DOI: 10.1002/asia.201100112

Physical Chemistry of Biological Interfaces: Generic and Specific Roles ofSoft Interlayers

Motomu Tanaka,*[a, b] Emanuel Schneck,[a] Hiroshi Y. Yoshikawa,[a] andFernanda F. Rossetti[a]

On the occasion of the 150th anniversary of the Department of Chemistry, The University of Tokyo

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Abstract: Nature defines the spatial boundaries betweendifferent phases using membranes, and the interfacial in-teractions are mediated by soft biopolymer interlayersthat contain various carbohydrates. This Review providesa comprehensive overview on the interplay of generic andspecific interactions at cell–cell and cell–tissue interfaces.

A focus will be put on the combination of defined modelsystems, experimental techniques in real- and reciprocalspace, and theoretical simulations.

Keywords: biopolymers · cells · interfaces · membranes ·X-ray/neutron diffraction

1. Introduction

Biological membranes, the major constituents of which arebilayer lipid membranes, define the outer boundary of cellsand internal cell organelles. Amphiphilic lipid molecules as-semble into bilayer membranes in water to minimize thecontact of hydrophobic chains to the surrounding water.Specific functions can be provided by the binding or incor-poration of various peripheral and integral proteins and car-bohydrates into 5 nm thick fluid matrices. In nature, mem-branes act as selective filters for nutrients, wastes, and spe-cific metabolites, as well as fundamental platforms for manyimportant biological processes, such as hormone transduc-tion and control of cell adhesion.

To date, phospholipid bilayers deposited onto macroscopi-cally large solid substrates, in the order of cm2 (called solid-supported membranes), have been the simplest and mostcommonly used experimental models of cell surfaces.[1–3]

Compared to other membrane models, such as suspensionsof spherical lipid vesicles and free-standing bilayer lipidmembranes, supported membranes retain not only the in-trinsic “fluid” nature to self-heal local defects but also theexcellent mechanical stability. This offers a unique advant-age to use various surface-sensitive techniques to probe thestructural and dynamic properties of membranes, includingattenuated total reflection Fourier-transform infrared spec-

troscopy (ATR-FTIR),[4] surface plasmon resonance,[5]

quartz crystal microbalance,[6,7] and neutron reflectivity.[8]

However, in spite of the great scientific achievements invarious key questions in biology (e.g., recognition of anti-gen-presenting cells by T cell lymphocytes and formation ofimmunological synapses), solid-supported membranes havethe drawback of being confined in close proximity of solidsubstrates. Here, the separation by a very thin water reser-voir (thickness: 5–20 �)[8,9] is not sufficient to prevent pro-teins from coming into direct contact with the bare sub-strate. For example, in our previous accounts, we reportedthat the direct spreading of proteoliposomes doped withhuman platelet integrin aIIbb3

[10,11] and F0F1-ATP synthase[12]

on solid substrates results in the pinning and denaturationof transmembrane proteins.

This can be overcome by separating membranes and solidsubstrates using “soft” interlayers based on hydrated poly-mers, which mimic the generic roles of native interlayers(Figure 1).[13,14] In nature, interactions between cells and tis-

sues are mediated by a complex interplay of short-range andlong-range forces across hydrated layers of carbohydrate-based biopolymers, such as extracellular matrix (ECM)[15]

and cell surface glycocalix.[16] They keep a finite distance(typically in the range of 10–100 nm) between neighboringcells to avoid direct, non-specific cell–cell contacts as well asto create hydrodynamic pathways for solute transport.

[a] Prof. Dr. M. Tanaka, Dr. E. Schneck,+ Dr. H. Y. Yoshikawa,#

Dr. F. F. RossettiPhysical Chemistry of BiosystemsInstitute of Physical ChemistryUniversity of Heidelberg69120 Heidelberg (Germany)Fax: (+49) 0622 1544918E-mail : tanaka@uni-heidelberg.de

[b] Prof. Dr. M. TanakaCell Biophysics LaboratoryInstitute of Toxicology and GeneticsKarlsruhe Institute of Technology76021 Karlsruhe (Germany)

[+] Present Address:Bio Soft Matter TheoryDepartment of PhysicsTU M�nchen85748 Garching (Germany)

[#] Present Address:Department of ChemistryFaculty of ScienceSaitama University338-8579 Saitama (Japan)

Figure 1. Models of cell–extracellular matrix contacts a) by the depositionof a two-dimensional cell membrane on a polymer support (polymer-sup-ported membrane; b).

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2. Thermodynamics of Biological Interfaces

From the general viewpoint of physical chemistry, the inter-actions of neighboring cell membranes, as well as the inter-actions of lipid membranes and solid substrates, can be de-scribed as two planes that keep a stable, distinct separationdistance by use of a thin interlayer. In case the middle ofthe interlayer under equilibrium retains its intrinsic bulkproperties (Figure 2 a), individual interfaces can be ex-

plained within the framework of the classical Gibbs capillarytheory. Here, a decrease in the interlayer thickness at a con-stant phase volume and contact area do not cost any changein the free energy of the system, and thus the pressure inthe interlayer is equal to zero. On the other hand, if thefields of the long-range forces in thin interlayers overlap(Figure 2 b), any change in the interlayer thickness willresult in positive or negative work.[17] This work originatesfrom the interplay of attractive and repulsive forces in theinterfacial region, which can have a range of more than sev-eral tens of nanometers.

To describe thermodynamics of thin liquid films analyti-cally, Derjaguin introduced the concept of a disjoining pres-sure, which is the sum of the net effect of the various inter-facial forces.[17,18] This approach is very helpful to simplifythe extremely complex calculation of each force contribu-tion, which is often found to be very difficult experimentally.The disjoining pressure P can be measured by applying ex-ternal pressures to keep the interlayer at mechanical equi-librium. Namely, it can be given as the difference betweenthe pressure in the interlayer P1 and the pressure in the bulklayer P0, as shown in Equation (1):

P ¼ P1 � P0 ð1Þ

The disjoining pressure can also be related to other ther-modynamic parameters. For example, a change in the inter-layer thickness d by dd at constant temperature T and chem-ical potential m, the work done by the external force�PðdÞ@d is equal to the increment of the Gibbs free energy

Abstract in Japanese:

Figure 2. a) Two parallel Gibbs layers, where the intrinsic properties ofthe bulk phase retain. Under equilibrium, there is no work required tochange the interlayer distance d. b) Two parallel planes, where the long-range force fields overlap within the interlayer. Here, either a positive ornegative work is required to change the interlayer distance, that is, thepressure within the interlayer differs from that in the bulk phase (disjoin-ing pressure).

Motomu Tanaka received his PhD fromthe Kyoto University in 1998, and joinedthe group of Prof. Erich Sackmann at theDepartment of Physics, Technical Univer-sity Munich (JSPS and Humboldt fellow-ships). From 2001 to 2005, he led an inde-pendent group funded by an EmmyNother fellowship, and earned a Habilita-tion degree in experimental physics at theTechnical University Munich. In 2005, hemoved to University of Heidelberg as aFull Professor in chemistry and physics.Since 2010, he has a joint appointment atthe Karlsruhe Institute of Technology. His

scientific interests include physics of biological matters, quantitative model-ling of development and disease, and design of bio-semiconductor hybridmaterials.

Fernanda Rossetti studied materials sci-ence at the ETH Zurich from 1995 to2001, where she also received her PhD in2005 under the guidance of Prof. MarcusTextor. In 2006, she joined the groupProf. Motomu Tanaka as a postdoctoralresearcher (DFG fellow). Since 2008, shehas been leading a junior group in thesame laboratory, supported by Heidel-berger Academy of Science.

Emanuel Schneck obtained his PhD inphysics at the University of Heidelbergunder the guidance of Prof. MotomuTanaka. During his PhD study, he was atInstitute Laue Langevin (France) andKyoto University (Japan) as a visiting stu-dent. Since 2010, he is a postdoctoral re-searcher in the group of Prof. RolandNetz at Technical University of Munich.His research interests include structureand mechanics of biomembranes, and theinteraction between biological surfaces.

Dr. Hiroshi Yoshikawa received his PhDdegree in applied physics from the OsakaUniversity in 2006 (Japan). After a shortpostdoctoral research in Osaka, he joinedthe laboratory of Prof. Tanaka at the Uni-versity of Heidelberg as a postdoctoral re-searcher (Humboldt fellow). In 2011, hemoved to Saitama University (Depart-ment of Chemistry) as an Assistant Pro-fessor. His research interests includequantitative regulation and characteriza-tion of biopolymers and cells.

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Physical Chemistry of Biological Interfaces

dG, given in Equation (2):

PðdÞ ¼ � @G=@dð ÞT;m ð2Þ

The minimum of the freeenergy that determines theequilibrium state can be foundwhen @2G

�@d2 > 0. Thus, to

keep a distinct equilibrium dis-

tance d between two planes (inbiology, plasma membrane contact mediated by a biopoly-mer interlayer), the disjoining pressure should fulfill thecondition shown in Equation (3):

@PðdÞ=@d < 0 ð3Þ

In case of cell adhesion, the trapping of membranes atthis local minimum can be referred to as “weak adhesion”,where the interaction potential may be approximated by aharmonic potential.[19,20]

On the other hand, the interlayer becomes unstable whenEquation (4) is satisfied:

@PðdÞ=@d > 0 ð4Þ

Continuous thinning of the wetting layer below the freeenergy minimum leads to a negative disjoining pressure,thus resulting in dewetting of the film. Typical examples arethe rupturing of polymer and surfactant films.[21–23] In biolog-ical systems, this can be found as the formation of tightmembrane contacts (adhesion plaque).[24,25]

3. Soft Interlayers Enhance Lubrication andDiffusion

From a thermodynamics viewpoint, the deposition of a lipidbilayer onto a hydrated polymer support should result in again of the Gibb�s free energy of the surface. Within thebasic framework of the physics of wetting, the prerequisitefor the formation of stable, uniform membranes on the sur-face is to achieve a positive spreading coefficient S, that is,the sum of the free energies from individual interfaces mustbe smaller than that of the solid/liquid interface, as shown inEquation (5).[26]

S ¼ gSL � gSP þ gPM þ gMLð Þ � 0 ð5Þ

Here, gSL is the free energy of the solid/liquid interface,gSP of the solid/polymer interface, gPM polymer/membraneinterface, and gML membrane/liquid interface. The polymersupports act as a lubricating layer between the membraneand the substrate,[27–29] and assist self-healing of local defectsin the membrane to cover macroscopically large substrates(~cm2) uniformly (Figure 3).

In 1975, Saffman and Delbr�ck described Brownianmotion of cylindrical particles in two-dimensional continu-

um.[30] Motion of particles (proteins) is driven by randomfluctuation forces that originate from unbiased collisionswith solvent (lipid) molecules. In their continuum treatment,the lateral diffusion coefficient is written as Equation (6):

D ¼ kBT4phm

lnhmh

hwRp� g

� �ð6Þ

hw and hm are the viscosities of water and membranegiven in [Pa s], and g= 0.5772 is an Euler�s constant. Such alogarithmic law explains well the relatively independentnature of D on the particle radius Rp reported for rhodopsin(D=3.0 mm2 s�1, Rp = 2.0 nm)[31] and acetylcholine receptortetramer (D =3.3 mm2 s�1, Rp = 6.0 nm)[32] in lipid vesicles.

Later, Hughes and White[33] introduced a dimensionlessparticle radius e as a measure to characterize the frictionalcoupling under asymmetric boundary conditions, as shownin Equation (7):

e ¼ Rp

hhw1 þ hw2

hm: ð7Þ

Here, hw1 and hw2 represent the viscosities of water onboth sides of the membrane. In contrast to the symmetricboundary condition assumed (hw1 =hw2, e�0.1) in Saffmanand Delbr�ck�s theory, membrane proteins are in contactwith viscous, asymmetric environments (e.g., glycocalyx andcytoskeleton). For example, in the case of solid-supportedmembranes, one has to consider a thin water reservoir (d=

5–20 �) between supported membranes and solid substrates.Evans and Sackmann modified this theory (see Figure 4) bytaking the interfacial drag s proportional to the velocity of adiffusant v into consideration: s=bsv.[34, 35] Note that bs isthe intrinsic frictional coefficient between the membraneand the substrate, which is dependent upon the viscosity hw2

and thickness d of the water reservoir between the mem-brane and substrate: bs = hw2/d. Within this framework, thediffusion coefficient D can be expressed as a function of thedimensionless particle radius of the diffusant e’ as in Equa-tion (8):

D ¼ kT4phm

14

e02þ e0K1 e0ð ÞK0 e0ð Þ

� ��1

ð8Þ

K0 and K1 are modified zero- and first-order Bessel func-tions of the second kind. Accordingly, the dimensionless par-ticle radius e� is written as a function of bs, e0 ¼ Rp

ffiffiffiffiffiffiffiffiffiffiffiffibs=hm

p. e

Figure 3. Microinterferometry images of a membrane on a 10 nm thick cellulose support, acting as a lubricat-ing interlayer. A macroscopic defect (shadowed) is healed by the spreading of the membrane within 120 s.

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can analytically be obtained from the dimensionless particlemobility m= 4pmm/f=4pmmD/kBT. Since the diffusion coeffi-cient D can be determined by fluorescence recovery afterphotobleaching (FRAP) or single molecule tracking, thefrictional coefficient bs can be calculated in a quantitativemanner if Rp is known. This enables one to quantitativelyadjust the frictional forces exerted to lipids and transmem-brane receptor proteins (such as integrin) by the thicknessand density of polymer interlayers.[36]

4. Modulation of Forces Perpendicular toInterfaces

To achieve a finite distance (of typically 10–100 nm) be-tween neighboring cells and between cells and tissue surfa-ces, biopolymer interlayers must keep the membrane awayfrom the deep potential minimum governed by van derWaals attraction (Note: in the case of direct membrane–sub-strate contacts, the predicted substrate–membrane distancelies around several �). Here, the interplay of long-rangeforce interactions should create another minimum in the in-terfacial interaction potential at which the disjoining pres-sure becomes zero.

The long-range forces operating in thin interlayers can bespecified into three major force contributors: a) van derWaals force, b) hydration repulsion, c) electrostatic force,and d) undulation repulsion originating from the thermody-namic fluctuation of the membrane. In the following, mem-branes consisting of zwitterionic lipids (e.g., phosphatidyl-choline) on uncharged polysaccharide supports (e.g., cellu-lose) are taken as a simple model case, as the complex con-tribution of electrostatic interaction in such a system wouldbe negligibly small.

First, the van der Waals force is calculated on the basis ofan asymmetric five layer model as a function of the polymerlayer thickness d.[37] If one takes a silicon wafer as a sub-strate, layer one and two are the bulk crystalline silicon andsilicon dioxide (thickness T1), respectively. Layer three con-sists of the polymer spacer, layer four is the lipid membranewith thickness T2, and layer five is bulk water. With thismodel, PvdW(d) can be written as Equation (9):

PvdW dð Þ ¼ 16p

A234

d3 �ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiA121A343

p

dþ T1ð Þ3�

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiA545A323

p

dþ T2ð Þ3�

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiA545A121

p

dþ T1 þ T3ð Þ3� �

ð9Þ

Axyz denotes the Hamaker constant of medium x interact-ing with medium z through medium y.

Second, hydration force was found to stabilize colloidaldispersions,[18,38] polar surfaces,[39] and soap films.[40] Thisforce is a consequence of the work necessary for removingwater from a hydrated layer to the infinitely thick, bulkliquid phase and thus Phyd(d) can be represented by an expo-nential decay function parameterized by a pressure constantP0 and a characteristic decay constant l, as shown in Equa-tion (10):[41]

Phyd dð Þ ¼ P0 exp � l

d

� �ð10Þ

The values for P0 and l are extracted from the force–dis-tance relationships, which were obtained by measuring theequilibrium thicknesses of the polymer layer at different os-motic pressures.

The repulsive force originating from thermodynamic un-dulations was first described by Helfrich (Figure 5 b).[42] Incase of a single supported membrane, the fluctuation pres-sure to the adjacent to the wall Pund(d) is given by Equa-tion (11):

Pund dð Þ ¼ a1kBTð Þ2kd3

ð11Þ

where k is the bending rigidity of the membrane. The pre-factor, a1 = (p2/128), has been found by analytical derivationand was confirmed by Monte Carlo simulations.[43,44]

The net force acting as a function of membrane–substratedistance showed excellent agreement with the correspondingvalue determined by elliposometry as well as specular X-rayand neutron reflectivity (Figure 5 a), thus demonstrating the

Figure 4. a) The model of lateral diffusion of lipids/proteins in a polymer-supported membrane. The frictional coupling between a cylindrical parti-cle and the substrate is reduced by the presence of a soft polymer sup-port. b) The dimensionless mobility plotted as a function of the dimen-sionless particle mobility for the continuum model (e<0.1), the stronglycoupled model (e>0.1), and the modified theory derived by Evans andSackmann. The experimental results from integrin receptors in polymer-tethered membranes at low and high tether densities are well explainedwith the modified theory.

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generic roles of polymer interlayers in modulating the mem-brane–substrate contact.[45]

5. “Native” Supported Membranes: Two-Dimensional Cell Membranes

One of the major challenges for polymer-supported mem-branes is to spread cells or vesicles extracted from cells andorganelles, instead of spreading vesicles with artificially re-constituted proteins. Compared to the spreading of proteoli-posomes, this opens a possibility to transfer onto planar sur-faces complex mixtures of membrane-associated proteinsthat nature uses. For this purpose, polymer supports are ad-vantageous over bare solid substrates, owing to their abilityto finely tune the cell–surface contacts.

Adult animal cells do not adhere onto bare glass/quartzslides, as they are negatively charged by sialic acid resi-dues.[46] In 2001, we have reported for the first time the dep-osition of human erythrocyte “ghosts” (red blood cells afterremoval of their cytoplasm) on regenerated cellulose sup-ports.[47] Incubation of ghost cells for 60 minutes resulted inpolymer-supported native membranes. The membranes arefree of local defects and expose their cytoplasmic domain,which was confirmed by selective immunofluorescence la-

beling (Figure 6 a). Moreover, we demonstrated that cellu-lose supports can also be used for spreading other nativemembrane extracts, such as sarcoplasmic reticulum mem-branes extracted from muscle cells and disrupted plasmamembrane extracts from human carcinoma cells.[48–50] Nativesupported membranes can offer unique advantages overintact cells for the structural characterizations using physicaltechniques such as scanning probe microscopy.[51] More re-cently, the vertical structures of human erythrocyte mem-branes spread on cellulose supports can be resolved by highenergy specular X-ray reflectivity (Figure 6 b).

6. Influence of Membrane-Bound Carbohydrateson Mechanics: Supported Multilayers

X-ray and neutron scattering techniques have been widelyused to investigate the physical characteristics of biologicalmembranes. In contrast to commonly used powder diffrac-tion experiments on lipid suspensions, the planar geometryof supported membranes allows for the identification of in-plane and out-of-plane momentum transfers.[52, 53] Interac-tions between membranes normal to the sample plane canbe detected from specular scattering, while the mechanicalproperties of membranes can be extracted from off-specularsignals. Thus, in contrast to single membrane supported onpolymer supports, supported multilayers of glycolipids canused as a model of inter-membrane interactions mediatedby membrane-bound glycocalyx (Figure 7).

Specular and off-specular neutron scattering is a powerfulexperimental method to investigate the influence of molecu-lar structures on intra- and inter-membrane interactions(Figure 8). Experiments at controlled humidity enables oneto examine the influence of the disjoining pressure on thesaccharide-mediated inter-membrane interactions,[54] whileexperiments in bulk buffers (i.e., in the absence of an exter-nal osmotic stress) reveal the effect of mono- and divalentions on membrane mechanics.[55]

In a kinematic approximation, the scattering from periodi-cal membrane stacks which possess correlated roughness

Figure 5. a) High energy specular X-ray reflectivity of a cellulose supportbefore and after the deposition of a phospholipid membrane. b) Thequantitative force–distance curves including van der Waals, hydration re-pulsion (hydration), and thermal fluctuation. The total force (disjoiningpressure) predicts that the equilibrium membrane-substrate distance (i.e.,point of zero force), which shows excellent agreement with the distancedetermined by X-ray and neutron reflectivity.

Figure 6. A native supported membrane spread on a polymer support.a) Immunofluorescence staining of a human erythrocyte membrane withthe anti-spectrin antibody denotes the exposure of the cytoplasmicdomain to the bulk. b) The scattering length density (SLD) profile recon-structed from the high energy specular X-ray scattering confirms the dep-osition of a single cell membrane that can be well represented by a slabmodel.

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can be expressed as a function of the displacement corre-lation function gk(r), shown in Equation (12):[56,57]

S qz; qjj� �

/ 1q2

z

NZ1

�1

e�q2zg0 rð Þ=2e�iqjjrdr þ 2

XN

k¼1

ðN � kÞ cos kqzdð ÞZ1

�1

e�q2zgk rð Þ=2e�iqjjrdr

2

4

3

5,

where gk rð Þ ¼ d2

p2 h

Z1

2p=R

1� Jo qjjr� �

exp �lkq2jjd

� h i

qjjffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1þ l2d2

4 q4jj

q dqjj

ð12Þ

Within the framework of the discrete smectic Hamiltoni-an,[58] the vertical inter-membrane interaction potential ischaracterized by the compression modulus B, while thebending elasticity of the membranes is characterized by themembrane bending modulus k. It should be noted that twokey parameters in the displacement correlation function,de Gennes parameter l and Caill� parameter h, are directlycorrelated to B and k, as shown in Equation (13):

h ¼ pkBT

2d2ffiffiffiffiffiffiffiffiffiffiffiffikB=d

p and l ¼ffiffiffiffiffiffiffik

Bd

r

ð13Þ

Therefore, the simulation of thescattering signal enables one to deter-mine the mechanical properties of themembranes. It has been demonstratedthat the synthetic or genetic modifica-tion of molecular structures signifi-cantly influences the mechanicalproperties of synthetic glycolipids[54]

and those of lipopolysaccharides (LPSs) purified from vari-ous bacteria mutants.[55]

Figure 7. Stacks of membranes coupled to carbohydrate head groups onplanar supports (supported multilayers) as the model of cell–cell contactsmediated by carbohydrates.

Figure 8. a) Planar geometry enables the discrimination of momentum transfers parallel and perpendicular to the membrane surface. b) A typical scatter-ing signal plotted in the reciprocal space coordinate. The second Bragg sheet (highlighted) was used for the analysis to ensure the kinematic approxima-tion. Both c) the integrated intensity along qz and d) the sheet width plotted along q j j can be well fitted within the framework of the smectic elasticitytheorem to calculate the mechanical properties of membranes.

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Physical Chemistry of Biological Interfaces

7. Interactions of Cells with EnvironmentsMediated by Carbohydrates

Carbohydrates expressed on cell surfaces interact not onlywith the neighboring cells and contact tissues, but also withtheir surrounding environments. For example, LPSs are amajor component of the outer leaflet of the outer mem-brane of Gram-negative bacteria, which stabilize the struc-tural integrity of bacteria and protect the membrane againstchemical attacks by intruders (e.g., cationic antibacterialpeptides, CAPs). To date, many in vivo studies demonstrat-ed that divalent cations (Ca2+ , Mg2+) significantly increasethe survival rate of bacteria,[59] but there have been no sys-tematic studies that account for the molecular mechanism.

Monolayers of lipopolysaccharide from various bacteriastrains deposited at the air/water interface can be used as arealistic model of bacterial outer membranes. The fine struc-tures perpendicular to the membrane surface can be studiedusing grazing incidence X-ray scattering out of specularplane (GIXOS; Figure 9 a).[60,61] In case the detector angle(azimuth angle) is very small (qxy ~0.03 ��1) and the inter-face roughness is conformal, the measured GIXOS signalscan be translated into the corresponding specular reflectivitycurves using Equation (14):

I qzð Þ / T kinð Þj j2 T koutð Þj j2 R qzð ÞRF qzð Þ

ð14Þ

I(qz) denotes the intensity measured in a GIXOS experi-ment, R(qz) is the corresponding specular reflectivity asmeasured in a q� 2q scan, RF(qz) denotes the specular re-flectivity from a flat (ideal) surface, and T(ki) and T ACHTUNGTRENNUNG(kout)represent the characteristic Vineyard Function for the graz-ing incidence configuration. GIXOS at the air/water inter-face offers a unique advantage over the specular reflectivitytechnique by significantly reducing the irradiation time (bya factor of 100), and thus minimizing the radiation damageto biological samples. Vertical electron density profiles nearthe surface can be reconstructed from GIXOS signals sug-gest the displacement of Na+ by Ca2+ as well as the collapseof saccharide chains in the presence of Ca2+ (Fig-ure 9 b).[62,63] Under this condition, the LPS monolayer re-mains intact even after injection of protamine near the mini-mum inhibitory concentration. Coarse-grained Monte Carlosimulations theoretically account for this finding in terms ofthe formation of an electrostatic energy barrier that pre-vents the approach of protamine to the hydrophobic region(Figure 9 c). This finding was in contrast to the intrusion ofprotamine in the absence of Ca2+ , which results in the com-plete destruction of the layered structure of LPS Ra mono-

layers.Despite of excellent agree-

ment between electron densityprofiles calculated from experi-ments (Figure 9 b) and simula-tions (Figure 9 c, inset), thequantitative determination ofion density profiles in � accura-cy is still a challenge. One ex-perimental breakthrough is tomeasure grazing-incidence X-ray fluorescence by placing afluorescence detector over thefilm.[64] Here, the penetrationdepth of the beam depends onthe incidence angle ai, given asEquation 15:

L ffi 1qc

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffia2

c

a2c � a2

i

s

ð15Þ

where ac and qc are the criticalangle of total reflection and thecorresponding momentumtransfer, respectively. This ena-bles one to calculate the densityprofiles of ions, thus provingthe almost complete displace-ment of K+ by Ca2+ . Moreover,the integration of the experi-mentally reconstructed excess

Figure 9. a) Schematic illustration of GIXOS setup. b) The electron density profiles reconstructed fromGIXOS results, indicating the collapse of saccharide head groups towards the air/water interface (z =0).c) The number density of saccharides, Na+ ions, and Ca2+ ions simulated by coarse-grained Monte Carlo simu-lations for the subphase in the absence (broken lines) and presence (solid lines) of Ca2+ ions. The electrondensity profiles calculated from the number density profiles (inset) show good agreement with the experimen-tal results.

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ion density profiles yields an accurate measurement of theeffective charge density of LPS monolayers.[65]

8. Active Control of Biological Interfaces UsingExternal Stimuli

Towards the active control of interactions at biological inter-faces, one of the sophisticated approaches would be the useof polymers whose properties can be modulated by externalstimuli.[66–68] For example, Okano and co-workers demon-strated non-invasive detachment of cell sheets from sub-strates using thermo-responsive, low critical solvation tem-perature polymers, such as poly(N-isopropylacrylamide).[68]

However, changes within a wide temperature range over along time could significantly interfere with cell viabilities. Inthis context, pH responsive polymers offer an advantage, iftheir physical properties (degrees of ionization, conforma-tional changes, viscoelasticities) are compatible to biologicalcells. Unfortunately, commonly used strong polyelectrolytescannot be used for such a purpose, as they carry extremelyhigh charge densities over wide pH ranges. Indeed, highlycharged surfaces lead to undesirably strong electrostatic in-teractions with cells and proteins. In a previous study wefound that adult mammal cell membranes (derived fromhuman erythrocytes) were strongly pinned to the surface ofa commonly used polycation (poly-l-lysine) owing to itshigh charge density; this problem can be interpreted interms of dewetting of the cell membranes.[47]

One possible solution is the use of weak polyelectrolytesthat change the degrees of ionization (d.i.) near physiologi-cal conditions. The reversible switching of the d.i. by pHmodulation also alters the polymer�s conformation andhence the interfacial interactions with membranes and cells.Previously, we deposited a diblock copolymer monolayerbased on poly(2-(dimethylamino)ethyl methacrylate-block-methyl methacrylate) (PDMAEMA-PMMA), which com-prises an equal number of monomer repeat units (n=36)for the hydrophobic PMMA and pH-responsive PDMAE-MA blocks, from the air/water interface onto a hydrophobicsolid substrate (Figure 10 a).[69,70] Specular neutron reflectivi-ty experiments in the presence and absence of a biologicalmembrane model (supported lipid membrane) demonstratedthat the PDMAEMA brushes reversibly change their con-formation by pH modulation,[69] which results in a significantvariation in the membrane–substrate interaction (Figure 10 band c).[70]

Such a strategy can be extended to biological cells byusing a copolymer hydrogel that exhibits a substantialchange in its viscoelastic response with subtle changes in so-lution pH.[71,72] To minimize the interference with cell viabil-ity, a highly biocompatible pH-responsive ABA-type tri-block copolymer (A=poly(2-(diisopropylamino)ethyl meth-acrylate)], (PDPA); B=poly(2-(methacryloyloxy)ethylphosphorylcholine), (PMPC); Figure 11 a).[73] As shown inFigure 11 b, PDPA50-PMPC250-PDPA50 triblock copolymerforms a free-standing transparent gel comprising a 3D net-

work of inter-connected “flower” micelles. At higher gelpH, the PDPA chains become more deprotonated andhence more hydrophobic. This leads to stronger inter-chaininteractions, thereby resulting in a highly elastic, physicallycross-linked hydrogel film. On lowering the gel pH, the hy-

Figure 10. a) Active control of membrane-substrate interactions usingstimuli-responsive polymer supports. b) Specular neutron reflectivitycurves of a supported membrane on a charged and uncharged polymersupport. c) The corresponding scattering density profiles indicate a clearswitching of the membrane–substrate distance.

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drophobic interaction between the now partially chargedPDPA blocks (and thus the physical cross-linking) becomesmuch weaker, which results in a softer gel film. The filmelasticity could be reversibly modulated by a factor of 40through careful pH adjustment without adversely affectingcell viability. Myoblast cells exhibited pronounced stressfiber formation and flattening on increasing the hydrogelelasticity (Figure 11 c). Furthermore, an abrupt jump in thehydrogel elasticity was found to result in a remarkable re-modeling of the cytoskeleton and the cell morphology (Fig-ure 11 d), which can be utilized to monitor how cells adapttheir morphology to time-dependent changes in their me-chanical environment.

9. Conclusions

The unique combination of well-defined model systems andexperimental techniques in real- and reciprocal space offerspossibilities to investigate the physical chemistry of complexbiological interfaces. Quantitative understanding of the in-terplay between generic specific interactions would enableone to apply such systems in versatile directions, including1) the regulation of the fate of cells and cell ensembles[73,74]

and 2) design of novel sensor platforms by transferringmembranes and proteins onto solid-based devices.[75–78]

Acknowledgements

M.T. thanks all the post-docs and graduate students in his laboratory fortheir enormous contributions, especially, S. Kaufmann, R. G. Oliveira, T.Schubert, O. Purrucker, F. Rehfeldt, and P. C. Seitz. We deeply appreci-

ate our collaborating partners for crossing the borders of disciplines totackle common scientific problems. We are thankful to ESRF and ILLfor synchrotron and neutron beam times, and to O. Konovalov (ESRF),B. Dem� (ILL), G. Fragneto (ILL) for their helpful support. The workspresented were supported by DFG, BMBF, JSPS, and Humboldt founda-tion. M.T. is a member of the German Excellence Cluster “Cell Net-work”, BIOQUANT, and Helmholtz Program “BioInterface”.

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Received: February 5, 2011Published online: && &&, 2011

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Physical Chemistry of Biological Interfaces

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Biophysics

M. Tanaka,* E. Schneck,H. Y. Yoshikawa,F. F. Rossetti &&&&—&&&&

Physical Chemistry of Biological Inter-faces: Generic and Specific Roles ofSoft Interlayers

Interplay at interfaces : Nature definesthe spatial boundaries between differ-ent phases using membranes. A com-prehensive overview on the interplayof generic and specific interactions at

cell–cell and cell–tissue interfacesprobed by the combination of variousexperimental techniques and theoreti-cal modeling is presented.

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FOCUS REVIEWSM. Tanaka et al.