Synthesis and characterization of antifouling poly(N - acryloylaminoethoxyethanol) with ultralow protein adsorption and cell attachment

Article abstract of DOI:10.1021/la502136q

Rational design of effective antifouling polymers is challenging but important for many fundamental and applied applications. Herein we synthesize and characterize an N-acryloylaminoethoxyethanol (AAEE) monomer, which integrates three hydrophilic groups of hydroxyl, amide, and ethylene glycol in the same material. AAEE monomers were further grafted and polymerized on gold substrates to form polyAAEE brushes with well-controlled thickness via surface-initiated atomic transfer radical polymerization (SI-ATRP), with particular attention to a better understanding of the molecular structure-antifouling property relationship of hydroxyl-acrylic-based polymers. The surface hydrophilicity and antifouling properties of polyAAEE brushes as a function of film thickness are studied by combined experimental and computational methods including surface plasmon resonance (SPR) sensors, atomic force microscopy (AFM), cell adhesion assay, and molecular dynamics (MD) simulations. With the optimal polymer film thicknesses (～10-40 nm), polyAAEE-grafted surfaces can effectively resist protein adsorption from single-protein solutions and undiluted human blood plasma and serum to a nonfouling level (i.e., <0.3 ng/cm²). The polyAAEE brushes also highly resist mammalian cell attachment up to 3 days. MD simulations confirm that the integration of three hydrophilic groups induce a stronger and closer hydration layer around polyAAEE, revealing a positive relationship between surface hydration and antifouling properties. The molecular structure- antifouling properties relationship of a series of hydroxyl-acrylic-based polymers is also discussed. This work hopefully provides a promising structural motif for the design of new effective antifouling materials beyond traditional ethylene glycol-based antifouling materials.

Full text of DOI:10.1021/la502136q

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Article
Synthesis and Characterization of Antifouling Poly (N-Acryloylamido-
Ethoxyethanol) with Ultralow Protein Adsorption and Cell Attachment
Hong Chen, Mingzhen Zhang, Jintao Yang, chao zhao,
Rundong Hu, Qiang Chen, Yung Chang, and Jie Zheng
Langmuir, Just Accepted Manuscript • DOI: 10.1021/la502136q • Publication Date (Web): 15 Aug 2014
Downloaded from http://pubs.acs.org on August 16, 2014
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Synthesis and Characterization of Antifouling Poly (N-Acryloylamino-
Ethoxyethanol) with Ultralow Protein Adsorption and Cell Attachment
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Hong Chen†, Mingzhen Zhang†, Jintao Yang†‡, Chao Zhao†, Rundong Hu†, Qiang Chenǁ, Yung
Chang¶, and Jie Zheng†*
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†Department of Chemical and Biomolecular Engineering
The University of Akron, Akron, Ohio 44325
‡College of Materials Science and Engineering
Zhejiang University of Technology, Hangzhou 310014, China
ǁ School of Material Science and Engineering
Henan Polytechnic University, Jiaozuo, China, 454003
¶ R&D Center for Membrane Technology and Department of Chemical Engineering
Chung Yuan University, Chung Li, Taoyuan 320, Taiwan
* Corresponding Author: Phone: 330ꢀ972ꢀ2096; Email: zhengj@uakron.edu
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ
Abstract: Rational design of effective antifouling polymers is challenging but important for many
fundamental and applied applications. Herein we synthesize and characterize an Nꢀ
acryloylaminoethoxyethanol (AAEE) monomer, which integrates three hydrophilic groups of
hydroxyl, amide, and ethylene glycol in the same material. AAEE monomers were further grafted
and polymerized on gold substrates to form polyAAEE brushes with wellꢀcontrolled thickness via
surfaceꢀinitiated atomic transfer radical polymerization (SIꢀATRP), with particular attention to a
better understanding of the molecular structureꢀantifouling property relationship of hydroxylꢀ
acrylicꢀbased polymers. The surface hydrophilicity and antifouling properties of polyAAEE brushes
as a function of film thickness are studied by combined experimental and computational methods
including surface plasmon resonance (SPR) sensors, atomic force microscopy (AFM), cell adhesion
assay, and molecular dynamics (MD) simulations. With the optimal polymer film thicknesses (~10ꢀ
40 nm), polyAAEEꢀgrafted surfaces can effectively resist protein adsorption from singleꢀprotein
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solutions and undiluted human blood plasma and serum to a nonfouling level (i.e. <0.3 ng/cm2). The
polyAAEE brushes also highly resist mammalian cell attachment up to three days. MD simulations
confirm that the integration of three hydrophilic groups induce a stronger and closer hydration layer
around polyAAEE, revealing a positive relationship between surface hydration and antifouling
properties. The molecular structureꢀantifouling properties relationship of a series of hydroxylꢀ
acrylicꢀbased polymers is also discussed. This work hopefully provides a promising structural motif
for the design of new effective antifouling materials beyond traditional ethylene glycolꢀbased
antifouling materials.
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Keywords: polyacrylamide; polyacrylate; antifouling polymer; protein resistance; proteinꢀpolymer
interaction.
Introduction
Antiꢀbiofouling surfaces are critical for many fundamental and industrial applications, ranging
from ship coatings, biomedical implants, bioseparation, biosensors, and carriers for drug and gene
delivery1ꢀ7. Development of effective antifouling materials against protein adsorption, blood
clogging, and bacterial film formation requires not only a precise design of materials with intrinsic
chemical structure and property alone (e.g. hydrophobicity, net charge, charge distribution), but also
a proper control of the physicochemical characteristics of its coating surface (e.g. film thickness,
packing density, surface roughness). Significant efforts have been made to design and synthesize
different antifouling polymers, which can be generally classified into two classes: hydrophilic
polymers including manniol8, poly(2ꢀmethyl2ꢀoxazoline)9, glycerol dendron10 and oligosaccarides11
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and zwitterionic polymers including poly(carboybeaine methacrylate (polyCBMA)12,
,
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poly(sulfobetaine
methacylate)
(polySBMA)14,
and
poly(2ꢀmethacryloyloxylethy
phosphorylcholine) (polyMPC)16, 17. Among hydrophilic antifouling materials, poly(ethylene glycol)
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(PEG)ꢀbased materials are well known for their superlow fouling capability against
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protein/cell/bacteria attachment 18, 19, thus they have been widely used to coat on different substrates
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13,
as a protective layer via physical adsorption
20, covalent immobilization 21, and plasma
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polymerization . PEGꢀbased polymers with ethylene glycol (—CH2CH2—O—) as repeat units
containing two carbon space lengths enable to form more hydrogen bonds with surrounding water
molecules as compared to (—CH2 —O—) or (—CH2CH2CH2 —O—). However, PEGꢀbased
polymers are readily subject to oxidization in complex media such as 100% blood plasma and
serum 12, 16. Among zwitterionic antifouling materials, poly(carboxybetaine) (pCB) is starting to
outperform other zwitterionic antifouling materials, because pCB is not only ultralow fouling, but
also has abundant functional groups for the direct immobilization of proteins, enabling both
ultralow fouling and functionalization in one material.
Considering that the existing antifouling polymers have very different chemical structures and
charge distributions, different antifouling mechanisms of “steric repulsion”, “surface hydration”,
and “foulingꢀrelease” have been proposed for their surfaceꢀresistance property9ꢀ11, 22ꢀ28. Some of
antifouling mechanisms are not exclusive to each other, implying that some common principles may
govern protein adsorption/resistance on the surfaces. It is generally accepted that surface resistance
to biofouling formation is largely attributed – as a first stage within the timescale of minutes to
hours – to early prevention of nonspecific protein adsorption on the polymerꢀcoated surfaces29, 30
.
Since all existing antifouling polymers are either hydrophilic or zwitterionic, the hydration layer at
different polymer interfaces can be achieved differently, i.e. hydrophilic polymers achieve surface
hydration via hydrogen bonds, while zwitterionic polymers achieve surface hydration via ionic
solvation 18, 19. In conjunction with surface hydration, high chain mobility, large exclusion volume,
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and steric hindrance of polymer coatings all likely contribute to antifouling performance
.
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However, the relation among molecular structure, surface hydration, and antifouling activity is still
not well understood, rendering more difficult for the structureꢀbased design of antifouling polymers.
For the hydroxylꢀacrylicꢀbased polymer materials, as shown in Scheme 1, chemical structures
of the monomers of 2ꢀhydroxyethyl methacrylate (HEMA), hydroxypropyl methacrylate(HPMA),
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2ꢀhydroxyethyl
acrylate
(HEA),
Nꢀhydroxyethyl
acrylamide)
(HEAA),
and
Nꢀ
acryloylaminoethoxyethanol (AAEE) contain one common group of a hydroxyl group (–OH),
meanwhile some monomers (HEMA, HPMA, and HEA) possess ester group (–COO–), while the
others (HEAA and AAEE) possess amide group (–CONH–) instead.
Acrylate
O
HEMA
OH
O
O
O
OH
HPMA
HEA
O
O
OH
Acrylamide
H
N
HEAA
AAEE
OH
O
O
H
N
OH
O
Scheme 1. Molecular structures of acrylateꢀbased monomers of HEMA, HPMA, and HEA, and
acrylamideꢀbased monomers of HEAA and AAEE.
These groups are all hydrophilic, enabling to associate with water around them to form a tightly
surfaceꢀbound water layer (“iceꢀlike” hydration layer) at polymer surface, which provide a strong
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barrier to prevent approaching proteins being adsorbed on the surface19, 25, 31, 32. Consequently, we
grafted and polymerized all of these monomers on the gold substrates to form polymer brushes
using surfaceꢀinitiated atomic transfer radical polymerization (SIꢀATRP). We demonstrated that at
an optimal range of film thicknesses, polyHEMA33 and polyHEAA34 brushes exhibited highly
surface resistance to nonspecific protein adsorption from undiluted blood plasma and serum at a
ultralow fouling level (i.e. < 5 ng/cm2 of adsorbed proteins), cell and bacterial adhesion. Under
more harsh conditions of cell culture or bacteria medium, polyHEMA suffered from a longꢀterm
stability issue to some extent similar to PEGꢀbased polymers. Differently, polyHEAA with amide
groups exhibited much longer stability and better antifouling performance in undiluted blood serum
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and plasma and bacteria medium than polyHEMA and polyHPMA without amide groups . The
shortꢀterm stability polyacrylateꢀbased polyHEMA and polyHPMA relative to polyacrylamideꢀ
based polyHEAA is likely attribute to ease to hydrolysis degradation of the ester bonds in
polyacrylate compared to the amide bonds in polyacrylamide. Further, we also found that two
polyacrylamide polymers with amide groups (polySBAA and polyHEAA) exhibited the improved
surface hydration and antifouling ability as compared to two corresponding polyacrylate polymers
with ester groups (polySBMA and polyHEA)35.
Our findings appear to suggest that the conjugation of acrylamide group with other
hydrophilic groups would present a better antifouling performance of materials. To test this
hypothesis, here we synthesized an Nꢀacryloylaminoethoxyethanol (AAEE) monomer (Scheme 1),
which contains three common hydrophilic groups of amide, hydroxyl and ethylene glycol (EG) on
the same monomer. More importantly, we used twoꢀcarbon space length to separate hydroxyl and
ethylene glycol groups, which are expected to optimize surface hydration like the EG unit. The twoꢀ
carbon space length allows surfaceꢀbound water molecules to form bridge hydrogen bonds with the
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neighboring hydrophilic groups. To further provide molecularꢀlevel understanding of the polymer
structureꢀantifouling activity relationship, we combined computational and experimental approaches
to study the surface hydration and antifouling properties of polyAAEE brushes as prepared by SIꢀ
ATRP 33, 34, 36ꢀ40. Surface plasmon resonance (SPR) and cell assay were used to realꢀtime monitor
surface resistance to proteins and cells on polymer brushes; atomic force microscopy and water
contact angle were used to characterize surface morphology and hydrophilicity; and molecular
dynamics (MD) simulations were used to quantify the structure and dynamics of water at polymer
interface. Effect of film thickness as a key factor on antifouling performance was also examined.
Collective data demonstrated that polyAAEE brushes highly resisted nonspecific protein adsorption
(<0.3 ng/cm2) from singleꢀprotein solutions and undiluted blood plasma and serum, and cell
adhesion over a 3ꢀday culture period.
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Materials and Methods
Materials. 2ꢀ(2ꢀaminoethoxy)ethanol(98%), acryloyl chloride(97.0%), hydroquinone,
copper(I) bromide(CuBr, 99.999%), human plasma fibrinogen(Fib), chicken egg white
lysozyme(Lyz), bovine serum albumin(BSA A7638), ethanol(absolute 200 proof), isopropanol,
chloroform, methanol, phosphate buffered saline (PBS,0.01M, pH=7.4, 0.138M NaCl, 0.0027M
KCl) were purchased from SigmaꢀAldrich Inc. (Milwaukee, WI). Tris [2-
(dimethylamino)ethyl)]amine (Me6TREN), silica gel (0.036ꢀ0.071 mesh) were purchased from Alfa
Aesar Inc. (Ward Hill, MA). Pooled human blood plasma and serum were obtained from
BioChemed Service (Winchester, VA). Water used in this work was purified by a Millipore water
purification system with a resistivity of 18.2 Mꢀcm. The ATRP initiator, ωꢀmercaptoundecyl
bromoisobutyrate, and bovine aortic endothelial cells (BAEC) were kindly provided by Professor
Shaoyi Jiang at the University of Washington.
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Synthesis of AAEE monomer. AAEE monomer was synthesized by the reaction of acryloyl
chloride (4.53g, 0.05mol) and 2ꢀ(2ꢀaminoethoxy) ethanol (10.5g, 0.1 mol) in ethanol (200 mL).
Acryloyl chloride dissolved in ethanol (100 mL) was added to a 3ꢀnecked flask equipped with a
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o
thermometer and a burette. The solution was cooled to ꢀ40 C (isopropanol/dry ice). 2ꢀ(2ꢀ
aminoethoxy) ethanol diluted in ethanol (100mL) was added to the flask in a dropwise way. The
reactant mixture was stirred for 2h at ꢀ40 C and then warmed to 2ꢀ4 oC overnight, as shown in
o
Scheme 2a. The product of AAEE was concentrated and suspended in acetone, which was gradually
evaporated after filtering precipitated salts. AAEE was further purified by a silica gel column,
washed with chloroform and eluted with acetone. 0.01wt% stabilizer hydroquinone was added to
the AAEE solution before solvent evaporation. AAEE was then stored in 25wt% aqueous solution
o
at 2ꢀ4 C to prevent selfꢀpolymerization. The yield was ~68%. Product identify and purity was
1
confirmed by H NMR (300 MHz, D2O): (ppm), 6.07ꢀ6.25 (m, 2H), 5.66ꢀ5.72(ddd, 1H), 3.53ꢀ
13
3.65(m, 6H), 3.38ꢀ3.42(m, 2H) and C NMR (500 MHz, D2O): (ppm), 168.6, 129.9, 127.3, 71.6,
68.7, 60.4, 39.1. AAEE was first reported and used for electrophoresis applications due to its high
hydrophilicity and hydrolytic stability by Chiari, M. et al.41 Most of the following studies have
employed AAEE for capillary electrophoresis, especially for genomics.42ꢀ44
(b)
O
O
S
Br
HS
Br
O
O
Au
Au
OH
H
N
OH
O
O
O
NH
Br
O
O
S
O
Au
Scheme 2. (a) Synthesis of AAEE monomer and (b) Schematic SIꢀATRP process for grafting
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polyAAEE onto a gold surface coated with immobilized initiators.
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SI-ATRP of AAEE on SPR chips. A SPR chip was prepared by coating a titanium adhesion
layer (2nm) and a gold layer (48nm) on a microscope glass slide using an electron beam evaporator.
The SPR chip was rinsed by ethanol and water sequentially, treated under UV/ozone for 20 min,
washed again with water and ethanol, and air dried. Before coating the polymer films, an initiator
selfꢀassembled monolayer (SAM) was first formed on the SPR chips by simply soaking preꢀcleaned
SPR chips into 1 mM ethanol solution of ωꢀmercaptoundecyl bromoisobutyrate (initiator) at room
temperature overnight. The chips were then washed with ethanol and dried in a stream of air prior to
use. Scheme 2b shows a general process for grafting polyAAEE onto a gold surface coated with
initiators via SIꢀATRP. To the first reaction tube, 25 wt% AAEE solution (1.5mL), Me6TREN
(80mg), and water (2mL) were sequentially added, followed by the removal of oxygen from the
tube by flowing through dry nitrogen stream for 20 min. Methanol added to the second tube was
also degassed by continuous nitrogen stream for 20 min. SPR gold chip coated with immobilized
initiators and CuBr (12.5 mg, 0087mmol) were placed in the third reaction tube sealed with a rubber
septum stopper, and degassed by vacuum and nitrogen injection for three cycles. The mixture of
AAEE and Me6TREN in the first tube and degassed methanol (2ml) in the second tube were
transferred to the third reaction tube with syringe under nitrogen protection. The SIꢀATRP reaction
was completed under nitrogen protection at room temperature or the reaction was stopped by
exposing to air. Upon grafting polyAAEE on the SPR chips, the chips were removed from the third
tube, soaked in PBS overnight to remove any unbounded polymer, washed with water, and dried in
a stream of air prior to later characterization. Different film thicknesses were simply achieved by
tuning the methanol/water ratio, monomer concentration, and polymerization time 33.
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Film thickness of polyAAEE brushes by ellipsometry. The thickness of polyAAEE brushes
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was measured by a multiple wavelength mode ellipsometer (J.A. Woollam Co., Lincoln, NE) with a
wavelength range of 400ꢀ800 nm at a fixed incident angle of 70o. A Cauchy model was used to fit
data for a bare SPR substrate first and then to determine the polyAAEE film thickness
simultaneously. Three different locations of each polyAAEE brush specimen were selected and
measured to obtain an average thickness with mean ± standard deviation. Different film thicknesses
of polyAAEE brushes can be obtained by tuning ATRP reaction condition.
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Contact angle measurement. Water contact angle of polyAAEE brush surface was obtained
by a sessile drop technique on a RameꢀHart goniometer (model 100ꢀ00, Mountain Lake, NJ), using
Millipore water under ambient conditions. Three different locations of each sample were selected
and measured to obtain an average contact angle with mean ± standard deviation.
Atomic force microscopy (AFM). Surface morphology and roughness of polyAAEE brushes
were examined by Nanoscope IIId AFM in a tapping mode (Veeco Inc. Goleta, CA) with a
commercial Si cantilever (NanoScience) with a spring constant of ~50 N/m. PolyAAEE films with
different thicknesses were scanned at a typical rate of 1.0ꢀ2.0 Hz with a vertical tip oscillation
frequency of`170~180 KHz. All images were acquired as 512×512 pixel images.
Protein Adsorption by SPR. A customꢀbuilt fourꢀchannel SPR sensor based on wavelength
interrogation was used to measure protein adsorption on polyAAEE brushes of various film
thicknesses. The liquid (protein solution or PBS solution) was flowed through four separated
channels under the pressure of a peristaltic pump (Ismatec). SPR chips coated with polyAAEE
brushes were attached to the base of the prism. A baseline signal was obtained by flowing PBS
buffer through the sensor to establish a stable baseline. Single protein solution in PBS (1mg/ml) of
Fib, BSA and Lyz and undiluted (100%) human blood plasma and serum were flowed through
independent channels for ~10 min, and SPR wavelength would shift if any protein adsorption
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occurred. Then, protein solution was replaced by PBS buffer solution to remove any unbound or
loosely bounded protein on the surface. All protein and PBS solutions were flowed through the
channels at the same rate of 0.05 mL/min. The wavelength shift between the PBS baselines obtained
before and after protein solution was used to quantify the amount of adsorbed proteins on polymer
brushes. A 1 nm SPR wavelength shift at 750 nm corresponds to ~15 ng/cm2 protein adsorption as
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reported by our previous works 33, 40
.
Cell adhesion. BAEC cell lines were cultured in Dulbecco’s modified Eagle’s media (DMEM)
with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% nonessential amino acids, and 2%
penicillin streptomycin solution as supplementation in a humidified incubator with 5% CO2 at 37 oC
on a tissue culture polystyrene flask for about 7 days to reach full confluence. BAEC were removed
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from the flask using a normal passaging protocol , and diluted in fresh media to a concentration of
105 cells/mL. Samples were rinsed with sterile PBS before exposing to UV light for 10 min, and
washed again with sterile PBS prior to be placed into 6ꢀwell flask. 3 mL cell suspension with a
concentration of 105 cells/mL was added to each well. The cells were incubated at 37 oC for 72
hours. Cell morphology and proliferation on polyAAEE surface were investigated by an EVOS xl
core inverted microscope (Advanced Microscopy Group, Bothell, WA) with a 10X objective after
gently rinsing with sterile PBS. Three typical images for each sample (Nsample=3) were selected and
the adhered cell number were counted and reported as mean ± standard deviation.
Molecular dynamics simulations of polyAAEE. Force field parameters for monomeric
AAEE were developed using the ParamChem tool (https://www.paramchem.org/), which is
compatible with the general CHARMM force field. After geometry optimization at MP2/6ꢀ31G*
level by Gaussian95, the partial charges were derived by fitting to the gasꢀphase electrostatic
potential using the restrained electrostatic potential (RESP) method. To validate the force field
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parameters of monomeric AAEE, short 2ꢀns simulations in the explicit TIP3P water were conducted
at 298 K. The bond lengths, bond angles, and torsion angles were well maintained in the MD
simulations as compared to quantum mechanism structures. MD simulations of a 16ꢀmer of
polyAAEE in a cubic TIP3P water box of 60 × 60 × 60 Å3 were performed using the NAMD
program 45 with CHARMM22 force fields 46. Each simulation system was first subject to 5000 steps
of steepest decent minimization with position constraints on polymers, followed by additional 5000
steps of conjugate gradient minimization without any position constraint. After energy minimization,
each system was gradually heated from 50 K to 298 K in 200 ps and equilibrated at 298 K for 1 ns
to adjust the system size and density under NPT ensemble with periodic boundary conditions in all
three dimensions. Then, 20ꢀns production MD run was conducted to examine the mutual dynamics
and interactions between polymers and water molecules. Shortꢀrange VDW interactions were
calculated by a switch function with a twin cutoff at 10 and 12 Å, while longꢀrange electrostatic
interactions were calculated by the particleꢀmesh Ewald method with a grid size of 1 Ǻ and a realꢀ
space cutoff of 14 Ǻ. The RATTLE algorithm was applied to constrain all covalent bonds involving
hydrogen atoms, so that a time step of 2 fs was used in velocity verlet integration.
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Results and Discussion
Surface characterization of polyAAEE brushes. Based on our previous studies of hydroxylꢀ
34,
acrylicꢀbased antifouling polymers polyHEMA, polyHPMA and polyHEAA33,
40, AAEE
monomer containing three hydrophilic groups separated by optimal carbonꢀspace length could be
developed into a promising antifouling material. The AAEE monomer was synthesized via the
reaction of acryloyl chloride and 2ꢀ(2ꢀaminoethoxy) ethanol in ethanol47, as shown in Scheme 2a.
Product identity and purity were confirmed by 1H and 13C NMR (Figure 1).
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Figure 1. (a) 1H NMR spectrum of monomer AAEE (300MHz, *=solvent signal) and (b) 13C NMR
spectrum of AAEE (500MHz).
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AAEE monomers were then initiated, polymerized, and grafted onto ωꢀmercaptoundecyl
bromoisobutyrate SAMꢀcoated gold substrates via the SIꢀATRP method (Scheme 2b). The SIꢀ
ATRP method has been widely used to graft a variety of antifouling polymers on different
substrates to form effective antifouling surfaces with wellꢀcontrolled film thickness and high
packing density, including polyzwitterinoicꢀbased polySBMA48 and polyCBMA13, polyacrylateꢀ
based polyHEMA33, polyHPMA40 and polyHEA35, and polyacrylamideꢀbased polyHEAA34.
Surface hydrophilicity or surface tension, commonly measured by water contact angles, is one of
the important physicochemical parameters affecting protein adsorption, cell and bacterial adhesion,
and even biofilm formation. Figure 2a shows the contact angles of a bare gold chip, an initiatorꢀ
coated chip, and a chip grafted with polyAAEE brush of ~23 nm thickness. It can be clearly seen
that water contact angles changed from ~58o on the bareꢀgold chip, to ~77o on the initiatorꢀcoated
chip due to the presence of hydrophobic Br atoms and alkyl groups, and to ~27o on the polyAAEEꢀ
grafted chip. Upon grafting polyAAEE onto the initiatorꢀcoated surface via the SIꢀATRP, water
contact angle was decreased dramatically, showing a strong evidence of enhanced surface
hydrophilicity. Since surface hydration is important for antifouling performance, the dependence of
film thickness of polyAAEE brushes on water contact angle was also examined. Figure 2b showed
that a small increase of film thickness from ~1 nm to ~6 nm led to a dramatic decrease of water
contact angle from ~55o to ~25o. When film thickness was larger than 6 nm and up to 40 nm, water
contact angle was not very sensitive to film thickness, retaining at a nearly stable value of ~25ꢀ30o.
This indicates that polyAAEE brushes of 6 nm or higher thickness enable to produce strong
hydrophilic surfaces. Considering that all antifouling surfaces are hydrophilic9, 10, thus water contact
angle may serve as a reasonable indicator, but may not be the sole one, for surface hydration or
even for bioadhesion resistance. The water contact angles results will be correlated with protein
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adsorption results later. We should note that some data points were indicated with relatively larger
error bars than others. This could be due to that contact angles change slightly as time upon each
measurement on different spots. Secondly, surface roughness varied in a small scale, particularly for
those thin polymer brushes of < 5 nm (see Fig. 5). Both effects could cause some contact angle data
to have the relatively large error bars.
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(a)
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Bare chip
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PolyAAEE brush
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Film Thickness (nm)
Figure 2. (a) Water contact angles for bare chip, initiatorꢀcoated chip, and polyAAEE brush
(~23nm) coated SPR chip. Error bars represent the standard deviation of the mean (n=3); (b)
dependence of water contact angle on the film thickness of polyAAEE brushes.
Protein adsorption on polyAAEE brushes. Our previous works have showed that the
optimal polymer film thicknesses to resist protein adsorption from single protein solution were ~20ꢀ
30 nm for polyHEMA brushes 33, ~25ꢀ45 nm for polyHPMA brushes 33, and ~10ꢀ40 nm for
polyHEAA brushes34. Film thickness of 20ꢀ30 nm seems to be a safe range to achieve effective
antifouling performance. As a proofꢀofꢀconcept, polyAAEE brushes of ~26 nm and ~24 nm were
prepared via SIꢀATRP method (Scheme 2b), and protein adsorption from three typical singleꢀ
protein solutions and undiluted blood plasma and serum was quantified by an inꢀhouse SPR sensor.
Figure 3a shows three typical SPR sensorgrams for lysozyme (14 kD, pI 12), fibrinogen (340kD, pI
5.5), and BSA (68kD, pI 4.8). Upon flowing freshly prepared 1 mg/mL protein solutions of
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lysozyme, fibrinogen, and BSA in PBS through independent SPR channels coated with polyAAEE
brushes (~28 nm film thickness), a very small amount of lysozyme (~10 ng/cm2), fibrinogen (~5
ng/cm2), and BSA (~8 ng/cm2) was immediately adsorbed on polyAAEE brushes, respectively.
However, when switching from protein solution to PBS solution, these initially adsorbed proteins
were immediately and almost completely removed from the brush surfaces, leading to almost
undetectable protein adsorption (i.e. below our SPR detection limit of 0.3 ng/cm2). This indicates
that initially adsorbed proteins were very loosely bound to the polyAAEE brushes. While protein
adsorption on polyAAEE brushes from singleꢀprotein solutions is undetectable by the SPR sensor,
undiluted human blood plasma and serum comprised of hundreds of proteins were used to challenge
polyAAEE brushes further. Figure 3b show the surface resistance of polyAAEE brushes to protein
adsorption from the undiluted human blood serum and plasma. The initial adsorption amount of
proteins was as high as ~1000 and ~680 ng/cm2 from undiluted blood plasma and serum. However,
when the plasma or serum solution was switched back to the PBS buffer, the adsorbed proteins on
polyAAEE brushes were almost completely washed away by PBS, leading to nonspecific protein
adsorption of < 0.3 ng/cm2 (Fig. 3b).
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Figure 3. (a) Single protein adsorption of 1mg/mL lysozyme (Lyz), fibrinogen (Fib), and bovine
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serum albumin (BSA) on the polyAAEE brush of ~26 nm thickness. For a better eye guidance, the
SPR curves of Lyz and Fib were shifted by 40 ng/cm2 and 20 ng/cm2, respectively; (b) protein
adsorption of 100% human blood plasma and serum on polyAAEE brush of ~24nm thickness.
Our and other prior works have showed that film thickness of polymer brushes could
dramatically affect surface resistance to protein adsorption, particularly from undiluted blood
plasma and serum. Here, polyAAEE was further studied for their film thickness dependence on
protein adsorption from undiluted blood plasma and serum (Figure 4). In Figure 4a, at a very thin
film thickness of < 6 nm, the nonspecific adsorption of plasma proteins was in a wide range of ~7ꢀ
100 ng/cm2, but with a rapid drop in adsorbed protein amount as film thickness. When film
thickness was between ~6 nm and ~10 nm, protein adsorption achieved to an ultralow fouling level
of ~5 ng/cm2. Further increase of film thickness to 10ꢀ40 nm led to protein adsorption to a
nonfouling level of ~0.3 ng/cm2.
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Figure 4. Dependence of protein adsorption from 100% human blood plasma (a) and serum (b) on
polyAAEE brushes of different film thickness.
Figure 4b shows similar trend of protein adsorption on polyAAEE brushes from undiluted blood
serum. The nonspecific protein adsorption was rapidly and greatly reduced from 60 ng/cm2 to ~5
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ng/cm2 as film thickness increased at a very narrow range of ~1ꢀ6 nm, then approached to an
ultralow fouling level at ~6ꢀ10 nm, and further minimized to a nonfouling level at ~10ꢀ40 nm. For
the polyAAEE system, protein adsorption data was correlated well with contact angle data, i.e. the
more surface hydrophilicity, the better antifouling performance. Film thickness of ~10 nm is likely a
threshold value to achieve excellent surface resistance to protein adsorption.
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Figure 5. Tappingꢀmode AFM image of polyAAEE brushes with a film thickness of (a) ~3 nm,
(b)~12 nm, (c) ~20 nm, and (d) ~29 nm.
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Surface morphology of polyAAEE brushes. Apart from surface hydrophilicity and film
thickness of polymer brushes, surface roughness is also a crucial parameter for antifouling
performance.49ꢀ51 While polymer has intrinsic antifouling capacity, rough polymerꢀcoated surface
could still kinetically trap approaching proteins in its heterogeneous valleys.40 52
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Here, tappingꢀmode AFM was employed to investigate the surface morphology and roughness
of polyAAEE brushes with different film thicknesses (Figure 5). As film thickness increased, the
surface roughness was generally deceased, with rootꢀmeanꢀsquare (rms) values of ~1.65 nm, ~0.57
nm, and ~0.50 nm for the film thicknesses of ~3 nm, ~12 nm, and ~20 nm, respectively. When the
film thickness was increased to ~29 nm, surface roughness as indicated by rms value was slightly
increased to ~0.78 nm. A close visual inspection of surface morphology by AFM showed that thin
polyAAEE brushes of ~3 nm thickness were covered by small isolated clusters, with the heights
fluctuated heavily. As film thickness increased to ~12 nm and ~20 nm, brush surfaces became
smoother and the heights were varied slightly. Further increase of film thickness to ~29 nm led to
several holeꢀlike structures on the surface, but the average height fluctuation was still minor. This
suggests that too low or too high film thicknesses tend to increase surface roughness to some extent.
Taken together of film thickness dependence on contact angle and protein adsorption, thin
polyAAEE brushes (<6 nm) seemingly exhibited poor surface resistance to protein adsorption due
to relatively low packing densities and insufficient surface hydration. It is likely that hydrophilic
carboxamideꢀethoxyethanol side chain (ꢀCONHꢀCH2CH2OCH2CH2OH) cannot completely
suppress hydrophobic Brꢀatoms and alkylꢀgroups to form a strong hydration layer as evidenced by
high contact angle of ~55ꢀ31o and relatively large surface roughness of ~1.7 nm rms value. The
thick polyAAEE brushes of >10 nm, the brushes became denser and smoother, and more
hydrophilic amide side chains and ethylene glycol groups were exposed to water to form a stable
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hydration layer with low contact angle (~30o), resulting in high surface resistance to nonꢀspecific
protein adsorption from undiluted blood plasma and serum. Long polymer chains in ~30 nm thick
polymer brushes may entangle with each other and cause slight increase of surface roughness, but
still retaining a relative low rms value of ~0.8 nm. Generally, the SIꢀATRP method enables to graft
polymer chains with high packing density and surface uniformity on the surface in a wide range of
film thicknesses.
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Cell adhesion on polyAAEE brushes. An effective antifouling surface should be able to
retain its antifouling properties in realꢀworld complex media. Adhesion of mammalian cells to a
surface can be achieved via specific interactions where adsorbed proteins act as cellꢀsurface
receptors to convey signals53, or nonspecific interactions where adsorbed precursor proteins of
BSA or fibrinogen promote subsequent cell and platelet adhesion and aggregation. It seems to be
plausible to suggest that surfaces resisting protein adsorption would be a possible candidate to resist
cell attachment. In real cell media, interactions between cells and surfaces are more complex than
previously understood. A surface that is highly resistant to nonspecific protein adsorption is not
necessarily sufficient to prevent cell adhesion11, 54. Herein, we further studied the adhesion of
bovine aortic endothelial cells onto the polyAAEE brushes of different thickness after three days
cell culture. As shown in Figure 6, optical images at magnification of 10× were taken on different
o
sample surfaces after cultured with BAEC for 72 h at 37 C. As controls, initiatorꢀcoated surface
were fully covered by BAEC as expected (Figure 6a), indicating that the initiator coated surfaces
have no ability to resist cell adhesion. For the polyAAEE brushes, a large number of cells were still
able to adhere on the thin polyAAEE brush of ~3 nm thickness (Figure 6b), with an adhered cell
density of ~3.4×104 cells/cm2 compared to the full confluence of ~7.5×104 cells/cm2. With the
increase of film thickness to ~7 nm, the adherent cells were decreased markedly, but still with nonꢀ
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negligible amounts of ~8.5×103 cells/cm2 (Figure 6c). There were almost no cells attached on
polyAAEE brushes of ~12 nm and ~23 nm (Figure 6dꢀe). This fact further confirms that polyAAEE
brushes highly resist cell adhesion when the film thickness is above 12 nm, showing similar trend of
protein adsorption as a function of film thickness. PolyAAEE is an excellent nonfouling material
against both protein adsorption and cell adhesion in a wide range of film thicknesses.
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Figure 6. Typical images of bovine aortic endothelial cells attachment on the surface of (a)
initiatorꢀcoated chip, polyAAEE brushes of (b) ~3nm, (c) ~7nm, (d) ~12nm and (e) ~23nm after 72
h culture with cells, and (f) the adhered BAEC cells density on the above surfaces (n=3).
Molecular dynamics simulations of polyAAEE hydration. To provide a better
understanding of the effect of molecular structure on the hydration of polyAAEE oligomers at
atomic level, we investigated the structural and dynamic properties of hydrated water molecules
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associated with polyAAEE 16ꢀmers using molecular dynamics (MD) simulations. Figure 7 shows
the radial distribution functions (RDFs, or g(r)) of water molecules around all hydrogenꢀbinding
atoms of polymer 16ꢀmers, which are often used to estimate the local water density around the
polymers. g(r) of polyHEAA was also included from our previous work for comparison 35. In Figure
7, polyAAEE showed displayed the first hydration peaks at the position of ~2.0 Å and the
second hydration peaks at ~3.0 Å. But, polyHEAA showed almost no peaks in these two
locations, instead displayed a relatively large peak at the far distance of >4.0 Å. The magnitude
of peak height for polyHEAA was higher than that of for polyAAEE, indicating that polyHEAA
tends to form a strong water layer at a relatively large distance as compared to polyAAEE. Due to
the presence of three hydrophilic groups in AAEE monomers, more water molecules are likely
to form intensive hydrogen bonds with polyAAEE at a closer distance, generating the more
stable polymerꢀwater association. We further analyzed the residence time of water molecules
around the two polymers. The residence times were 13.5 ps for polyHEAA and 12.7 ps for
polyAAEE, respectively, which reveals the similar tendency for water molecules to stay near
both polymers. Comparing the corresponding g(r) and residence time of water between
polyAAEE and polyHEAA, we found different spatial distribution and association of water
molecules around the two polymers, but with similar hydration properties.
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Figure 7. Radial distribution function (RDF) between oxygen atoms (Ow) of water molecules and
polyHEAA and polyAAEE.
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Structure-antifouling activity relationship of hydroxy-acrylic-based polymers. Here we aimed
to summarize and discuss the structureꢀdependent antifouling performance of hydroxylꢀacrylicꢀ
based polymers, including polyHEMA, polyHPMA, polyHEAA, polyAAEE, and polyHEA. These
polymers only differ by a pendant group on the same monomeric backbone (Scheme 1). We have
systematically studied these polymers for their antifouling performance using the same
experimental protocols33ꢀ35, 40. To make fair comparison, here we confined the antifouling
comparison to nonspecific protein adsorption on different polymer brushes under their optimal film
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thicknesses. In this way, every step ̶ from polymer brush preparation by SIꢀATRP, to protein
adsorption measurement by SPR, to film thickness measurement by ellipsometry
controlled using the wellꢀestablished experimental techniques.
̶ can be well
When grafting these polymers on the substrate to form brushes via SIꢀATRP, all polymer
brushes exhibit almost undetectable nonspecific protein adsorption (<0.3 ng/cm2) from singleꢀ
protein solutions. Protein adsorption from singleꢀprotein solution is not very sensitive to film
thickness of these polymer brushes; however protein adsorption from undiluted human blood
plasma and serum strongly depends on the film thickness. Similar film thickness effect was also
observed for other antifouling polymer brushes of polySBMA55. For the acrylateꢀbased polymers
we examined, polyHEMA brushes exhibit <5ng/cm2 adsorbed protein from undiluted blood plasma
and serum at an optimal thickness range of 20ꢀ30 nm33. However, polyHPMA brushes with an
optimal film thickness of ~30 nm induce minimal protein adsorption of ~13.5 and ~50 ng/cm2 from
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undiluted human blood serum and plasma, respectively . Structural comparison between HEMA
and HPMA monomers reveals the importance of carbonꢀspacer length for antifouling properties.
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HPMA monomer with threeꢀcarbon spacer has one extra methylene (ꢀCH2ꢀ) group than HEMA with
twoꢀcarbon spacer. The hydroxylꢀ and acrylateꢀrich groups in both polyHEMA and polyHPMA
enable to achieve surface hydration through the formation of hydrogen bond network with
interfacial water molecules, which make both polymer brushes to possess antifouling capacity.
Meanwhile, the extra methylene group also makes polyHPMA more hydrophobic than polyHEMA,
resulting in the less antifouling ability to resist protein adsorption from complex human blood
plasma and serum.
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Different from ester group (–COO–) in HPMA and HEMA monomers, acrylamideꢀbased
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HEAA and AAEE monomers have amide (–CONH–) on pendant group. Both polyHEAA and
polyAAEE brushes show undetectable protein adsorption of <0.3 ng/cm2 from singleꢀprotein
solutions, similar to polyacrylateꢀbased polyHEMA and polyHPMA brushes. Under the optimal
film thicknesses between ~10ꢀ40 nm, both polyHEAA and polyAAEE brushes show undetectable
protein adsorption from undiluted human blood plasma and serum. Comparing AAEE monomer
with HEAA monomer shows that additional ethylene glycol (ꢀCH2CH2Oꢀ) functional group being
introduced between amide group and hydroxyl group in AAEE monomer should have higher
tendency to interact with water and form more hydrogen bonds, but this effect is largely
compensated by polymer film thickness on surface. Our result of fibrinogen adsorption on
hydrogels by enzymeꢀlinked immunosorbent assay (ELISA), indicated that polyAAEE adsorbed
less fibrinogen protein than polyHEAA, exhibiting a better antifouling activity under more complex
conditions (data not shown). On the basis of above analysis and comparison, while polyacrylamideꢀ
based polyHEAA and polyAAEE exhibit comparable antifouling properties to polyacrylateꢀbased
polyHEMA, polyHEAA and polyAAEE enable to retain their nonfouling properties in a wider
range of film thicknesses as compared to polyHEMA. As for stability, since amide is more stable to
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hydrolysis degradation than ester group, brushes of polyHEAA and polyAAEE as polyacrylamide
would retain antifouling property longer in aqueous media than that of polyHEMA and polyHPMA.
PolyHEAA and polyHEA provide another straightforward example for direct comparison
between polyacrylamide and polyacrylate, in which HEAA contains an amide group while HEA
contains an ester group. At an optimal film thickness, polyHEAA had undetectable protein
adsorption from undiluted blood plasma and serum, while polyHEA had protein adsorption of ~4.9
and ~5.0 ng/cm2 from undiluted blood plasma and serum, respectively.35 Such minor difference in
protein adsorption could likely be attributed to their subtle structural difference. It was further
shown that polyHEAA hydrogels always had higher equilibrium water content than polyHEA
hydrogels. Amide group coupled with hydroxyl group in polyHEAA shows stronger tendency to
interact with water molecules and adsorb more water around polymer chains than ester group with
hydroxyl group in polyHEA. This could imply that polyacrylamideꢀbased polymers with amide
groups could have better antifouling capability than polyacrylateꢀbased polymers with ester groups.
Conclusions
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In this work, we synthesized and characterized a hydrophilic monomer of AAEE with
integration of three hydrophilic groups of hydroxyl, amide, and ethylene glycol groups, followed by
grafting and polymerizing AAEE monomers on a gold substrate to form polymer brushes of varied
film thicknesses using the SIꢀATRP. Surface hydration and antifouling property of polyAAEE
brushes showed a strong dependence of film thickness. Collective data from SPR and water contact
angle revealed that under their optimal film thickness of 10ꢀ40 nm, polyAAEE brushes exhibited
the lowest water contact angle of ~30o and almost undetectable protein adsorption (<0.3 ng/cm2)
from singleꢀprotein solutions and undiluted protein plasma and serum. The enhanced surface
hydrophilicity appears to positively correlate with proteinꢀresistant ability. It was further shown that
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polyAAEEꢀgrafted surfaces can effective resist cell adhesion up to 3 days. This work provides a
new direction to prepare hydroxylꢀacrylamideꢀbased antifouling polymers beyond traditional PEGꢀ
based materials.
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Acknowledgement. We thank financial support from NSF CREER Award CBETꢀ0952624 and
NSF CBETꢀ1158447.
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