Facile and universal immobilization of l-lysine inspired by mussels

Article abstract of DOI:10.1039/c2jm16598h

A novel functional molecule lysine-dopamine (LDA) was successfully synthesized and explored as a universal modifier for different types of surfaces, inspired by mussel adhesive moiety dopamine and bio-functional moiety l-lysine. The universal, robust, and efficient surface modification based on LDA was achieved through a facile and cost-effective dip-coating process, confirmed by Fourier transform infrared spectroscopy (FTIR), contact angle, X-ray photoelectron spectroscopy (XPS) and scanning electron microscope-energy dispersive spectroscopy (SEM-EDS) measurements. Meanwhile, LDA modification improved cell adhesion, promoted cell growth, and accelerated endothelialization on the substrate surface and provided plasma clot lysis activity. The results indicated that the surface biocompatibility was obviously improved by the one-step modification method for the immobilized l-lysine.

Full text of DOI:10.1039/c2jm16598h

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PAPER
Facile and universal immobilization of L-lysine inspired by mussels
a
a
a
a
a
Peiyu Sun, Haoxiang Lu, Xiong Yao, Xiaoxiong Tu, Zhen Zheng and Xinling Wang
ab
*
Received 14th December 2011, Accepted 23rd March 2012
DOI: 10.1039/c2jm16598h
A novel functional molecule lysine-dopamine (LDA) was successfully synthesized and explored as
a universal modifier for different types of surfaces, inspired by mussel adhesive moiety dopamine and
bio-functional moiety L-lysine. The universal, robust, and efficient surface modification based on LDA
was achieved through a facile and cost-effective dip-coating process, confirmed by Fourier transform
infrared spectroscopy (FTIR), contact angle, X-ray photoelectron spectroscopy (XPS) and scanning
electron microscope-energy dispersive spectroscopy (SEM-EDS) measurements. Meanwhile, LDA
modification improved cell adhesion, promoted cell growth, and accelerated endothelialization on the
substrate surface and provided plasma clot lysis activity. The results indicated that the surface
biocompatibility was obviously improved by the one-step modification method for the immobilized
L-lysine.
group of grafted PAA onto a membrane served as a bioactive
molecule and mimics the behavior of the very expensive PLL.
Many available surface modification techniques have limita-
tions such as substrate specificity (self-assembled monolayer and
silylation), requirements for complex instrumentation and
expensive equipment (Langmuir–Blodgett and plasma), compli-
cated processes (layer-by-layer assembly and grafting polymeri-
zation), or inherent instability (physical absorption). Therefore,
development of facile and versatile strategies for surface modi-
fication of widely different materials has proven challenging.
Inert and hydrophobic surfaces such as polytetrafluoroethylene
(PTFE), polyethylene (PE), polypropylene (PP), and poly-
(vinylidene fluoride) (PVDF) often suffer from poor cell adhe-
sion due to low surface energy, lack of functional groups, and the
absence of cell recognition sites. The immobilization of func-
tional molecules on PTFE surfaces through simple methods to
promote endothelial cell growth can be very challenging.
Introduction
Surface functionality plays a critical role in chemistry, materials
science, physics, biology and many other sciences and technol-
ogies. In biomedical yield, surface properties strongly influence
the performance of implanted biomaterials. A variety of strate-
gies have been pursued to create biocompatible surfaces. For
blood contacting materials, anti-thrombogenic surfaces can be
created by functionalizing materials with bio-inert molecules
(e.g. anti-platelet PEG),1,2 immobilizing bio-active molecules
(e.g. anticoagulant heparin or fibrinolytic lysine),3,4 or seeding
with endothelial cells.1,5 For cell culture and tissue engineering,
cell-adhesion surfaces can be achieved by immobilization of
extracellular matrix (ECM) molecules (e.g. fibronectin),4,6 cell
recognition peptides (e.g. RGD),4,5,7,8 polylysine (PLL, cationic
adhesion mechanism),8–10 and mussel proteins (Cell-Tak, DOPA-
mediated adhesion).8,11
Lysine and PLL are widely used as biocompatible materials
and as a platform for bioactive molecule conjugation to create
biocompatible surfaces.9,12–15 The immobilization of these func-
tional moieties on a surface is crucial for their use in biomedical
applications. Yavin, et al.13 immobilized PLL on substrates by
physical adsorption to improve biocompatibility and used it as
a platform for conjugation with bioactive molecules.14 However,
the binding strength of adsorbed PLL is relatively weak, so the
PLL can detach from the surface during the course of use.
Young, et al.15 reported that lysine immobilized through the acid
Mussels can adhere tightly to all types of inorganic and
organic surfaces in a wet and turbulent environment by excreting
adhesive proteins that are incredibly strong and durable.16 Waite,
et al.17 discovered that the strong adhesion relies on the repeated
DOPA-lysine motif in mussel adhesive proteins. Deming, et al.18
identified that the catechol group in DOPA plays a key role in the
universal adhesion property due to its chemical versatility and
diversity of affinity. Messersmith, et al.19 showed that low
molecular weight dopamine containing catechol moiety can also
mimic the powerful adhesive ability of mussel proteins and can
be used as a universal coating for different surfaces. For cell
adhesion promotion, cell-takꢀ (polyphenolic proteins extracted
from mussels)8,11 is used to attach cells to many types of different
surfaces. DOPA-lysine co-polypeptides20 have also been reported
to promote cell adhesion and growth on EVA. However, the cell-
tak and DOPA-lysine co-polypeptides are very expensive.
aSchool of Chemistry and Chemical Engineering, Shanghai Jiao Tong
University, Shanghai 200240, China. E-mail: xlwang@sjtu.edu.cn; Fax:
+86 21 54741297; Tel: +86 21 54745817
bState Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong
University, Shanghai 200240
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Herein, we report a newly designed small molecule, lysine-
dopamine (LDA), as a universal, robust, and efficient modifier
for different surfaces. LDA can be applied by a simple, conve-
nient, and cost-effective method on various substrates, and
obviously improve the biocompatibility of substrate surface. The
modified surface shows good durability, improves cell adhesion,
promotes cell growth, and accelerates endothelialization.
was added drop-wise into the flask. The reaction was carried out
ꢀ
orator was used to remove the solvent and by-product. 1 mol Lꢁ1
HCl aqueous solution was added drop-wise into the flask until
the pH was approximately 3. Ethyl acetate was applied to extract
the product, then evaporated to isolate the product. Finally, the
at 30 C for 24 h under a nitrogen atmosphere. A rotary evap-
ꢀ
product was dried in an oven (60 C) for 24 h. The yield of BL
was 92%. 1H-NMR (DMSO-d6, 300 MHz, d in ppm): 12.42 (br.
s, 1H, COOH), 6.98 (d, 1H, –OCONH–CH<), 6.78 (t, 1H,
–OCONH–CH2–), 3.78 (m, 1H, –OCONH–CH<), 2.85 (m, 2H,
–OCONH–CH2–), 1.85–1.05 (m, 24H, –CH2– and –CH3).
Experimental
Materials
All reagents used were available from commercial sources.
Lysine, N-hydroxysuccinimide (NHS), di-t-butyl dicarbonate
((Boc)2O), dopamine hydrochloride (DA-HCl), and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl)
were purchased from Aldrich and used without further purifi-
cation. Tetrahydrofuran (THF), dichloromethane (DCM), ethyl
acetate (EA), methanol, triethylamine (TEA), HCl, and
NaHCO3 were purchased from Sinopharm Chemical
Reagent Co.
(2) Synthesis of (Boc)2-lysine-NHS (BLN). The BL synthe-
sized in the first step was dissolved in 125 mL dichloromethane,
then N-hydroxysuccinimide (NHS) was added into the flask
(NHS in excess). 1-Ethyl-3-(3-dimethyllaminopropyl)carbodii-
mide hydrochloride (EDC-HCl) was dissolved in the dichloro-
ꢀ
methane at 0 C (2 mol equivalents relative to NHS). After 30
min, the reaction was warmed to room temperature and allowed
to proceed for another 5.5 h under a nitrogen atmosphere. Pure
water was used to wash the solution 3 times; the solvent was
removed, and the product dried in an oven (60 ꢀC). The yield of
BLN was about 88%. 1H-NMR (DMSO-d6, 300 MHz, d in
ppm): 7.55 (d, 1H, –OCONH–CH<), 6.78 (t, 1H, –OCONH–
CH2–), 4.27 (m, 1H, –OCONH–CH<), 2.96–2.65 (m, 6H,
–N(CO–CH2)2 and –OCONH–CH2–), 1.85–1.05 (m, 24H,
–CH2– and –CH3).
Titanium discs (grade 5, Ti-6Al-4V, Institute of Metal
Research, Chinese Academy of Sciences), copper foil, mono-
crystalline silicon (Si), ceramic (Al2O3, TA Instruments), quartz
(SiO2), and glass discs were cleaned ultrasonically in 2-propanol
for ten minutes before use. Microporous membranes such as
polyethylene (PE, ET20-26, Entek), polypropylene (PP, Celgard
2500), polyvinylidene fluoride (PVDF, Dow), polytetrafluoro-
ethylene (PTFE, Millipore), and dense films such as polyethylene
terephthalate (PET, Toray), polyvinyl chloride (PVC), LDPE
(Sinopec Corp.), PVDF (Shanghai 3F New Material Co., Ltd),
and PTFE (Shanghai 3F New Material Co., Ltd) were used
without treatment.
(3) Synthesis of (Boc)2-lysine-dopamine (BLDA). The BLN
synthesized in the second step was dissolved in 125 mL methanol.
Dopamine hydrochloride (dopamine-HCl) was added into the
flask (dopamine-HCl in excess). After the dopamine-HCl dis-
solved, triethylamine (TEA) was added into the solution (the
mole ratio of dopamine-HCl : TEA was 1.5 : 1.3). The reaction
proceeded for 12 h at room temperature under a nitrogen
atmosphere. The solvent and TEA were removed. Ethyl acetate
was applied to dissolve the product, followed by washing with
deionized water 3 times. The ethyl acetate was evaporated and
Synthesis of lysine-dopamine
Lysine-dopamine (LDA) was synthesized from lysine and
dopamine. The detailed conditions are shown in Scheme 1.
(1) Synthesis of (Boc)2-lysine-OH (BL). L-Lysine (10 mmol)
was dissolved in the mixed solvent (150 mL deionized water and
50 mL tetrahydrofuran) in a flask and 12.6 g NaHCO3 were
added. Then di-t-butyl dicarbonate (Boc anhydride) (150 mmol)
ꢀ
the product was dried in an oven (45 C) for 24 h. The yield of
1
BLDA was about 71%. H-NMR (DMSO-d6, 300 MHz, d in
ppm): 8.60–8.90 (d, 2H, –Ar(OH)2), 7.76 (t, 1H, –CONH–CH2–),
6.85–6.65 (m, 3H, –OCONH–CH< and –OCONH–CH2–),
6.64–6.30 (m, 3H, –Ar), 3.78 (m, 1H, –OCONH–CH<), 3.28–
2.98 (m, 2H, –CONH–CH2–), 2.85 (m, 2H, –OCONH–CH2–),
2.45 (m, 2H, –CONH–CH2–CH2–), 1.85–1.05 (m, 24H, –CH2–
and –CH3).
(4) Synthesis of lysine-dopamine (LDA). The BLDA synthe-
sized above was dissolved in 100 mL ethyl acetate. 50 mL HCl/
ethyl acetate solution was then added, and the reaction was
carried out for 3 h at room temperature under a nitrogen
atmosphere. After reaction, the solvent was removed and the
product was oven-dried (45 ꢀC) for 24 h. The final product is
a bis-hydrochloride of LDA. The yield of LDA was about 99%.
1H-NMR (DMSO-d6, 300 MHz, d in ppm): 8.60–8.90 (d, 2H,
–Ar(OH)2), 8.46 (t, 1H, –CONH–CH2), 8.13 (s, 2H, >CH–NH2),
7.79 (s, 2H, –CH2–NH2), 6.64–6.30 (m, 3H, –Ar), 3.64 (m, 1H,
>CH–NH2), 3.42–3.10 (m, 2H, –CONH–CH2–), 2.73 (m, 2H,
Scheme 1 Synthesis of lysine-dopamine.
10036 | J. Mater. Chem., 2012, 22, 10035–10041
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–CH2–NH2), 2.53 (m, 2H, –CONH–CH2–CH2–), 1.85–1.05 (m,
6H, –CH2–). ES MS m/z 282.18 [M + H]+.
on
a
Hitachi (S-2150) field-emission scanning electron
microscope.
Cell adhesion and growth experiments
Surface modification for different substrates by LDA
(1) Cell culture. Human umbilical vein endothelial cells
(HUVEC) were obtained from Shanghai Veterinary Research
Institute. Cells were sub-cultured twice a week and maintained in
a 37 ꢀC incubator containing a humidified 5% CO2/95% air
atmosphere. Dulbecco’s modified eagles medium (DMEM)
supplemented with 10% (v/v) fetal bovine serum (FBS) was
changed every 2 days.
Many types of substrates (microporous membranes, films, etc.)
were modified by LDA through a simple dip-coating process
(Fig. 1). Substrates modified by dopamine (DA) were used for
comparison. The substrates were immersed into 0.02 mol Lꢁ1
LDA or DA solution (pH ¼ 8.5) and agitated at 30 ꢀC for 40 h.
The membrane was then removed and washedꢀ with deionized
water. After drying to a constant weight at 40 C in a vacuum
oven, the resulting modified substrate was used for surface
characterization and cell growth/adhesion experiments. In order
to assess durability, the modified membranesꢀwere agitated with
deionized water in a shaker (150 rpm) at 45 C for 3 days. The
membranes were then removed, washed with deionized water,
and dried for contact angle characterization.
(2) Cell seeding of the membrane. Membranes were cut into
a circular shape (1.5 cm diameter) and sterilized with 75% alcohol
and ultraviolet light. Then, membranes were transferred into
a 24-well tissue culture polystyrene plate (TCPS, Corning), and
secured by a silicon rubber ring. 500 ml cell suspension (about
30 000 cells per well) was used for culture seeding.
Characterization
(3) Evaluation of cell viability and membrane cytotoxicity.
The WST-1 (Beyondtime Bio-Tech, China) assay was used to
measure cell viability. Briefly, after cells were cultured on PTFE
surface or LDA modified PTFE surface for 24 h, the culture
medium was changed and then 10% (v/v) WST-1 reagent was
added to each well. The cells were incubated for another 1 h, and
150 ml of the cell suspension in each well was transferred into
a 96-well flat plate (Corning). The absorbance was measured at
450 nm using a microplate reader.
FTIR and attenuated total reflection (ATR) spectra were
measured with a Perkin-Elmer (Spectrum 1000) FTIR spec-
trometer at room temperature. Powdered samples were studied
(KBr pellets) by FTIR. The film or membrane samples were
studied by FTIR or ATR-FTIR.
1H-NMR spectra were obtained with a Varian (Mercury plus-
400) NMR spectrometer at room temperature in DMSO-d6, and
chemical shifts were reported in ppm relative to tetramethylsilane
(TMS).
(4) Evaluation of cell adhesion and cell proliferation. After
24 h of cell incubation, the medium was discarded. Poorly
adhered cells were washed with 3 ꢂ PBS. The cells attached to
the membranes were then stained with acridine orange/
ethidium bromide (AO/EB) and observed with a fluorescence
microscope.
The UV-vis spectrum of LDA in water was recorded on
a Perkin Elmer (Lambda 750S) UV-vis spectrometer. All
measurements were performed in quartz cuvettes. Water was
used as a blank.
Contact angles were measured on a Contact Angle System
OCA20 (Data Physics Instruments GmbH, Germany) using the
sessile drop method with deionized water; the drop volume was
3.5 ml. All of the measurements were taken at ambient temper-
ature and the values reported in this paper are the average of five
measurements.
Plasma clot lysis assay
The plasma clot lysis assay was carried out according to the
literature reported by Li et al.4 Briefly, samples were incubated in
pooled normal human plasma (PNP) for 2 h at room tempera-
ture. The samples were rinsed in tris-buffered saline (TBS, pH
7.4) for three times, and placed in clean wells. 0.1 mg mlꢁ1 of
tissue plasminogen activator (t-PA) in TBS was added and
incubated for 30 min. The samples were rinsed extensively with
buffer. Then the clot-lysing potential was assessed using
a modified plasma recalcification assay. A 100 ml PNP was added
to the wells containing the surfaces. After a 5 min equilibration
The surface chemical composition was analyzed using a Shi-
madzu-Kratos (AXIS Ultra) X-ray photoelectron spectroscopy
(XPS). The XPS measurements were carried out at a base pres-
sure of about 5 ꢂ 10ꢁ10 mbar using an Mg X-ray (1253.6 eV)
source.
The surface elemental distribution was analyzed by scanning
electron microscope-energy dispersive spectroscopy (SEM-EDS)
ꢀ
period at 37 C, 100 ml CaCl2 (0.025 M) was injected into the
wells. Absorbance at 405 nm was measured at 30 s or 60 s
intervals over a 80 min period.
Results and discussion
Characterization of LDA
The functional bio-molecule LDA is a newly synthesized small
molecule containing a mussel-derived adhesive moiety (catechol)
and a biofunctional moiety (lysine). It was synthesized from
Fig. 1 Lysine-dopamine (LDA): chemical structure (a) and schematic
representation (b); surface modification of LDA by dip-coating (c).
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lysine and dopamine, both of which are non-toxic and biocom-
patible. The structures of LDA and intermediate products
including BL, BLN, and BLDA were confirmed by FTIR,
1H-NMR and UV-vis spectroscopy.
In the FTIR spectra (Fig. 2), for BL, the characteristic N–H
stretching vibration appear at 3348 cmꢁ1; the 1710 cmꢁ1 band is
associated with the stretching vibration of the carbonyl C]O.
Bands at 2975, 2933, 2869, 1456, 1392 and 1365 cmꢁ1 are due to
the alkyl groups, the latter two bands are Boc C–(CH3)3 group
overtone bands. The characteristic absorption of C–O–C appears
at 1170 cmꢁ1. Compared to BL, there are some new absorption
bands at 1816, 1784, 1212 and 1070 cmꢁ1 in BLN, which can be
assigned to the N-hydroxysuccinimide (NHS) group. Further-
more, compared to BL and BLN, the shift of the carbonyl from
1710 cmꢁ1 to 1673 cmꢁ1 in BLDA and LDA, and the appearance
of a band at 1603 cmꢁ1 corresponding to the phenyl ring implies
the successful amidation and grafting of dopamine. For LDA,
the amine absorption peak broadens and the alkyl
absorption weakens, indicating the removal of the amine-pro-
tecting groups.
In the 1H-NMR spectra (Fig. 3), for BL, the chemical shifts at
12.42 ppm, 6.98 ppm, 6.78 ppm, 3.78 ppm, 2.85 ppm and 1.36
ppm correspond to protons on the –COOH, –CH(COOH)–NH-
Boc, –CH2–NH-Boc, –CH(COOH)–NH-Boc, –CH2–NH-Boc,
and –O–C(CH3)3 moieties. Compared with BL, new chemical
shifts in BLDA at 8.60–8.90 ppm, 7.76 ppm, and 6.64–6.30 ppm
correspond to protons on –Ar(–OH)2, –CO–NH–CH2–, and the
Ar of dopamine, due to the successful amidation reaction.
Compared with BLDA, there was no peak at 1.36 ppm in LDA,
indicating successful deprotection.
Fig. 3 1H-NMR spectra of BL, BLN, BLDA and LDA.
remarkable adhesion. The newly designed small molecule LDA
can mimic the universal and strong adhesion of the mussel
adhesive protein (Fig. 1), and it can be used as a biocompatible
modification to membranes since lysine is a bio-functional
molecule and dopamine is an excellent anchoring moiety.
As shown in Fig. 5, the brown color indicated that surfaces
had been successfully modified by LDA. L-Lysine immobiliza-
tion can be achieved through simple dip coating in a LDA/PBS
solution for all different kinds of substrates including metal (Ti
alloy, Cu), ceramic (Al2O3, glass), inorganic (silicon, quartz), and
polymer (PTFE, PVDF, PE, PP, and PET). The modified
surfaces all show similar water contact angles near 60ꢀ, though
the different original surfaces show quite different water contact
angles (Fig. 6).
In the UV-vis spectrum of LDA (Fig. 4), there is an absor-
bance peak for the catechol group at 280 nm, which confirms the
conjugation of dopamine with lysine.
The successful surface modification on the surfaces by LDA
can be also confirmed by FTIR, XPS, and SEM-EDS measure-
ments. The FTIR spectra of a PP microporous membrane (PP-
pore) before and after modification by LDA show the different
surface chemical structures. Compared with the PP-pore
membrane, new bands at 1660 cmꢁ1, 1560 cmꢁ1 (amide I, II
1
The FTIR spectra, H-NMR spectra and UV-vis spectrum
confirm the successful synthesis of LDA.
Surface modification for different substrates by LDA
+
stretching vibration) and 1520 cmꢁ1 (NH3 bending vibration)
are observed in LDA modified PP porous membrane (PP-pore-
LDA) (Fig. 7).
It is well known that the universal adhesion ability of mussels
relies on the repeated DOPA-lysine motif in mussel foot adhesive
proteins. In the adhesive protein, the pendant catechol from
DOPA and the amine from lysine are responsible for the
Compared with the unmodified membrane, a LDA modified
PTFE porous membrane (PTFE-pore-LDA) shows an obvious
increase in surface oxygen content, the appearance of surface
nitrogen, and a sharp decrease in surface fluorine content (Fig. 8)
by XPS. These results indicate that LDA was indeed introduced
onto the PTFE membrane surface.
Fig. 2 FTIR spectra of Boc2-lysine-OH (BL), Boc2-lysine-NHS (BLN),
Boc2-lysine-DA (BLDA) and lysine-dopamine (LDA).
Fig. 4 UV-vis spectrum of lysine-dopamine (LDA).
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Fig. 5 Facile and universal surface modification by LDA: photograph
of substrates before (upper row) and after immersion in LDA (lower
row).
Fig. 8 XPS and elemental analysis of PTFE-pore and PTPE-pore-LDA.
shown in Fig. 10, no obvious change of water contact angle (near
60ꢀ) was observed for PTFE-pore-LDA membrane before or
after testing, which suggests that the LDA modification of the
PTFE-pore membrane is durable.
Cell viability, cell adhesion and cell proliferation
Since lysine is a bio-functional molecule and dopamine is an
excellent anchoring moiety, a biocompatible surface can be
created by the one-step modification method for the immobili-
zation of lysine via the mussel adhesive moiety. Surfaces
modified by LDA show potential for cell culture and cell
immobilization.
As shown in Fig. 11, the PTFE-pore-LDA substrate has
remarkable cell affinity compared to the corresponding control
PTFE membrane (PTFE-pore). LDA modified PTFE membrane
shows a much higher WST reduction activity (+300%) than that
of PTFE membrane. The PTFE-pore-LDA substrate is favorable
for the culture of HUVEC. Therefore, a LDA modified surface
improves the cell viability and shows potential in promoting cell
proliferation.
Fig. 6 Water contact angle of original and LDA modified substrates.
LDA also improves adhesion to HUVEC and accelerates
endothelialization. As shown in Fig. 12, few cells were observed
on the surface PTFE-pore membrane after poorly adhered cells
were removed by washing with PBS, while lots of live cells (in
green color) can adhere to and grow on the surface of the
PTFE-pore-LDA membrane. As is well known, HUVEC can
barely adhere to an inert PTFE surface. The good cell adhesion
and cell proliferation on the PTFE-pore-LDA surface are
attributed to the biocompatibility of LDA through two
Fig. 7 FTIR spectra of original and LDA modified PP porous
membranes.
Thus, classically adhesion-resistant materials, such as PTFE,
were successfully modified by LDA through a simple dip-coating
process. The LDA modified PTFE surface shows an even
distribution of N and O as shown in Fig. 9.
Stability of surface modification
In order to confirm the stability of surface modification by LDA,
durability testing on PTFE-pore-LDA was carried out. As
Fig.
9 Elemental distribution on PTFE-pore-LDA surfaces by
SEM-EDS.
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Fig. 10 Water contact angle of PTFE-pore-LDA before (a) and after (b)
agitation with deionized water in a shaker (150 rpm) at 45 ꢀC for 3 days.
Fig. 13 Clot formation in plasma expressed as absorbance at 405 nm
versus time for PP-pore, PP-pore-DA and PP-pore-LDA.
Conclusions
Inspired by mussel adhesive moiety dopamine and bio-functional
moiety lysine, a novel molecule LDA was successfully synthe-
sized and explored as a universal and robust surface modifier for
different materials including metal, ceramic, inorganic, and
polymer. Impressively, inert surfaces (even PTFE) can be also
well modified by LDA through a facile and cost-effective dip-
coating process. More importantly, lysine was immobilized on
the surface via mussel adhesive moiety dopamine during the
approach. Furthermore, the PTFE membrane modified by LDA
showed excellent durability, improved cell adhesion, promoted
cell growth, and accelerated endothelialization. Also, for the
immobilization of fibrinolytic lysine, LDA modified surface
shows plasma clot lysis activity. For the unique properties,
functional molecule LDA shows potential in surface chemistry,
materials science, bio-technology, tissue engineering and many
other applications.
Fig. 11 Cell viability of human umbilical vein endothelial cells
(HUVEC) after culturing for 24 h.
Acknowledgements
Fig. 12 Photomicrograph of HUVEC on membranes after culturing for
The authors are grateful for project support from The Dow
Chemical Company, the National Natural Science Foundation
of China (20974061) and the Shanghai Leading Academic
Discipline Project (No. B202). The authors thank Dr Thomas
H. Kalantar of The Dow Chemical Company for helpful
comments on this manuscript.
24 h (AO/EB staining): (a) PTFE-pore, (b) PTFE-pore-LDA.
adhesion mechanisms (cationic adhesion and DOPA-mediated
adhesion).8
Plasma clot lysis
For the fibrinolytic ability of lysine,4 the substrate modified by
LDA shows potential for creating a clot-lysing surface. In the
plasma clot lysis assay, clot formation was expressed as absor-
bance at 405 nm vs. time. The onset of coagulation is indicated by
a steep rise in the absorbance vs. time curve following recalcifi-
cation of the plasma. As shown in Fig. 13, the PP-pore and PP-
pore-DA surface showed a typical clot formation curve:
a plateau in absorbance was reached and maintained indicating
a fully formed, stable clot. In contrast, for the PP-pore-LDA
surface, the absorbance returned to baseline, indicating that the
clot formed and then was lysed. This result indicates that the
LDA modified surface is bioactive and shows specific binding
capacity of plasminogen, based on the immobilized fibrinolytic
lysine through a simple coating process.
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