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Colloids and Surfaces B: Biointerfaces 62 (2008) 250–257

Influence of assembling pH on the stability of poly(l-glutamic acid) andpoly(l-lysine) multilayers against urea treatment

Jie Zhou a, Bo Wang a,b, Weijun Tong a,b, Elena Maltseva b, Gang Zhang b, Rumen Krastev b,Changyou Gao a,∗, Helmuth Mohwald b, Jiacong Shen a

a Key Laboratory of Macromolecular Synthesis and Functionalization, Ministry of Education, and Department of Polymer Science and Engineering,Zhejiang University, Hangzhou 310027, China

b Max-Planck-Institute of Colloids and Interfaces, 14424 Potsdam, Germany

Received 29 May 2007; received in revised form 30 September 2007; accepted 22 October 2007Available online 4 November 2007

bstract

Polyelectrolyte multilayers of poly(l-glutamic acid) (PGA) and poly(l-lysine) (PLL) were built up using the layer-by-layer (LbL) technique inow pH (3.6, PM3.6) and in neutral pH (7.4, PM7.4) solutions. The multilayers were then treated with a concentrated urea (one kind of denaturant forroteins and polypeptides) solution (8 M) and rinsed with corresponding buffer. The buildup and treatment processes were investigated by ultravioletisible spectroscopy and ellipsometry. The surface morphology was observed by scanning force microscopy (SFM). The inner structures wereetermined by X-ray reflectometry and circular dichroism spectroscopy (CD). An exponential growth of the optical mass and the layer thicknessas observed for both PM3.6 and PM7.4. After urea treatment, a significant mass loss for PM3.6 was found, while no mass change was recorded

or PM7.4. The dominant driving force for PM7.4 is electrostatic interaction, resulting in multilayers with an abundant �-sheet structure, which has

igher stability against urea treatment. By contrast, the dominant driving force for PM3.6 is hydrogen bonding and hydrophobic interaction, whichre sensitive to the urea treatment. The mechanism is substantiated by molecular mechanics calculation. This has offered a convenient pathway toediate the multilayer properties, which is of great importance for potential applications. 2007 Elsevier B.V. All rights reserved.

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eywords: Polypeptide; Multilayer; Stability; Hydrogen bonding; Electron inte

. Introduction

Layer-by-layer (LbL) assembly has been diversely used touild up multilayer thin films since it was introduced a decadego [1–8]. The properties of the assembled films such as layerhickness, film strength, rigidity, stability, permeability andermselectivity are all dependent on the assembly conditions,otably on ionic strength [9–11], pH [11–15], and temperature16] of the solutions. Besides the electrostatic interaction, hydro-en bonding is also a kind of frequently employed force to drivehe multilayer growth [17–20].

The traditionally employed building blocks for LbL areolyelectrolytes and nanoparticles, etc., which are usually non-iodegradable and less biocompatible. More recently, much

∗ Corresponding author. Tel.: +86 571 87951108; fax: +86 571 87951108.E-mail address: cygao@mail.hz.zj.cn (C. Gao).

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927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2007.10.017

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ore attention has been paid to the biocompatible componentsncluding proteins, DNAs, polysaccharides and polypeptides.mong which, multilayer films and microcapsules assembled

rom poly (l-glutamic acid) (PGA) and poly (l-lysine) (PLL)re known to have potential applications for biosensors, guidedellular adhesion [21,22], and drug delivery. Some researchersave found that the (PGA/PLL)i film exhibits an exponentialrowth regime [23,24] other than linear growth often observedor the traditional pairs. To explain this phenomenon, Lavalle etl. proposed that these polyelectrolytes may diffuse in and outf the film during each assembly circle, leading to formationf polyanion/polycation complexes in the superficial layer [23].t has been proved that the LbL buildup process is exponentialnly if the diffusion rate is fast enough to carry the polymers

ithin the entire thickness of the layer [25–27]. A fraction of

he diffused species cannot be released during the rinsing stepnd thus participate in the following film growth. Otherwise, itould follow linear or slight superlinear growth mode [28,29].

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J. Zhou et al. / Colloids and Surfac

althur et al. investigated the influence of drying, temperaturend pH cycling on properties of the (PLL/PGA)i multilayer filmssembled at pH 7.4, and found that the film would retain itsroperties after cycling [30]. Zhi and Haynie found that thePLL/PGA)i film assembled at neutral pH has a predominant-sheet character. Transformation of the secondary structuresf the polypeptides in the film from �-sheet to �-helix wouldesult in a more open and loose film morphology after treatedith low pH buffer solution [31].On the other hand, polypeptides, a category of weak polyelec-

rolytes, interact with each other not only through electrostaticnteraction but also through hydrogen bonding and hydropho-ic interaction [32]. Their chemical nature allows them toorm secondary structures such as �-helices and �-sheets32]. For example, previous results showed that there are sec-ndary structures in PLL solution [33,34], PLL–PGA complex28,29,35–38] and (PLL/PGA)i films [11,29,31,33,39]. Whenhe multilayer films are built up at different pH, the intra- andntermolecular interactions are changed correspondingly, result-ng in different inner structure of the film. The pH of assemblyetermines the charge density of polymer chains and structure ofolypeptides in the bulk [37]. While the former has major effectn the amount of polyelectrolytes adsorbed, the later plays aore important role for constructing the inner structure of thelm. When the polypeptides in the bulk are structured already,

he molecular conformation in the multilayers can be in a kinet-cally trapped state, since the surface effect imposes a highctivation barrier to the conformational relaxation [40]. On thether hand, unstructured polypeptides still remains the ability toeorient in the adsorption step to minimize the free energy of theystem. Furthermore, the molecular conformation of polypep-ides in the multilayers also depends on the molecular weightnd bulk concentration, which are expected from a kinetic sce-ario [41]. Basically, the secondary structure in the films prefers-sheet at neutral pH, while � helix can be deposited from theulk as well [17].

Up to present, most of the assembly was implemented ateutral pH. Assembly of PLL/PGA at low pH, more contri-ution from secondary interactions, i.e. hydrogen bonding andydrophobic interaction can be expected, since the polyacidecomes electrostatic neutral near its pKa [31,39]. Therefore,n this work, we shall report the fabrication of the PLL/PGA

ultilayers at a pH smaller than the pKa of PGA (pKa 4.9) [42],nd the variation of the multilayer stability against treatmentf concentrated urea. As a comparison, the PLL/PGA multilay-rs are also assembled at neutral pH. The results show that theLL/PGA multilayers assembled at low pH can be destroyed byrea solution of the same pH, while the PLL/PGA multilayersssembled at neutral pH are stable after treated by urea solutionf the same pH.

. Materials and methods

.1. Materials

Poly (l-lysine) (PLL, Mw 50–100 kDa), poly (l-glutamiccid) (PGA, Mw 30–70 kDa), polyethyleneimine (PEI, Mw

2

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Biointerfaces 62 (2008) 250–257 251

50 kDa), and buffer salts tris(hydroxymethyl)-aminomethaneTRIS) and 2-(N-morpholino)ethanesulfonic acid (MES) werell purchased from Sigma–Aldrich and used as received. Allolutions were prepared using ultrapure water (Milli-Q-plus sys-em, Millipore) with a resistivity of 18.2 M� cm. The buffer

edia at pH 3.6 was prepared by MES (25 mM), NaCl (0.1 M)nd adjusted by HCl solution. The buffer media at pH 7.4 wasrepared by TRIS (25 mM), MES (25 mM), and NaCl (0.1 M).

.2. Preparation of polypeptide multilayer films

A polypeptide solution with a concentration of 1 mg/mL wasrepared with pH 3.6 or pH 7.4 buffer. Quartz slides, siliconafers or quartz cuvettes (for CD measurement) were cleaned in0% H2SO4 (concentrated)/30% H2O2(aq) (“piranha”) and thenn hot H2O2/ammonia/water, 1:1:5 (vol/vol), rinsed in water, andlown dried with a stream of N2. After treated with 10 mg/mLEI to adsorb a PEI layer, the substrates were sequentially dipped

nto PGA and PLL solutions, each for 20 min, followed by threeashings with the buffer of the same pH, each for 3 min. Twoinds of polypeptide multilayers with the same layer number, i.e.GA/(PLL/PGA)6, were fabricated at pH 3.6 and pH 7.4 solu-

ions and were designated as PM3.6 and PM7.4, respectively.

.3. Monitoring the film growth

The building up process for the PGA/(PLL/PGA)6 multilay-rs at pH 3.6 (PM3.6) or pH 7.4 (PM7.4) was independentlyollowed by UV–vis spectroscopy (Cary 50 UV–vis spectropho-ometer) and ellipsometry. The absorbance at 221 nm, which isssigned to the polypeptides and is approximately independento the backbone conformation [43], was recorded after eachssembly. The amount of peptides absorbed can be directlyorrelated to the absorbance at this wavelength, thanks to itsnsensitivity to molecular conformation.

Ellipsometry is an optical method that measures the changesn phase difference (Δ) and amplitude difference (Ψ ) betweenhe parallel component and the perpendicular component uponeam reflection. The instrument used in this study is an opti-al null ellipsometer (Multiskop Ellipsometer, Optrel GmbH),quipped with a He–Ne laser. Measurements were performedt a wavelength of 632.8 nm and an incidence angle of 70◦.he raw data were fitted by a four-layer model, considering theontribution from silicon, silicon oxide, polymer (polypeptideultilayer), and air to obtain the refractive index and thickness

f the films. When the film is thick enough (over 3–5 nm), theefractive index can be determined from Δ to Ψ trajectories,hich are separate enough for films with different indices. With

hickness lower than that threshold, the index of the polymerayer was set as 1.5, which does not have distinguishable effectsn the thickness measurement. The samples were blown driedn a nitrogen stream for 3 min before measurement.

.4. Urea treatment and characterization

The PM3.6 or PM7.4 were treated with 8 M urea solutionith a pH value of 3.6 or 7.4 buffer for 1 h, then rinsed for

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mpeg(aaaaobtiaabiActwPiresults [24]. Ellipsometry measured simultaneously the refrac-tive index of the film, which can give additional information forthe film structure. Fig. 2b shows that for the first few layers therefractive index was considerably high, implying that the multi-

52 J. Zhou et al. / Colloids and Sur

min with the same buffer, respectively. After treatment, thebsorbance at 221 nm was recorded by UV–vis spectroscopynd the film thickness d and the reflex index n were determinedy ellipsometry.

The surface morphology of the multilayer films before andfter urea treatment was measured by scanning force microscopyDigital Instruments nanoscope IIIa Multimode SFM, Santa Bar-ara, CA) in air at room temperature with a tapping mode.

The changes in the film structure were followed by specular-ray reflectometry. The techniques provide information about

he electron density of thin films in direction z perpendicular tohe film surface over a length scale of 1–500 nm with a resolu-ion down to some tens of a nanometer [44,45]. Furthermore, itan provide the information on interfacial roughness in a rela-ively large area, which substantiates the observation of SFM.he information that can be extracted in a single reflectivityxperiment includes the film thickness h, the surface rough-ess σ, and the scattering density profile ρ(z) across the film.he latter can be converted into a composition profile and thusives information about the species distribution across the film.he experiments were performed at the triple axis diffractome-

er built at the Hahn-Meitner Institute (HMI), Berlin, Germany.he instrument has a horizontal scattering plane. The primaryeam geometry is defined by the line focus of a sealed X-ray tube0.04–8 mm2, Cu anode) and a diaphragm (0.1 mm × 8 mm) at aistance of 500 mm. The reflected beam is made monochromaticy a pyrolytic graphite crystal and the pulse height discriminatorf the scintillation detector. A Ni absorber was inserted in theeflected beam path for measurements at high intensities, e.g.rimary beam and region of total external reflection.

The reflectivity curves R(Q) (Q = 4π sin θ/λ, is the wave vec-or transfer, θ the angle of incidence and λ is the wavelength ofhe incoming radiation) were analyzed by applying the standardtting routine Parratt 32 [46]. The program calculates the opticaleflectivity of X-rays from flat surfaces based on Parratt’s recur-ion scheme for stratified media [47]. The film was modeled asonsisting of layers of specific thickness and scattering lengthensity. The experimental data were always fitted with the sim-lest one box model which assumes that the film represents aingle layer with a homogeneous scattering density distributionnd a finite thickness. The thickness, the scattering density andhe roughness of each layer were obtained from the fit.

The inner structures of the multilayers were also charac-erized by CD spectra. CD spectra, a moderate-resolution butighly informative technique that can reveal structural informa-ion on chiral molecules, would appear to be a useful alternativeo study molecular structure. It has been used successfullynd extensively to determine the conformation of proteins andolypeptides in solution [11,48–51]. To measure the CD spectraJasco model J-715 Circular Dichroism spectrometer, Japan),he measurement cell was used as the substrate to deposit theolypeptide multilayers. The film area of the beam passinghrough is ∼80 mm2. The CD instrument was set at 100 mdeg

ensitivity, 1 nm bandwidth, 1 s response time, 1 nm data pitch,nd 100 nm min−1 scan rate. Twenty to thirty scans were accu-ulated and averaged for each measurement. The measured

llipticity values recorded in mdeg could not be normalized

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B: Biointerfaces 62 (2008) 250–257

or path length or polymer concentration. Baseline spectra wereollected by measuring the CD signal of the quartz substraterior to film assembly. Final spectra were obtained by sub-racting the baseline spectrum from the corresponding samplepectra.

. Results and discussion

.1. Buildup and morphology of the PGA/PLL multilayers

To compare the influence of adsorbing pH on the assembledultilayers, the PGA/PLL multilayers were assembled from

olypeptide solution with a pH value of 3.6 and 7.4 (multilay-rs PM3.6 and PM7.4), respectively. Firstly, the stepwise layerrowth was monitored independently by UV–vis spectroscopyFig. 1) and ellipsometry (Fig. 2). The UV–vis absorbancet a definite wavelength represents the “optical mass” of thedsorbed materials. Fig. 1 shows that for both multilayers thedsorbed mass was increased along with the layer number withn exponential growth regime which has been observed previ-usly [23,24]. No big difference on the absorbance was observedetween the PM3.6 and PM7.4. However, the absorbance ofhe PM3.6 shows a zigzag increase compared to a monotonousncrease of PM7.4 (Fig. 1), namely a big increase after PGAssembly but keeping constant or even slight decrease after PLLssembly. The same alteration tendency has also been observedy ellipsometry (Fig. 2a), where the multilayer thickness wasncreased also exponentially as a function of layer number.gain the PM3.6 shows a zigzag increase in thickness which

oincides with the style of mass change (Fig. 1). Although thehickness of PM3.6 is more or less larger than that of PM7.4hen the layer number is smaller, the final thickness for bothGA/(PLL/PGA)6 multilayers assembled at pH 3.6 and pH 7.4

s almost same. This observation is in agreement with Gergely’s

ig. 1. The change of UV–vis absorbance of (PLL/PGA)6 films buildup at dif-erent pH (3.6 and 7.4), and treated with a concentrated urea solution then rinsedy buffer, respectively.

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ig. 2. (a and b) Ellipsometry results of (PLL/PGA)6 films buildup at pH 3.6nd 7.4, and treated with urea and rinsed with buffer, respectively.

ayers are porous and have lots of defects. Along with the layerumber increase, the refractive index decreased gradually andeached to a constant value of 1.5 for both PM3.6 and PM7.4,hich is consistent with the literature results [21,29,52]. At this

tage, all the substrate is covered by the multilayers with fewefects. After deposition for three circles, the refractive indexor PM3.6 shows a zigzag change again, with larger value whenGA was the outmost layer.

At neutral pH, both kinds of the polypeptides are highlyharged, thus can form multilayers via electrostatic interaction.

hen the assembly (for PM3.6) is conducted at a pH lowerhan the pKa of PGA, because of the protonation effect, it willf course decrease the electrostatic interaction between PGAnd PLL. Indeed, at a still lower pH (1.5), no multilayers cane built up from the PGA and PLL pair [39]. At pH 3.6, apartrom the electrostatic force, hydrogen bonding and hydropho-ic interaction should also exist which shall be discussed in theext section. More PGA molecules (more structured at pH 3.6)ay be adsorbed in comparison with PLL owing to the sec-

ndary interactions. Meanwhile, due to the relatively weakernteractions, when a film ending with PGA is further broughtnto contact with a PLL solution, some of the loosely attachedGA molecules on the super-surface will interact with PLL in

he solution to form PLL/PGA complex, which will release fromhe surface of the multilayers [24]. This results in the overallonstant layer mass or thickness but building block and charge

eversal. The overall effect is the zigzag growth behavior. Simi-ar phenomenon has been observed at high pH, in which the lossf PLL molecules was observed instead of PGA [39]. Since the

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Biointerfaces 62 (2008) 250–257 253

LL molecules at pH 3.6 is highly charged, they can adhere moretrongly that are hardly removed by the next PGA adsorption.

There are some other papers [11,22–24,30,31] mentioned themount or thickness of the PLL/PGA multilayers, but theseesults are hard to compare directly because of the differentssembly conditions. The amount or thickness of the PLL/GA multilayers is affected not only by pH value but also byoncentration of the polypeptides, properties of the substrate,nvironment humidity [29], molecular weight, ionic strength11], and even different measurement method [22].

The intrinsic difference in molecular state at pH 3.6 and pH.4 also results in huge difference in the film morphology, ashown in Fig. 3a and c. A lot of large grain particles with a sizef 100–200 nm are found on the film surface fabricated at pH 3.6Fig. 3c), resulting in a high roughness (RMS) of 11.3 nm. Therain size and the RMS of the film are even larger than those oflm assembled at pH 7.4 followed by acid treatment [24,31,53].y contrast to the PM3.6, the surface of PM7.4 is smoother andompact with a RMS of 2.3 nm (Fig. 3a). This type of morphol-gy is rather common for multilayers assembled from chargedolymers, since the strong charge attraction or repulsion usuallyields flatter surface with a uniform structure [31,54]. When theolypeptide multilayers are assembled at low pH, as aforemen-ioned relatively large amount of PGA will loosely adsorbednto the fully ionized PLL layer. In the next PLL assembly partf the over adsorbed PGA may form complex with PLL, leadingo the formation of large grains. Similar phenomenon was alsobserved when the assembly was conducted at high pH [53] dueo the same reason.

.2. Variation of PGA/PLL multilayer structure in responseo urea treatment

Because of its structural similarity to a peptide group, the ureas able to form stronger hydrogen bonds with peptide groups thanater does. Therefore, it is able to “break” interpeptide hydro-en bonds which water cannot break. Furthermore, urea caneaken the hydrophobic interaction of nonpolar parts of a pro-

ein or polypeptide molecule, and thus stabilize the denaturedorms [55–57]. This effect is mild, and the proteins and peptidesan regain their native conformation after removal of the urea58,59]. By contrast, the electrostatic interaction is less sensitiveo urea treatment. Figs. 1–3 (the right sides) present the alterationf mass, layer thickness and surface morphology of PM3.6 andM7.4 after the multilayers were treated with a concentratedrea solution (8 M) for 1 h and rinsed with buffer for 1 min.o significant difference was found for PM7.4 in terms of theolypeptide amount (Fig. 1), layer thickness (Fig. 2a), refractivendex (Fig. 2b), and surface morphology (Fig. 3b, RMS 1.8 nm).owever, ∼23% mass of PM3.6 was lost after the urea treat-ent, and further loss of 57% after rinsing, altogether resulting

n 80% mass loss (Fig. 1). Ellipsometry measured ∼29% thick-ess reduction after urea treatment and further 53% reduction

ssembled at pH 4.4 were ruffled by some ions like chloride,hosphate, and calcium in the culture media [22]. Meanwhile,he refractive index was improved slightly with a value compa-

254 J. Zhou et al. / Colloids and Surfaces B: Biointerfaces 62 (2008) 250–257

F (c), then treated with urea and buffer at pH 7.4 (b) and pH 3.6 (d), respectively.

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able to three bilayers of PGA/PLL, implying the exposure ofhe substrate due to incomplete coverage. These results would

ean that the PGA/PLL multilayers assembled at pH 3.6, whichhould be largely driven by hydrogen bonding and hydrophobicnteraction can be erased mostly in a pH 3.6-concentrated ureaolution.

Parallel to the mass loss, the surface morphology of the PM3.6as also altered significantly by the urea treatment (Fig. 3d).he huge grains disappeared, and smaller and more homoge-ously distributed particles emerged, although the grain size istill larger than that of the PM7.4. This change leads to theecrease of RMS from 11.3 to 2.9 nm.

.3. Inner structures of the PLL/PGA multilayer film

To understand the mechanism of the responsivity to the ureareatment, X-ray reflectometry, CD spectrometer, and molecular

echanics [60,61] calculation were used to explore the innertructure of the PLL/PGA films.

Fig. 4 presents the X-ray reflectometry results of (PLL/PGA)6lms built at different pH. The thickness and the roughness at their/film interface of PM7.4 were measured as 18.9 and 1.0 nm,espectively, which are in good agreement with the ellipsometrynd SFM results. The Si/film interface roughness was 2.8 nm,nd the electron density was 1.00 × 10−5 A−1. By contrast, theM3.6 is too rough to get any meaningful data from the fit. These

esults indicate that the structure of PM7.4 is more compact thanM3.6.

After urea treatment of PM7.4, no significant changesere found in terms of thickness (20.4 nm), electron density

Fig. 4. X-ray reflectometry results of (PLL/PGA)6 films buildup at pH 7.4 (a)and pH 3.6 (b). Curves (1) were collected from the as-prepared samples, whilecurves (2) were collected after the urea treatment. The curves are shifted forclarity. The solid lines present the best fits to the experimental data.

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ig. 5. CD Spectra of (PLL/PGA)6 films buildup at pH 3.6 (1), then treated withrea solution at pH 3.6 (2), and of (PLL/PGA)6 films buildup at pH 7.4 (3), thenreated with urea solution at pH 7.4 (4) followed by pH 7.4 buffer rinse (5).

1.15 × 10−5 A−1), and air/film (0.9 nm) or Si/film roughness2.8 nm), demonstrating that the mass on the substrate wastill remained. The XRR curve obtained after urea treatmentf PM3.6 can be fitted only assuming deposition of very thinlm with low electron density. This finding demonstrates again

he removal of the polypeptide multilayers.The secondary structure of the polypeptide multilayers was

hecked by CD spectra. All the films were fabricated on quartzuvettes. The weak signals did not allow extracting quantita-ive information on the secondary structures. However, the peakssignment can be more valid for peptides than proteins, as theatter often have aromatic chromophores interfering the spectra.he present analysis is verified by widely reported data in litera-

ures as listed below. Thus, one can distinguish a strong positive�–�*)perpendicular band at ca. 191 nm, a negative (�–�*)paralleland at ca. 208 nm, and a negative n–�* band at ca. 222 nm inhe spectrum of the PM3.6 (curve 1 in Fig. 5), which indicate an-helix-like structure [11,49–51]. Contrast to PM3.6, for PM7.4

curve 3 in Fig. 5) the negative �–�* transition peak shifted toa. 216 nm and the positive n–�* band shifted to ca. 197 nm,uggesting a mainly �-sheet-like structure [50,51].

After urea treatment, the CD signal of PM3.6 was dramati-ally decreased (curve 2 in Fig. 5). Since only 23% mass lossas detected by UV–vis spectroscopy and ellipsometry, one

an conclude that the signal decrease should not be causedy the reduced mass, but by the change of the inner struc-ure, i.e. the �-helix. Contrast to this observation, no significanthange was found for PM7.4 after urea treatment (curve 4 inig. 5), even with buffer rinsing again (curve 5 in Fig. 5). Thisbservation proves that the �-sheet-like structure of the PM7.4as still remained with no sign of apparent mass loss, which

s consistent with the UV–vis spectroscopy and ellipsometryeasurements.The secondary structures of polypeptides in solution are

ighly affected by pH and related with pKa of the polypep-

ides. Due to electrostatic repulsion between the side groups, it isery difficult to form helical or extended structures in a chargedtate. For example, PLL (pKa = 9.4) [59] forms random coil-liketructure in a pH 3.6 or 7.4 solution due to the high charge repul-

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Biointerfaces 62 (2008) 250–257 255

ion between the ionized lysine side chains [33,34,43,62,63]. AtH above the pKa of PGA (4.9), the molecular chain is highlyharged as well, leading to a high degree of random coil-liketructure. �-helix structure appears in PGA solution at a low pHalue (e.g. 3.6) [39], because protonation of COO− groups willreatly reduce the charge repulsion.

Different with the PGA in a pH 7.4 solution, in the multilayersost of the random coil-like structure changes to �-sheet-like

tructure due to the charge compensation [31]. For instance,oulmedais et al. found that at neutral pH deposition of a PLL

ayer on the first PGA layer induced an important increase ofhe �-sheet content, implying that this change is a result ofhe PGA/PLL interaction [28–30,35–38]. They found that whenolyanions and polycations are mixed, electrostatic interactionsan also stabilize the structure of �-sheets [30,39]. Hence, inhe PM7.4, the strong electrostatic interaction between adjacentide chains can stabilize this structure [30,39]. This can explainhy the PM7.4 is stable in concentrated urea solution in termsf film thickness and secondary structure.

The results of molecular mechanics calculation (MM2),alculated by Chem3D, also confirm that ionization of the car-oxylic group of the PGA molecule destroys the helical structurehich exists in the protonated form, and thus increases the sys-

em energy. The end-to-end distance of a chain extends morehan 50% caused by the electrostatic repulsion and the helicaltructure is diminished. Presence of PLL molecules effectivelyowers down the system energy by charge compensation andorming �-sheet like structures. At pH 7.4, the charge com-ensation can stabilize the PLL/PGA pair by ∼200 kcal/molonomer, while the stabilization energy at pH 3.6 is only10 kcal/mol monomer. At pH 3.6, the intramolecular heli-

al structure of PGA chains is disturbed by pairing with PLL,hich costs extra energy. This difference in energy should be

esponsible for variation of the film stability.As aforementioned, in the formed PM3.6, PGA has an appar-

nt �-helix-like structure. The change of assembly pH valueauses the self-assembly switching from a regime in whichntermolecular adhesion is predominantly driven by electrostaticnteraction (pH 7.4) to a regime where hydrogen bonding andydrophobic interaction are dominant (pH 3.6). These obser-ations are consistent with the knowledge that formation ofelical structures is mainly stabilized by intraamide hydrogenonds [34]. These weaker interactions result in a less stableultilayer structure, which are then sensitive to the treatment

f urea, a reagent being able to destroy the �-helix structureas indicated by the CD spectrum) and hydrophobic interaction.onsequently, the PM3.6 was mostly removed after urea treat-ent and buffer rinsing as detected by UV–vis and ellipsometryeasurements.

. Conclusions

The PLL/PGA multilayers were assembled at pH 3.6 and

.4, which are below and above the pKa (4.9) of PGA, respec-ively. Both the polypeptide multilayers exhibit an exponentialrowth regime along with the layer number. While the multi-ayers buildup at neutral pH have a monotonous increase, the

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ultilayers built up at 3.6 show a zigzag increase manner: a bigncrease after PGA assembly but keeping constant or even slightecrease after PLL assembly. This phenomenon is explained byhe lower charge degree of PGA. When a film ending with PGAs further brought into contact with a PLL solution, some of theoosely attached PGA molecules will be replaced by the PLL

olecules, most possibly in a format of PGA/PLL complex,eading to their detachment from the multilayer surface. Thisesults in the overall constant layer mass or thickness but build-ng block and charge reversal. Since the PLL molecules at pH.6 is highly charged, they can adhere more strongly and areardly removed by the next PGA adsorption. The PM3.6 showsery rough surface with grains ranging 100–200 nm, while theM7.4 surface is rather smooth.

After the multilayers were treated with a urea solution (8 M)nd rinsed with buffer, more than 80% mass of the PM3.6 wasost, while no mass loss was found for PM7.4. CD spectra con-orm that the secondary structure of PM3.6 is abundant with-helix, which is a result of protonation of the carboxylic groupsf PGA. In such kind of polypeptide multilayers, the dominantriving force is hydrogen bonding and hydrophobic interaction,hich is less stable than the electrostatic interaction. Togetherith the sensitivity of �-helix and hydrophobic interaction to the

oncentrated urea, the PM3.6 is thus erasable at the experimentalonditions. Contrast to this, CD spectra record abundant �-sheettructure in the PM7.4, indicating that the dominant driving forcen this case is electrostatic interaction. This endows the multi-ayers with higher stability against urea treatment, leading to nohange in terms of optical mass, layer thickness, surface rough-ess and secondary structure. This study has offered a convenientathway to mediate the properties of polypeptide multilayers,hich is of great importance for their applications in fields ofiomaterials, tissue engineering and biosensors.

cknowledgements

We thank Dr. L. Ma and Z.W. Mao for their continuousupport and stimulating discussions. This study is finan-ially supported by the Natural Science Foundation of China20434030), the Major State Basic Research Program of China2005CB623902) and the National Science Fund for Distin-uished Young Scholars of China (50425311).

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