surface modification to control protein/surface interactions

Feature Article

Surface Modification to Control Protein/Surface Interactions

Lin Yuan, Qian Yu, Dan Li, Hong Chen*

Protein/surface interactions are well known to play an important role in various biologicalphenomena and to determine the ultimate biofunctionality of a given material once it is incontact with a biological environment. Control over the interactions between proteins andmaterial surfaces are not only of great theoretical inter-est but also of crucial importance for many biomedicalapplications. In this Feature Article, we summarize var-ious successful approaches used in our laboratory andother groups for controlling protein adsorption throughchemical modification of the surface and/or the intro-duction of specific topographic features. Some perspect-ives on future research in these areas are also presented.

Introduction

In the biological environment, biomaterials interact with

many different components. Among all of these compo-

nents, proteins are of prime importance for the biointerface

since most cell structures and functions depend on protein

assembly and activity.[1,2] The interactions between

proteins and material surfaces involve a variety of

intermolecular interactions (e.g., hydrophobic interactions,

electrostatic interactions, coordination bonding, molecular

recognition, etc.).[3,4] In addition, protein adsorption is

influenced by surface topography.[5] Sincematerial proper-

ties are generally not ‘‘designed’’ to control protein

adsorption and activity, it is necessary to modify the

surface chemically and physically to produce functional

polymeric biomaterials. Incorporating hydrophilic poly-

mers to weaken hydrophobic interactions with proteins,

L. Yuan, Q. Yu, D. Li, H. ChenCollege of Chemistry, Chemical Engineering and MaterialsScience, Jiangsu Key Laboratory of Advanced Functional PolymerDesign and Application, SoochowUniversity, Suzhou 215123, ChinaE-mail: [email protected]. Yu, D. LiSchool of Material Science and Engineering, Wuhan University ofTechnology, Wuhan 430070, China

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conjugating special ligands for specific protein recognition,

grafting stimuli-sensitive polymers to regulate protein

adsorption in response to environmental factors, and

fabricating polymer surfaces with specific topographic

features to control spatial protein distribution or protein

amount are all successful methods for controlling protein

interactionswith surfaces. Such control using these various

approaches is the overall topic of our research.

Hydrophilic Polymers for SurfaceModification to Repel Proteins

It is generally observed that protein adsorption increases

with increasing surface hydrophobicity.[6] Therefore, in our

laboratory several hydrophilic polymers have been inves-

tigated as surface modifiers to reduce hydrophobicity and

protein adsorption. Poly(ethylene oxide)/poly(ethylene

glycol) (PEO/PEG) chains have been shown to resist protein

adsorption via two probable mechanisms: steric repulsion

due to chain compression and a ‘‘barrier’’ created by

structured water associated with the PEO/PEG.[7,8] There-

fore many groups have developed PEO/PEG-based materi-

als as antifouling surfaces.[9–12]

elibrary.com DOI: 10.1002/mabi.201000464 1031

Lin Yuan received his PhD from Shanghai Insti-tute of Plant Physiology and Ecology, ChineseAcademy of Sciences in 2001. After a post docat Johns Hopkins University, he joined WuhanUniversity of Technology as an associate pro-fessor in 2003. Since 2010, he has been a pro-fessor in the College of Chemistry, ChemicalEngineering and Materials Science at SoochowUniversity. Currently, his research interests arefocused on nanomaterials and biotechnology.

Qian Yu received his BS degree from WuhanUniversity of Technology in 2005. He is currentlya PhD candidate in the School ofMaterial Scienceand Engineering at Wuhan University of Tech-nology. His research is focused on the stimuli-responsible polymer modified surfaces and theirinteractions with proteins.

Dan Li received her BS degree from WuhanUniversity of Technology in 2006. She iscurrently a PhD candidate in the School ofMaterial Science and Engineering at WuhanUniversity of Technology. Her research is focusedon the surface modification for improving bloodcompatibility of biomaterials.

Hong Chen received her PhD degree from Nanj-ing University in 2001. During 2001 to 2004, shewas a postdoctoral researcher in McMaster Uni-versity. From 2005 to 2009, she has been aprofessor in the School of Materials Scienceand Engineering at Wuhan University of Tech-nology. Since 2010, her research group moved toSuzhou and joined SoochowUniversity. Hermainresearch interests are focused on biointerface.

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L. Yuan, Q. Yu, D. Li, H. Chen

Polydimethylsiloxane (PDMS) has a long history of use in

biological and biomedical applications but its strong

hydrophobicity makes it susceptible to nonspecific protein

adsorption. We prepared protein resistant PDMS-based

elastomers either by incorporating mono- or bis(triethoxy-

silyl)PEO (TES-PEO-Me or TES-PEO-TES) during rubber

formation by classic room temperature vulcanization

chemistry (Scheme 1A).[13,14] Through the TES groups PEO

was covalently bound to the backbone of the silicone; and

the PEO chains migrated to the surface when exposed to

water. Both resulting surfaces exhibited increased hydro-

philicity and resistance to protein adsorption. The mono-

functional PEO was found to be much more protein

repellent probably due to the easy migration of the free

end to the aqueous interface, while the loop structure

formed by the bifunctional PEOwasmore constrained. This

method offers a facile and highly efficient approach for

surface modification of PDMS. Moreover, novel properties

of PDMS surfaces can also be achievedby thismethodusing

other oligomers containing TES groups. For example, Ma’s

group developed an initiator-integrated PDMS (iPDMS)

using a similar approach, which could initiate polymeriza-

tion of various functional monomers allowing further

‘‘tunability’’ of PDMS surface properties.[15]

Although bulk modification is an easy way to improve

surface bioinertness, it has some ‘‘side’’ effects on bulk

mechanical properties. Covalent grafting of PEO to a

material surface is also an efficient way to prepare a

protein-repellent surface. We have described an effective

methodfor functionalizingPDMSsurface, inwhichPEOwas

grafted to pre-functionalized PDMS (PDMS-Si-H) through

hydrosilylation (Scheme 1B).[16] Fibrinogen adsorption

from buffer to the PEO-modified surfaces was reduced by

more than 90% compared with controls. We adopted a

similar ‘‘grafting to’’ strategy to immobilize PEGon another

well-known biomaterial, polyurethane (PU). The effect of

PEG graft density and conformation on protein adsorption

was also investigated.[17] PU was firstly modified with

monobenzyloxypoly(ethylene glycol) (BPEG) and then

backfilled with PEG. The graft density was increased by

backfilling PEG while fibrinogen and albumin adsorption

significantly increased. We proposed that the benzyloxy

end groups of preconstructed BPEG chains stretch to the

surface after PEG backfilling, which possibly accounts for

the observed increase in protein adsorption. Therefore, the

conformation of the surface-grafted PEG appears to make

an important contribution to the protein-repellent proper-

ties of the modified surfaces. The results indicate that if

we design a surface modified by polymers containing

segments with different hydrophobicity, it is possible to

alter surface hydrophilicity and further influence the

interaction of materials with biomolecules, through

regulating the conformational change of polymer chains

in aqueous solution.

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Further, we have developed these methods to prepare a

variety of bioactive surfaces by attaching specific biomo-

lecules distally to the surface-immobilized PEO/PEG as

described in the following section. ThePEO/PEGthenactsas

a spacer as well as a protein resistant component.

An inherent disadvantage of the ‘‘grafting to’’ strategy

that must be used with PEG is that the grafting density

is limited due to steric restrictions on the attachment of

long, preformed chains; these low densities may be

insufficient for the achievement of antifouling proper-

ties.[18] Recently, we introduced a facile, high yield method

for surface modification of polyurethane (PU) with any

double-bond containing monomer (Scheme 1C).[19] In this

method, C¼C double bonds are incorporated into the

PU surface, which are subsequently copolymerized with

hydrophilic monomers such as 2-hydroxyethyl methacry-

late (HEMA) andN-isopropylacrylamide (NIPAAm). PHEMA

and PNIPAAm grafts are expected to give protein resistant

surfaces. After modification, the hydrophobic PU surfaces

become relatively hydrophilic due to the bound water

molecules associated with the polymer chains. We

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Scheme 1. Surface modification with hydrophilic polymers for reduction of proteinadsorption.


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showed that fibrinogen adsorption was reduced by more

than 90% on these modified surfaces by both PHEMA and

PNIPAAm.

Compared with conventional radical polymerization

methods, surface-initiated atom-transfer radical polymer-

ization (SI-ATRP) is amore effectiveway to obtain a surface

having uniform properties that can control protein

adsorption behavior (Scheme 1D).[20] Several typical

hydrophilic polymer brushes [21–25] have been prepared

by SI-ATRP and these surfaces have exhibited excellent

antifouling properties. Very recently, we prepared well-

controlled poly(N-vinylpyrrolidone) (PVP)-grafted silicon

surfaces for the first time using SI-ATRP.[26] It was found

that surfaces with thick grafted layers showed a dramatic

reduction in the level of adsorption of threemodel proteins

(fibrinogen, human serum albumin, and lysozyme) with

varying properties. As the thickness of the PVP layer

increased, the water contact angle (WCA) gradually

decreased and then remained constant beyond a ‘‘critical’’

thickness of �13nm. Fibrinogen adsorption on the PVP

modified surfaces followed the same trend, suggesting

that to a large extent protein adsorption was determined

by surface wettability. Based on the literature [22,23]

and our findings, we proposed that the thickness of a

grafted polymer layer is important for resistance to

nonspecific protein adsorption and that there is a ‘‘critical

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thickness’’ for most hydrophilic poly-

mers. When the grafted layer is above

its ‘‘critical thickness’’, proteinadsorption

on the modified surface will be indepen-

dent of the layer thickness. When the

polymer layer is thick enough on the

surface, the surface properties will

depend largely on the bulk properties of

the grafted polymer, since the substrate

will be fully covered.

Wealso designed amphiphilic polymer

modified surfaces for controlling protein

adsorption by hydrophobic interactions.

BlockcopolymersofPVPandhydrophobic

polystyrene (PS) were grafted on the

surface by consecutive ATRP.[27] It was

found that as the surface wettability

decreased (WCA increased by about 208fromPVP toPVP-b-PS surface), theadsorp-

tion of fibrinogen and lysozyme

increased. So it seemed possibly that

protein adsorption could be regulated

by tuning the chemical composition of

diblock copolymers tethered to the sur-

face. This concept is being pursued in our

ongoing work.

In addition to the neutral polymers

discussed above, another major class

of hydrophilic polymers are the zwitterionic polymers

including poly(2-methacryloyloxyethyl phosphorylcholine)

(PMPC),[22] poly(sulfobetainemethacrylate) (PSBMA),[24] and

poly(carboxybetaine methacrylate) (PCBMA).[25] As the

neutral polymers form hydration layers via hydrogen

bonding, the zwitterionic polymers can bind water

molecules even more strongly via electrostatically

inducedhydration. Fenget al. compared theanti-biofouling

properties of PMPC and poly(oligo(ethylene glycol) metha-

crylate) (POEGMA) brushes of different thickness and

graft density.[23] They found that the hydrophilicity of

the modified surfaces increased and the non-specific

protein resistance increased as polymer thickness and

graft density increased. Jiang’s group focused on the

preparation of zwitterionic polybetaine-based materials

to resist protein adsorption and biofilm formation.[28–30]

PSBMA and PCBMA grafted surfaces prepared via SI-ATRP

have been shown to be highly resistant to non-specific

protein adsorption in both single protein solution and

complex media (such as human blood plasma or serum).

Recently, they developed a new strategy to prepare

nonfouling surfaces using mixed-charge self-assembled

monolayers (SAMs) and mixed-charge polymer coatings.

They indicate that ultralow-fouling surfaces can be

achieved by introducing a nanometer scale homogeneous

mixture of balanced charge groups on the surface.[31,32]

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Regulation of Specific Protein/SurfaceInteractions by Surface Modification

Specific binding of a biomolecule to promote a favorable

biological response is of crucial importance for the

development of implant materials, tissue engineering,

protein purification and biosensors. Proteins can be

specifically recognized and bound by several kinds of

biological ‘‘ligands’’ including antibodies, enzymes, pep-

tides, polysaccharides, and nucleic acids. Many mechan-

isms of specific protein recognition have been exploited in

solution; their translation to interfacial systems may

provide surfaces having targeted bioactivities. However,

the binding of proteins from solution to specific sites on a

surface is complicated bynonspecific protein adsorption. In

addition, surface bound proteins are often structurally

altered leading to loss of biological activity. Therefore, the

development of better methods to introduce specific

protein binding ligands into a material surface is central

to the development of new bioactive surfaces.

Activating a surface with functional groups and binding

bioactive molecules via reaction with these groups is a

convenient method to produce a bioactive surface. For

example, fibrinolytic surfaces were developed by coupling

lysine to surface-localized chlorosulfonyl groups such that

Scheme 2. Various strategies of surface modification for specific protein binding.

both the e-NH2 and �COOH groups of

lysine were free (e-lysine).[33,34] These

surfaces were found to adsorb plasmino-

gen from plasma with some degree of

selectivity and were subsequently able

to lyse fibrin clots in vitro following

exposure to tissue plasminogen activator

(t-PA). However, for further development

of afibrinolytic surface for clinical applica-

tions, a higher surface density of immo-

bilized e-lysine is needed. In this regard, a

polyurethane material was covalently

coated with polyacrylamide bearing

pendant e-lysine and benzophenone

moieties byphotochemicalmethods.[35,36]

The e-lysine density achieved by this

methodrangedfrom0.2to3.2nmol � cm�2

depending on the concentration of the

coating reagent. Plasminogen adsorption

from plasma increased with increasing

lysine content and reached a value of

1.2mg � cm�2 for the surface with the

highest lysine content.

While high specificity for the target

proteins in some cases could be achieved

by surface grafting of designated bioac-

tive molecules with high graft density,

elimination of all nonspecific protein

adsorption is not an easy task given that



typical environments, such as blood, always contain

surface-active components. From this consideration our

group has made great efforts on both suppression of

nonspecific protein adsorption and promotion of specific

protein adsorption in a single surface design (Scheme 2B).

Since PEG-grafted surfaces have been shown to be

particularly effective in minimizing protein adsorption

as described above, immobilization of bioactive mole-

cules on the surface through a PEG spacer should be

expected to reduce nonspecific protein adsorption and

thus enhance selectivity of specific proteins. In addition,

when used as a spacer, PEG tends to move the bioactive

moiety away from the surface, making it potentially more

effective than if coupled directly. We synthesized an

asymmetric PEG with an allyl group and an N-succini-

midyl carbonate (NSC) group at the respective termini.

The PEG was hydrosilylated onto a Si�H functional PDMS

surface with the NSC group distal to the surface and

available for covalent immobilization of amine-contain-

ing bioactive molecules. Three series of biomolecules

were successfully immobilized including oligopeptides,

proteins, and glycosaminoglycans, demonstrating the

generality of the method.[37] Based on this strategy, a

heparinized silicone surface was developed with a

heparin surface density of 0.68mg � cm�2. High specificity

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for antithrombin with little fibrinogen adsorption was

noted in plasma studies and thrombin inhibition by the

heparin modified surfaces was demonstrably greater as

measured by a chromogenic thrombin generation

assay.[38] These results confirmed the validity of the

combination of ‘‘bioinert’’ and ‘‘bioactive’’ strategies to

improve protein selectivity. Recently, we used such a

strategy with PDMS and PU as substrates. e-lysine was

conjugated to the distal terminus of surface-grafted

PEG [39,40] and it was shown that these lysinated surfaces

reduce nonspecific protein adsorption while binding

plasminogen from plasma with a high degree of

selectivity. When activated by t-PA the plasmin generated

at the surface was shown to lyse fibrin in vitro. The PEG

spacer was also shown to assist in the prevention of

platelet adhesion and activation in flowing whole blood.

Neither surface-bound lysine nor adsorbed plasminogen

promoted platelet adhesion. However, the PEG spacer,

while effectively resisting nonspecific protein adsorption,

was a deterrent to the specific binding of plasminogen

due to the protein repellent properties of the PEG and the

enhanced mobility of the chain-end conjugated lysines.

It is well known that the repellent effect of PEG is

dependent on chain length; therefore we investigated

the effects of PEG chain length on plasminogen binding

to lysine at the PEG distal terminus.[41] It was concluded

that lysinated surfaces with PEG spacers of relatively

short length adsorb plasminogen more rapidly than those

with longer PEG, although theultimate adsorbedquantities

are the same, and correspondingly, the surface with the

greater plasminogen binding capacity lyzed clot more

rapidly.

Asmentioned in theprevious section, the surfacedensity

of PEG achievable by ‘‘grafting to’’ strategy is limited due

to steric hindrance,[42] and the density of chain-end

conjugated bioactivemolecules is correspondingly limited.

An alternative approach to generate biologically active

interfaces via surface-initiated polymerization (SIP) is to

use bioinert polymer brushes as a platform that can

be functionalized with bioactive molecules to introduce

defined biological functions.[42] SIP is a well-known

‘‘grafting from’’ method to generate much denser polymer

layers, and, more importantly, if the surface-grafted

polymers have abundant side chains with active chain

ends, this permits the generation of a high concentration of

chemically active sites on the surface for binding bioactive

molecules (Scheme 2C). Of the variousmonomers available

to form such comb-like polymers, OEGMA and HEMA

have been the most widely used due to their excellent

bioinertness. Raynor et al described an approach tomodify

the surface of titanium with dense polymer brushes of

POEGMA by SI-ATRP, and then convert the hydroxyl end

groups to 4-nitrophenyl carbonate groups allowing for

the tethering of bioadhesive peptides.[43] This approach




provides a robustmethod to generate coatings that prevent

fouling while selectively promoting cell adhesion. Simi-

larly, Xu et al prepared a series of POEGMA or PHEMA

modified silicon surfaces by SI-ATRP and coupled bioactive

molecules including collagen (for cell adhesion), heparin

(for antithrombogenicity), and IgG (for antigen binding) to

the pendant hydroxyl groups by chemically bifunctional

molecules such as thionyl chloride, 1,10-carbonyldiimid-

azoleandsuccinicanhydride.[44–46] This strategyalso favors

a high signal-to-noise ratio in biosensor and microarray

applications.Xuetal. reported recentlyonbinarybrushesof

poly(poly(ethylene glycol) methacrylate-co-poly(ethylene

glycol)methyl ether methacrylate) (P(PEGMA-co-PEG-

MEMA)) and poly(poly(ethylene glycol)methyl ether

methacrylate)(PPEGMEMA) prepared by consecutive ATRP

from a resist-microdomained silicon wafer surface.[47] The

terminalhydroxylgroupsonthesidechainsofPEGMAunits

were subsequently activated to allow coupling of IgG. The

resulting IgG-modified PEG microdomains interacted only

and specifically with the target anti-IgG protein while

the other PEG microregions inhibited nonspecific protein

adsorption.

More recently, we developed a PHEMA modified PU

surface, on which high lysine density and fibrinolytic

activity were achieved by coupling lysine to the hydroxyl

groups of the tethered PHEMA such that the e-amino

group was free.[48] The lysine density reached a value of

2.81comparedto0.76nmol � cm�2onacomparablePU-PEG-

lysine surface. This surface was shown to reduce non-

specific (fibrinogen) adsorptionwhilebindingplasminogen

from plasma with high affinity. With increased plasmino-

gen binding capacity, this surface showed more rapid clot

lysis (20min) in a standard in vitro assay than the

corresponding PEG-lysine system (40min). This method

of modification may also provide a generic approach for

preparing bioactive PU surfaces of high activity and low

nonspecific protein adsorption.

We have also pursued a strategy for the design of

bioactive surfacesusingadiblock copolymerofOEGMAand

N-hydroxysuccinimidyl methacrylate (NHSMA), POEGMA-

b-PNHSMA.[49] These materials have shown both non-

specific protein resistance (due to POEGMA) and high

loading capacities for immobilized bioactive molecules

(due to PNHSMA) (Scheme 2D). This approach avoids the

activation process and reduces losses due to multistep

procedures and side-reactions. After immobilization of

appropriate biomolecules (e.g., biotin, heparin) these

bioactive surfaces are expected to enhance specific

biological recognition with high selectivity of their target

molecules (avidin, antithrombin). The underlying bioinert

POEGMA layer prevents the adsorption of other proteins

and provides a relatively hydrophilic environment which

maintains the activity of the immobilized bioactive

molecules.

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Stimuli-Responsive Polymers and DiblockCopolymers for Surface Modification toRegulate Protein Adsorption and CellAdhesion

In recent years, stimuli-responsive polymers, also called

smart polymers,which canundergo conformation or phase

changes in response to variations in conditions (tempera-

ture, pH, ionic strength, electric field, light) have received

considerable attention.[50–52] When such polymers are

immobilized on a solid surface, they give rise to stimuli-

responsive changes in surface properties including wett-

ability, roughness, and biomolecule adhesion. Our work in

this area is briefly discussed in this section. We are

interested in using smart polymers to control protein

adsorption by change of environmental stimulus factors.

Poly(NIPAAm) (PNIPAAm) is the best known of the

thermo-responsive polymers, and has been extensively

studied for awide range of biomedical applications such as

diagnostics, affinity precipitation, and controlled drug

release.[53,54] When PNIPAAm is immobilized on a solid

surface, it can undergo an expanded/collapsed conforma-

tional transition at the lower critical solution temperature

(LCST, �32 8C) resulting in temperature controlled surface

wettability and biomolecule adhesion properties.[55,56]

Considerable efforts have been made to utilize PNIPAAm

coated surfaces as cell culture substrates.[57,58] In our group,

we have focused on the factors influencing protein

adsorption to PNIPAAm modified surfaces (Scheme 3A).

In recent work, we have found that the thermo-respon-

siveness of protein adsorption on PNIPAAm-modified

silicon surfaces is thickness dependent.[59] In the thickness

range below 15nm, changes in adsorption in response to

temperature are not great. However, it is interesting that

these surfaces are generally protein resistant. On the other

hand, for thicker PNIPAAm grafted surfaces, the tempera-

ture sensitivity of protein adsorption is significant.

Adsorption data for proteins of different size indicate that

PNIPAAmmodified surfaces are protein size-sensitive; this

Scheme 3. (A) Thermo-responsive polymer brushes for control over prprotein adsorption and cell adhesion.



may be exploited in applications such as protein purifica-

tion and biosensors.

Wehavealso prepared PNIPAAmmodifiedPUsurfaces as

cell-culture substrates and found that their thermo-

responsive properties were critically dependent on the

molecular weight (MW) of the PNIPAAm. We found that

only surfaces grafted with PNIPAAm of an appropriate

intermediate MW (61 000 to 100 000) can facilitate adhe-

sion and detachment of cells by control of temperature.[60]

This phenomenonwas also observed for PNIPAAmbrushes

grafted on silicon and tissue-culture polystyrene (TCPS)

surfaces.[61,62] Thermo-responsive cell adhesion/detach-

ment behavior was found to weaken or even disappear

when the PNIPAAm layer thicknesswas beyond a ‘‘critical’’

value.

To developmore advanced systems thatmight avoid the

thickness limitation noted above, we designed a novel

strategy using diblock copolymer brush surfaces with

blocks of PNIPAAm (or other functional polymers) as the

first block. PSwas chosen as the second block because it is a

typical hydrophobic polymer and is widely used in cell

culture applications (Scheme 3B).[63] As expected, we found

that the adhesion and detachment of L929 cells could be

more finely ‘‘tuned’’ using these materials. Thermo-

responsive control of the adhesion/detachment of cells

was again seen on block copolymer surfaces where initial

PNIPAAm blocks (attached to the surface) were coupled to

distal PS blocks. It should be noted that the MW (and

thickness) of thePNIPAAmblockwas thesameas in thecase

of the homoPNIPAAmwhichwas not thermo-responsive to

cells. Thisdesign strategyhasbeenapplied toother systems

including PVP-b-PS[27] and POEGMA-b-PS[64] copolymer

brushes. These surfaces are able to promote cell adhesion

and proliferation while maintaining good protein resis-

tance.

Surface roughness, especially nano-scale roughness can

dramatically enhance environment-responsive wettabil-

ity. For example, Jiangandcoworkershaveobtained several

smart surfaces based on nanoscale topography with

otein adsorption and (B) diblock copolymer brushes for regulation of

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reversible switching between superhydrophilicity and

superhydrophobicity.[65,66] Motivated by this unique prop-

erty and the known general trend that protein adsorption

increases with increasing surface hydrophobicity, we

investigated protein adsorption on smart polymer grafted

surfaces with nanoscale topography. We fabricated silicon

nanowire arrays (SiNWAs) using a wet chemical etching

method and then grafted PNIPAAm onto the arrays. The

thermoresponsive wettability was enhanced significantly

by introducing nanostructure. However, it is interesting

that the SiNWAs-PNIPAAm surfaces exhibited good non-

specificprotein resistance regardlessof temperature (below

and above LCST), i.e. the thermo-responsive behavior was

eliminated. The protein-resistant properties may be due to

water molecules trapped in the interstices of the nanowire

arrays, forming a hydration layer that blocks protein

contact. We also grafted a typical pH-responsive polymer,

poly(methacrylic acid) (PMAA), onto SiNWAs to investigate

the influence of pH on protein adsorption. Surprisingly,

lysozyme adsorption at pH¼ 4.0 was nearly 700% higher

than at pH¼ 7.4, and the lysozyme adsorbed at lower pH

couldbedesorbed simplyby increasing thepH.This result is

of interest and may be the basis for new biomedical

applications of SiNWAs.[67]

Protein Adsorption on ‘‘Topographic’’ and‘‘Patterned’’ Surfaces

Protein adsorption depends not only on surface chemistry

but also on surface topography.[68–71] Polymeric biomater-

ials generally have multiple microscale or nanoscale

surface topographies or patterns. These surface features

are either unknowingly introduced duringmaterial proces-

Scheme 4. Surface topography based on the interaction of surfaceslabeled fibrinogen and (B) adhered platelets on a dot-like protrusioadhered L929 cells on a patterned PDMS-PEG surface. (E) Quantity oaround 7.29mm; error bar: SD, n¼ 3). See text for definition of PU,




sing or intentionally designed into the material for specific

biomedical applications such as scaffolds for tissue

engineering. It has been observed that different topogra-

phies or patterns can influence the distribution and/or

amount of protein adsorption, and can affect subsequent

biological reactions such as cell adhesion and proliferation.

In general it is difficult to separate the effects of surface

topographyand surface chemistry. Recently,weproposed a

simplemethod to investigate the exclusive effect of surface

microtopography on protein adsorption and subsequent

biological responses using PDMS with specific topographi-

cal patterns but homogeneous chemical composition.[72] It

was found that fibrinogen adsorbed randomly on a flat

surface whereas it was specifically distributed on a

patterned surface (Scheme 4A). Furthermore, data on

platelet adhesion from platelet rich plasma and flowing

whole blood showed that most of the adherent platelets

were spatially ‘‘in register’’ with the adsorbed fibrinogen

(Scheme 4B). These results indicate that microtopography

can affect the distribution of adsorbed protein and

influence the location of platelets, suggesting that it is

possible to distribute protein adsorption and cell adhesion

to appropriate regions by introducing particular patterns

onto biomaterial surfaces.

As well as introducing specific topographies onto

chemically homogeneous surfaces, fabricating chemically

heterogeneous patterns is also an effective method to

regulate protein adsorption and subsequent cellular

responses. We prepared a chemically heterogeneous

patterned PDMS-PEG surface by ultraviolet (UV) lithogra-

phy.[73] Due to the different chemical properties of the UV

exposed domains and non-exposed domains, proteins and

cells were found to attach selectively to specific regions

(Scheme 4C, D). This indicates that chemical differences

with biomolecules. (A) Adsorbed fluorescein isothiocyanate (FITC)-n patterned surface. (C) Adsorbed FITC-labeled fibrinogen, and (D)f adsorbed fibrinogen on sample surfaces (diameter of one disk isPUL etc.

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induced by surface treatment can also introduce topogra-

phical features, leading to specific cell patterns. It is

important to note that this approach provides a simple

and convenient method for cell patterning without pre-

adsorption of cell adhesive molecules prior to cell culture;

this approach should be attractive for applications in

biosensors and cell based devices.

Surface nano-topography influences not only the dis-

tribution but also the amount of protein adsorption.

Natural material surfaces have multiple and special

topographic structures.[65] Surface wettability and surface

energy are significantly affected by these structures,[74]

which will certainly further influence the behavior of

biological molecules contacting the material surface. For

example, we used natural lotus leaf as a template to

replicate its uniquemicrostructure on a PU surface (Scheme

4E).[75] It was found that compared with a flat PU surface, a

PU surface with lotus leaf-like topography (PUL) adsorbed

proteins extensively, possibly due to the increase in surface

area. Furthermore, these surfaceswere chemicallymodified

with Pluronics (PEO-PPO-PEO block copolymers) (surfaces

referred to as PU/P and PUL/P). The modified surfaces

became more hydrophilic and the PUL/P surface showed

water contact angles less than 108, classifying it as

superhydrophilic. Moreover, fibrinogen adsorption on the

PU/P surface was reduced by �90% compared with the PU

surface. When lotus leaf-like topography was constructed

on the PU/P surface, adsorption was reduced further (97%

reduction) although the surface area was increased. Since

proteins did not readily adsorb on the PUL/P surface, cell

(L929) attachment also decreased significantly. The

attached cells did not shownormalmorphology: theywere

spherical and without pseudopodia extensions. We also

investigated protein bovine serum albumin (BSA) adsorp-

tion from the cell culture medium and found adsorption

behavior similar to that of fibrinogen from buffer,

indicating that protein adsorption did indeed influence

cell adhesion behavior.

It has been suggested that blood compatibility could be

improved by fabrication of biointerfacial topography at

micro/nano scales.[76–78] For example, to mimic the inner

surface of blood vessels, Fan et al. developed a multiscale

structuredPDMSsurfacewith interlacedsub-mmridgesand

nano-protuberances. Platelet adhesion assays showed that

the resulting surface exhibited good platelet resistance

underflow.[79]Milner et al. built nanoscale square pillars on

a polyether(urethane urea) (PUU) surface and found that at

low shear stress platelet adhesion on this surface was

significantly suppressed.[80] It has also been suggested that

optimization of chemical properties together with topo-

graphycouldprovidesurfaceswithnovel functionality. Sun

et al. fabricated a nanostructured biomaterial surface with

low surface free energy by coating carbon nanotube arrays

(CNTA) with fluorinated poly(carbonate urethane) (FPCU).



The resulting surface exhibited superhydrophobicity and

greatly reduced platelet adhesion and activation.[81]

These results illustrate that surfaces with significant

resistance to nonspecific protein adsorption and cell

adhesion can be achieved by combining the effects of both

surface chemical composition and topography, and that, in

general, this may be an effective approach to the design of

surfaces of improved biocompatibility.

Future Perspectives

Although considerable progress has been made in control-

ling protein adsorption on biomaterial surfaces, many

challenges remain, including thepreparationof surfaces for

specific protein binding, maintenance of protein structure

andactivity in theadsorbed state, andexploitationof smart

material approaches. Future research in the following areas

is required.

Novelmodificationmethods for various surfaces are still

required to inhibit non-specific protein adsorption and

enable ligand conjugation to be made conveniently and

effectively. The resulting surfaces should promote specific

protein adsorption and functional activity. Such materials

should also allow the initiation of specific physiological

processes and should find applications inmicrofluidics and

other biological detection systems.

Smart surfaces that can regulate specific protein

adsorption or control adsorbed protein conformation are

alsoneeded. Thesesurfacesmayserveas switches tocontrol

the quantity of adsorbed protein and catalytic activity in

the case of enzymes.

In addition to chemical properties, surface topography

plays an important role in protein adsorption, and

nanoscale roughness significantly affects surface proper-

ties. Surfaces that combine topographic and chemical

properties should be further developed for the control of

protein interfacial behavior.

Acknowledgements: This work was supported by the NationalNatural Science Foundation of China (20634030, 20974086, and20920102035). We thank Prof. John Brash for helpful discussions.

Received: November 25, 2010; Revised: January 6, 2011;Published online: February 17, 2011; DOI: 10.1002/mabi.201000464

Keywords: biocompatibility; interactions; interfaces; proteins;surface modification

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surface modification to control protein/surface interactions

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