK dimer)/n(lipoic acid)
MW (Mw)
PDI
Yield (%)
20 : 1
40 : 1
200 : 1
2.8 kDa
5.4 kDa
12.0 kDa
1.13
1.31
1.90
44.2
47.6
51.4
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Table 2 Element contents of the polypeptide SAMs determined by
XPS (Mw ¼ 2.8 kDa)a
Theoretical
value (%)
Measured value
(%)
Relative deviation
(%)
Element
C
N
O
70.8
12.5
16.7
67.3
11.9
20.8
ꢁ5.0
ꢁ4.9
24.8
a
The composition of the substrate is not included and the relative
element content values are calculated as follows: element% ¼ specic
element tting area/(C + N + O) tting area ꢃ 100%.
Fig. 2 ATR-FTIR spectroscopy of EK polypeptide SAMs at different
MWs. The main characteristic peaks of the secondary amide in band I
(nC]O, 1600–1750 cmꢁ1) and band II (dN–H, 1500–1560 cmꢁ1) can be
seen from the spectra, indicating the formation of the EK polypeptide
SAMs on the gold surface.
format, which indicates that the conformation of the 2.8 kDa EK
polypeptide on the gold surface is not in the a-helix format. It is
believed that the conformation of these polypeptides might be
close to randomly coiled since the diameter of the random coil
increases more slowly than the increase rate of the MW. Also,
the small random coils could pack more densely than large
ones, which leads to fewer surface defects for the 2.8 kDa EK
polypeptide SAMs.24 The nonspecic protein adsorption could
be reduced consequently since defects are the key reason for
protein adsorption on the zwitterionic polymer coated surface.
Thus, the lowest nonspecic protein adsorption is achieved by
the SAMs formed by the 2.8 kDa EK polypeptide as shown in
Fig. 5.
contents of the SAMs (Table 2). The XPS-measured carbon and
nitrogen contents were slightly lower than their theoretical
values while the oxygen content was much higher than its
theoretical value. The deviation of the oxygen content from its
theoretical value is about 24.8%, which is similar to the obser-
vation by S. F. Chen and Y. C. Chung, et al.1,22,23 According to
their previous results, the high content of oxygen mainly came
from the hydration water on the zwitterionic polymer, which
will also lead to lower contents of carbon and nitrogen in the
XPS assay than the theoretical ones. Moreover, such an obser-
vation also agrees with the peaks over 3500 cmꢁ1 in the FTIR
spectra. All this information suggests that the zwitterionic
polypeptide SAMs should obviously contain water due to all
samples being exposed to an ambient environment.
Film thickness (or packing density) is an essential parameter
which inuences the nonspecic protein adsorption of the
SAMs. In Fig. 4, when the MWs increase from 2.8 kDa to 12.0
kDa, the corresponding thicknesses of the SAMs are 2.45 ꢀ 0.02
nm, 3.12 ꢀ 0.02 nm and 3.82 ꢀ 0.03 nm, respectively. It is clear
that the thickness does not linearly increase with the MW. As
the MW increases, the increase rate of the SAM thickness drops.
It is worth mentioning that the thickness of the 2.8 kDa EK
polypeptide SAMs (2.45 ꢀ 0.02 nm) is rather reasonable. The
thickness of the 2.8 kDa EK polypeptide SAMs should be about
3.3 nm or 7.7 nm, for an a-helix format or an all-extending state,
respectively. The measured thickness is shorter than the a-helix
Nonspecic protein adsorption assay
The resistance of the polypeptide SAMs to nonspecic protein
adsorption was evaluated from the amounts of conjugated HRP
on the surface. The adsorption of anti-IgG/HRP was used as the
antibody adsorption, while the specic adsorption of anti-Fg/
HRP on the physically adsorbed Fg was used to represent the
adsorption of Fg. The relative adsorption values were obtained
from the comparison between the protein adsorption on the
samples and on the TCPS. As shown in Fig. 5, with the increase
of the MW from 2.8 kDa to 12.0 kDa, the relative nonspecic
protein adsorption for the antibody and Fg increases from 5.1 ꢀ
1.6% and 7.3 ꢀ 1.8% to 11.6 ꢀ 5.6% and 15.2 ꢀ 3.2%, respec-
tively. Moreover, the relative adsorption amounts of Fg were
higher than the antibody on these three EK polypeptide SAMs. It
is believed that these phenomena are mainly caused by the
Fig. 4 Film thicknesses of polypeptide SAMs at different MWs. When
the MWs increase from 2.8 kDa to 12.0 kDa, the film thicknesses
Fig. 3 XPS survey spectra of the samples and bare gold surface. The increase from 2.45 ꢀ 0.02 nm to 3.82 ꢀ 0.03 nm. The gradient of the
emergence of N 1s and obvious intensity increase of C 1s and O 1s film thickness versus MW decreases when the MW increases. The
indicate the formation of the EK polypeptide SAMs on the gold surface. results are shown as means ꢀ standard deviations (SD) (n ¼ 3).
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Fig. 6 Cell images of HUVECs on (a) representative EK polypeptide
SAM (Mw ¼ 2.8 kDa) surface and (b) TCPS surface. Cells were stained
with FDA. No cell attachment was detected on the SAM surfaces and a
large amount of cells were found on the TCPS surface.
Fig. 5 Relative nonspecific antibody and Fg adsorption on polypeptide
SAMs formed by different MW EK polypeptides, where the nonspecific
protein adsorption of the TCPS surface was set at 100%. The relative
nonspecific protein adsorptions of the antibody and Fg increase from
5.1 ꢀ 1.6% and 7.3 ꢀ 1.8% to 11.6 ꢀ 5.6% and 15.2 ꢀ 3.2%, when the MW
of the EK polypeptides increases from 2.8 kDa to 12.0 kDa. The results
are means ꢀ standard deviations (SD) (n ¼ 3).
and engineering applications.26 Staphylococcus aureus and E.
coli are commonly used model bacteria in scientic research.
Staphylococcus aureus can cause skin infections, endocarditis,
osteomyelitis and pneumonia. E. coli can cause urinary tract
infections or other infections. Thus, microbial adhesion assays
were performed to further investigate the fouling resistance of
the SAMs to Gram positive (G+) Staphylococcus aureus and Gram
negative (Gꢁ) E. coli. The results show that there was no obvious
attached Staphylococcus aureus and E. coli (data not shown)
observed on the polypeptide SAM surfaces, while a large
amount of attached Staphylococcus aureus was present on the
TCPS surface (Fig. 8). In short, all results indicate that the
polypeptide SAM surface showed good resistance to nonspecic
protein adsorption, cell attachment and bacterial adhesion.
higher tendency of Fg to denature due to its exible structure.12
Compared to the pMPC or PEG coated surface, it is believed that
the main reason for the high protein adsorption on the EK
polypeptide SAMs is the defects on the EK polypeptide SAMs.
There are two factors which might contribute to the increase
of the protein adsorption as the MW increase. The large size of
high MW polypeptide might cause big cavities among the
immobilized polypeptide chains, where proteins can adsorb.
The second possible factor is protein adsorption through
multiple contact points between protein molecules and the EK
polypeptide chains. Long-chain polypeptides are exible and
have more contact points available with protein molecules,
which might cause nonspecic protein adsorption.25 On the
contrary, these two issues can be avoided in the SAMs formed by
the short EK polypeptide. In short, the SAMs formed by the
short EK polypeptide could result in better resistance through a
dense monolayer structure and strong hydration of zwitterionic
molecular structures.
MTT and haemolytic activity assay
In order to demonstrate the good biocompatibility of the EK
polypeptides, in vitro cytotoxicity and haemolytic activity of the
EK polypeptides were investigated. The EK polypeptides show
no toxicity up to 5 mg mLꢁ1 concentration aer 24 h incubation.
The cell viabilities vary between 87.4% and 107.6% when the
polypeptide concentrations vary within 0.01–5 mg mLꢁ1 (Fig. 9).
Even when the concentration is up to 5 mg mLꢁ1, the corre-
sponding cell viability can be maintained at 87.4 ꢀ 4.5% of the
normal cell value, indicating no cytotoxicity in vitro. Moreover,
the cell morphology in the 5 mg mLꢁ1 (Mw ¼ 2.8 kDa) EK
polypeptide solution shows no apparent difference to the
control solution.
Cell attachment and bacterial adhesion assay
In general, it is believed that the protein resistance of surfaces
could also exhibit resistance to cell attachment and bacterial
adhesion. Here, HUVECs, derived from the endothelium of
veins from the umbilical cord, were use to test the resistance to
cell attachment. In this experiment, FDA is chosen to stain
HUVECs to observe cell attachment and cytoplasmic spreading.
Thus, we examined cell attachment on various SAM surfaces
with different MWs aer 24 h incubation (images are not
shown). The representative morphology of cells on the EK
polypeptide SAM and its control surface (TCPS) are presented in
Fig. 6. The results show no HUVEC attachment on the SAMs of
various EK polypeptides, whereas the HUVECs grow normally in
the same culture well. Furthermore, HUVECs display good
attachment and spreading conditions on the TCPS surface with
an additional 5 mg mLꢁ1 of polypeptide (Mw ¼ 2.8 kDa) in the
culture medium (Fig. 7), indicating there is good compatibility
between the polypeptide and the cells.
Fig. 7 Representative morphology of cells cultured in (a) RPMI 1640
growth media and (b) with an additional 5 mg mLꢁ1 EK polypeptide
(Mw ¼ 2.8 kDa). Almost no apparent differences between the sample
and its control were found even though the polypeptide feed
concentration is up to 5 mg mLꢁ1, indicating no cytotoxicity in vitro for
Microbial adhesion onto surfaces and the subsequent
formation of a biolm are critical issues for many biomedical the polypeptides.
582 | J. Mater. Chem. B, 2014, 2, 577–584
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Fig. 8 Bacterial adhesion images of Gram positive (G+) Staphylo-
coccus aureus on (a) the representative polypeptide SAMs (Mw ¼ 2.8
kDa) and (b) the TCPS surface. No obvious adhered bacteria were
detected on the SAMs and a large amount of bacteria were found on
the TCPS surface.
Fig. 10 Haemolytic activities for the polypeptides with various MWs at
a concentration of 5 mg mLꢁ1. For samples with MWs of 2.8 kDa, 5.4
kDa and 12.0 kDa, no haemolytic activity was detected. The results are
shown as means ꢀ standard deviations (SD) (n ¼ 3).
Furthermore, the haemolytic activity assay (Fig. 10) also
indicates the low interaction between EK polypeptides and the
cell membrane. It was found that the light absorbance of the
supernatants aer removal of red blood cells with 2.8 kDa,
5.4 kDa and 12.0 kDa EK polypeptides was very low. The hae-
molytic activity of the 2.8 kDa and 5.4 kDa EK polypeptides was
even slightly lower than that of the negative control (PBS solu-
tion). Such a phenomenon might be due to the protection
imparted by the EK polypeptides to red blood cells. The highest
haemolytic activity is observed for 12.0 kDa EK polypeptides, for
which the haemolytic activity is just 2.4 ꢀ 1.1%. Such a low
haemolytic activity agrees with the cytotoxicity results and also
suggests that the low cytotoxicity should be attributed to the low
interactions of the EK polypeptides with the protein and cell
membrane. In short, all the EK polypeptides exhibit very good
biocompatibility and are very good candidates for biodegrad-
able nonfouling materials.
Furthermore, they also have the inherent biodegradability of
natural polypeptides. The new synthesis route through EK
dimer polycondensation solves the nonuniformity problem in
the NCA ring opening polymerization of mixed E and K mono-
mers. Moreover, good control of the MW of EK polypeptides is
achieved by this new method. The results indicate that a rather
short polypeptide (2.8 kDa) is sufficient to resist nonspecic
protein adsorption. It is expected that this kind of polypeptide
could be used as an alternative to other synthetic nonfouling
materials, such as PEG, to avoid non-biodegradability in
biomedical applications, especially for nano drug carriers.
Acknowledgements
This work is supported by the National Natural Science Foun-
dation of China (20974095, 20936005, 21174127) and the Ph.D.
Programs Foundation of the Ministry of Education of China
(20110101110034).
Conclusions
In this work, nonfouling polypeptides with uniform alternating
charges were prepared by the polycondensation of a covalently
bonded EK dimer using facile EDC$HCl/HOBt chemistry. The
results show that the EK polypeptides can have excellent resis-
tance to nonspecic protein adsorption and biocompatibility
the same as other nonfouling zwitterionic polymers.
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