Protein-Triggered Instant Disassembly of Biomimetic Layer-by-Layer

ARTICLE pubs.acs.org/Langmuir

Protein-Triggered Instant Disassembly of Biomimetic Layer-by-Layer Films Khalil Abdelkebir, Fabien Gaudi
ere, Sandrine Morin-Grognet, Gerard Coquerel, Hassan Atmani, Beatrice Labat, and Guy Ladam* Laboratoire de Biophysique et Biomateriaux (La2B), SMS EA 3233, IMR FED 4114, Universite de Rouen, Centre Universitaire d’Evreux, 1 rue du 7
eme Chasseurs, BP 281, 27002 Evreux Cedex, France ABSTRACT: Layer-by-Layer (LbL) coatings are promising tools for the biofunctionalization of biomaterials, as they allow stress-free immobilization of proteins. Here, we explore the possibility to immobilize phosvitin, a highly phosphorylated protein viewed as a model of bone phosphoproteins and, as such, a potential promotive agent of surface-directed biomineralization, into biomimetic LbL architectures. Two immobilization protocols are attempted, first, using phosvitin as the polyanionic component of phosvitin/poly-(L-lysine) films and, second, adsorbing it onto preformed chondroitin sulfate/poly-(L-lysine) films. Surprisingly, it is neither possible to embed phosvitin as the constitutive polyanion of the LbL architectures nor to adsorb it atop preformed films. Instead, phosvitin triggers instant massive film disassembly. This unexpected, incidentally detected behavior constitutes the first example of destructive interactions between LbL films and a third polyelectrolyte, a fortiori a protein, which might open a route toward new stimuliresponsive films for biosensing or drug delivery applications. Interestingly, additional preliminary results still indicate a promotive effect of phosvitin-containing remnant films on calcium phosphate deposition.

’ INTRODUCTION Layer-by-Layer (LbL) films (or “polyelectrolyte multilayers”) are supramolecular nano- to micrometric architectures which are easily prepared by sequential adsorptions of oppositely charged polyelectrolytes and/or nanoparticles onto substrates of almost any chemical nature and shape.1 For the past decade, these systems have become of topmost interest for the design of biofunctional coatings, due to the possibility to incorporate a variety of biomimetic and/or biological, thus biocompatible, polyelectrolytes (polypeptides, polysaccharides) and bioactive species therein.2,3 Of particular interest is the use of LbL films as matrices for stress-free immobilization of proteins atop biomaterials. In spite of rare examples of protein-resistant LbL architectures obtained following specific strategies,4,5 the general rule is that proteins readily adsorb onto LbL films, even when the protein and the external layer of the film are similarly charged, through combination of various attractive (electrostatic, H-bonding, van der Waals, hydrophobic) interactions.6,7 As soft hydrated matrices, LbL can also stabilize the structure, maintain the surface mobility, and, in turn, keep the activity of immobilized proteins.8,9 Another, more specific, destructive mode of interaction exists between a particular class of proteins, namely enzymes, and LbL films comprised of targeted biological polymers, leading to slow enzymatic degradation of the films.10
12 More generally, the controlled disassembly of LbL films in physiological media is a growing field of interest, especially in biotechnology, as a promising strategy to release active compounds from surfaces or capsules.13 The most versatile destructive physicochemical treatments are based on (i) pH changes in the case of LbL r 2011 American Chemical Society

systems comprised of weak polyelectrolytes or (ii) ionic strength increases destabilizing attractive interactions between polyelectrolytes.14
16 Destructive effects of electric fields17 and surfactants18 were also reported. Besides enzymatic degradation, targeted strategies based on hydrolytic or redox degradation of specific film components are also developed.13 Another specific approach consists of destabilizing LbL films built up through receptor
ligand biomolecular interactions, with competitive small biological molecules like biotin19 or carbohydrates.20,21 Here, we report on the peculiar destructive mode of interaction of a protein, namely phosvitin (PhV), with biomimetic LbL films. Phosvitin (PhV) is a 35 kDa phosphoglycoprotein from egg yolk comprised of 217 amino acid residues, half of which are phosphoserine residues.22 With an isoelectric point of 4.0, PhV has a highly negative global charge of
179 at physiological pH. Due to its polyanionic character, PhV is a strong chelatant of metal, in particular calcium, ions.23 Also, in physiological conditions, PhV is highly flexible and poorly organized into α-helices or β-sheets because of repulsive electrostatic interactions within large sequences of contiguous phosphoserine residues.24 The initial purpose of this work was to study the possibility to use the LbL method to enrich surfaces with PhV, as a model of the phosphoproteins involved in calcium phosphate (CaP) biomineralization processes and, as such, a possible biological promotive agent for heterogeneous nucleation of CaP.25
28 Received: August 23, 2011 Revised: October 17, 2011 Published: October 18, 2011 14370

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Langmuir Within the field of bone surgery, CaP coatings are known to improve implant osseointegration by fostering cellular adhesion, proliferation, and differentiation.29 So-called “biomimetic” methods of CaP deposition consisting of substrate immersion into pseudophysiological supersaturated calcium phosphate solutions have been increasingly addressed because they are more versatile than usual deposition methods like plasma spraying. They are less restrictive in term of substrate geometry, and they allow incorporation of bioactive organic compounds (cytokines, antibiotics, growth factors, proteins, polypeptides, DNA) within CaP coatings.30 Such approach requires surfaces governing the heterogeneous nucleation of CaP. In vivo, biomineralization is governed by noncollagenous proteins,25,31 through numerous phosphate, carboxylate, and/ or sulfate moieties having strong affinity for calcium ions. Based on this principle, surfaces enriched with these groups favored the heterogeneous nucleation of CaP, by increasing the local calcium concentration.32 Surfaces enriched with cationic amine groups, through the deposition of poly-(L-lysine) (PLL) for instance, are presumed to exert similar influence on local phosphate ions concentration.33 A few previous works managed to promote the heterogeneous nucleation of CaP by means of LbL coatings comprised of synthetic and/or biomimetic components, but further developments are still needed to improve the uniformity and polymorphism control of CaP coatings.33
38 To our knowledge, very few studies have inspected the interactions of phosphoproteins with LbL films: Szyk-Warszy nska et al. and Halthur et al. described the effective buildup of films comprised of caseins39 and amelogenin,40 respectively, both in association with PLL, and only recently, Grohmann et al. reported on the buildup of PhV/PLL films.41 Here, we attempted two immobilization modes of PhV into LbL films, (i) using PhV as the polyanionic component of PhV/PLL architectures and (ii) bringing preformed chondroitin sulfate (ChS)/PLL films in contact with PhV. ChS is a linear, very hydrophilic, both sulfated and carboxylated polyanionic glycosaminoglycan present in bone and cartilage tissues,25,31 where it is presumed to participate also in biomineralization, with its sulfate and carboxylate moieties as nucleation sites. We used complementary techniques to characterize the buildup and structure of LbL films: quartz crystal microbalance with dissipation monitoring (QCM-D), optical waveguide light-mode spectroscopy (OWLS), atomic force microscopy (AFM), Fourier transform infrared spectroscopy in the attenuated total reflectance mode (ATR-FTIR), Raman confocal microscopy (MCRam), and X-ray photoelectron spectroscopy (XPS).

’ EXPERIMENTAL SECTION Chemicals. Tris(hydroxymethyl)aminomethane (Tris) was purchased from Sigma. CaCl2 3 2H2O and Na2HPO4 3 2H2O were purchased from Fluka. NaH2PO4 was purchased from Aldrich. Analytical grade NaCl, HCl, H2O2 (30% in water), and H2SO4 were purchased from Acros. All solutions were prepared with ultrapure water (18.3 MΩ cm) obtained from a Barnstead EasyPure system. All substrates (except the ATRFTIR ZnSe crystal) were cleaned in Piranha solution (3:1 mixture of concentrated H2SO4 and H2O2 30%; Piranha solution is a powerful oxidizer and reacts violently with organic materials or solvents and should be handled with extreme care) for 20 min, rinsed with H2O, and then dried with argon prior to film buildup. Solutions of anionic chondroitin sulfate A (ChS, 20
30 kDa,

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C9819 from Sigma), cationic poly-(L-lysine) (PLL, 40
60 kDa, P3995 from Sigma), and phosvitin (PhV, P1253 from Sigma) at 1 mg mL
1, solutions of cationic poly(ethyleneimine) (PEI, 60 kDa, 18197-8 from Aldrich) at 5 mg mL
1, and equimolar supersaturated calcium phosphate solution at 7.5 mM (7.5 mM CaCl2, 3.75 mM NaH2PO4, and 3.75 mM Na2HPO4) were all prepared in saline Tris/HCl buffered conditions (“Tris buffer”: 10 mM Tris, 150 mM NaCl, pH 7.4). PEI was always adsorbed as a precursor layer prior to LbL film buildup. It is widely admitted that this uniform anchoring layer limits the influence of possibly different initial states of the substrates between distinct buildups.42 QCM-D. Experiments were carried out with a D300 system (QSense, Sweden) using a QAFC302 flow chamber and QSX301 gold-coated quartz crystal sensors. This technique consists of measuring the resonance frequency shifts Δf and the dissipation factor changes ΔD of a quartz crystal sensor upon material deposition.43 Δf depends on the total oscillating mass, including coupled water, therefore providing a “wet mass” measurement. The LbL buildup was performed by successive injections of polyelectrolyte or protein solutions (5 mL) and rinsing solution (5 mL) through the flow chamber and monitored in situ. Δf was measured at the fundamental frequency around 5 MHz and at the third (15 MHz), fifth (25 MHz), and seventh (35 MHz) overtones. The fundamental frequency was discarded for it is often affected by contact with the mounting frame.44 Data analysis was carried out using the Q-Tools software provided with the instrument. The Sauerbrey equation, which proportionally relates resonance frequency shifts to adsorbed masses in the case of rigid deposits, and a Voigt viscoelastic model44 were used to derive adsorbed masses per unit area and thicknesses, assuming a film density of 1.1 g cm
3.5 OWLS. Experiments were performed with an OWLS110 system (MicroVacuum, Hungary) with OW2400 sensors. A detailed description of the technique can be found elsewhere.45 Briefly, OWLS is based on the incoupling of a laser beam into a waveguide by an optical grating. Incoupling angles of the transverse electric (TE) and magnetic (TM) modes are sensitive to refractive index changes in the contacting medium. OWLS continuously measures these in-coupling angles and converts them into effective refractive indices NTE and NTM. The solvent molecules trapped into adsorbed layers do not contribute to OWLS signal variations; therefore, OWLS provides “dry mass” values.4 LbL films were built up as follows: a polyelectrolyte solution (200 μL) was injected in the measurement cell and let at rest in contact with the sensor for about 15 min. The surface was then rinsed for another 15 min by flowing Tris buffer through the cell. This protocol was repeated alternatively with the cationic and the anionic polyelectrolytes. Data analysis was performed according to the extended homogeneous and isotropic adlayer model developed by Picart et al.,46 assuming a dn/dC value of 0.18 mL g
1.45 AFM. A PicoSPM AFM setup from Molecular Imaging with silicon nitride cantilevers of spring constant 0.38 N m
1 was used to characterize the morphology of LbL films in their native hydrated state, i.e., in contact with Tris buffer. LbL films were built up onto glass substrates using an automated dip-robot (DR-3, from Riegler & Kirstein GmbH). Each adsorption step consisted of a 15-min immersion of the substrate into a polyelectrolyte solution, followed by repeated immersions into five different rinsing Tris buffer solutions. Height images were taken in constant force contact mode at a fixed scan rate of 1 Hz with a 14371

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Langmuir resolution of 512 pixels  512 pixels. Micrographs were analyzed with SPIP (Scanning Probe Image Processor) Software to determine the arithmetical mean roughness Ra. ATR-FTIR. Measurements were performed with a PerkinElmer Spectrum BX system. LbL films were assembled directly onto the zinc selenide (ZnSe) crystal (cleaned with an optical wipe and acetone). For each adsorption step, a liquid film of solution was deposited using a Pasteur pipet and maintained at rest in contact with the ZnSe crystal for 15 min, and then the solution was removed and replaced by the rinsing solution. Then the rinsing solution was removed, and the surface was dried with argon for 5 min, prior to recording the surface absorbance spectrum.5 Characteristic IR bands of interest were those identified from the respective reference spectra of pure ChS, PLL, and PhV deposits from water solutions. MCRam. Raman measurements were carried out with a confocal Raman microscope composed of a Raman spectrometer (LabRam HR by Jobin-Yvon Horiba with a 600 lines mm
1 grating) coupled to a microscope (model BX41, Olympus) via optical fibers. The excitation of Raman scattering is operated with a helium
neon laser at wavelength 632.8 nm. The laser beam is focused on the sample by means of a 100 microscope objective. A L 200 μm confocal pinhole placed before the entrance slit rejects Raman signal from out-of-focus planes. Raman spectra with good signal-to-noise ratio were recorded with an integration time of 120 s. XPS. Surface elemental analyses were performed with a Gammadata Scienta SES 200 XPS spectrometer equipped with a monochromatized Kα1,2 anode (14.866 eV) and a concentric hemispherical analyzer. Photoemitted electrons were collected at a takeoff angle of 90° from the substrate, with electron detection in the constant analyzer energy mode. With this geometry, the depth of analysis, which depends on the inelastic mean free path of photoelectrons traveling in the top layer, can be evaluated to 9 nm. Survey spectrum signal was recorded with a pass energy of 500 eV, and for high resolution areas (C 1s, O 1s, and N 1s) pass energy was set to 200 eV. In this last mode, the energetic resolution of the spectrometer is estimated to be 0.46 eV. Peak fitting was made with mixed Gaussian
Lorentzian (30%) components with equal full-width-at-half-maximum (fwhm) using CASAXPS software. The surface composition expressed in atom % was determined using integrated peak areas of each component and taking into account transmission factor of the spectrometer, mean free path, and Scofield sensitivity factors of each atom.

’ RESULTS Considering its highly negative charge and flexible and disordered conformation,22,24 PhV constitutes, at first sight, a good candidate as the polyanionic component of LbL films. Moreover, we observed that PhV and PLL readily precipitated in solution under physiological Tris buffer conditions, suggesting that they should self-assemble into LbL films. Therefore, we first explored the buildup of PhV/PLL films. Figure 1 reports the QCM-D data obtained during the buildup of a PEI/(PhV/PLL)7 architecture. It comes out that, following the initial effective adsorption of PhV onto the PEI precursor layer, the successive PLL and PhV injections resulted in cyclical variations of the QCM-D parameters, consisting respectively of decreases and increases of the wet mass. Both effects almost compensate each other, such that the buildup is ineffective. Recently, Grohmann et al. also reported

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Figure 1. (A) Evolution of the normalized resonance frequencies and dissipation factors for the third, fifth, and seventh overtones (n = 3, 5, 7) during the buildup of a PEI/(PhV/PLL)7 film at pH 7.4 and 150 mM NaCl (Tris buffer) onto a 5 MHz QCM-D substrate. (B) Extract of (A) showing the continuous evolution of the QCM-D parameters for the third overtone during the buildup of the fourth and fifth layer pairs. “R” stands for “rinsing steps”. (C) Conversion of the raw data shown in (A) into hydrodynamic thickness dQCM‑D and hydrated mass QQCM‑D according to the Voigt viscoelastic model applied to the third, fifth, and seventh overtones (full line) and to the Sauerbrey relation applied to the third overtone (dotted line).

cyclical evolution of the dry mass by means of an optical technique (reflectometric interference spectroscopy, RIfS), during the buildup of PhV/PLL films built up in close conditions, but the presence of a PEI precursor layer.41 At first sight, however, respective evolutions of the dry mass described by these authors and of the wet mass described here appear to be contradictory, as they varied in the opposite way for a given PLL or PhV injection. This point will be elucidated later in this report, when discussing the PEI/(PhV/PLL)7 buildup profile in the Discussion section. At this stage, since PLL is the most versatile and widely used polycationic component of biomimetic LbL films, we limit ourselves to conclude that PhV does not constitute a choice polyanionic component for the design of such films. This conclusion is all the more surprising since Szyk-Warszy nska et al. succeeded in building up LbL films comprised of PLL and β-casein, a highly phosphorylated, flexible, and disordered protein with similar features to PhV.39 Since proteins are known to readily adsorb on top of LbL coatings,6,7 we next explored the immobilization of PhV atop preexisting ChS/PLL films. Ten PEI/(ChS/PLL)6 films were built up, then treated with PhV solutions at 1 mg mL
1 until stable QCM-D parameters were obtained, and finally rinsed with Tris buffer. As illustrated by the typical buildup described in Figure 2A, B, PhV always generated an instantaneous significant increase of the resonance frequencies, accompanied by a decrease 14372

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Figure 2. Evolution of (A) the normalized resonance frequencies and (B) the dissipation factors for the third, fifth, and seventh overtones (n = 3, 5, 7) during the buildup of a PEI/(ChS/PLL)6/PhV film at pH 7.4 and 150 mM NaCl (Tris buffer) onto a 5 MHz QCM-D resonator. (C) Conversion of the raw data shown in (A) and (B) into hydrodynamic thickness dQCM‑D and hydrated mass Q QCM‑D according to the Voigt viscoelastic model applied to the third, fifth, and seventh overtones (full line) or according to the Sauerbrey relation applied to the third overtone (dotted line). (D) Mean step-by-step evolution of dQCM‑D and Q QCM‑D (according to the Voigt model) over 10 distinct PEI/(ChS/PLL)6/PhV films. Bars represent standard errors.

Figure 3. (A) Evolution of the effective NTE and NTM indices during the buildup of a typical PEI/(ChS/PLL)6/PhV film. (B
D) Evolution of the mean optical parameters for the three similar architectures studied by OWLS: (B) refractive index nOWLS; (C) optical thickness dOWLS; (D) optical “dry” mass QOWLS. Bars represent standard errors. Inset in (D) shows an optical microscopic image of one of the three films after drying. The surface was scratched with a needle before drying.

of the dissipation factors. The conversion of raw QCM-D data into hydrodynamic thickness (dQCM‑D) and wet mass (Q QCM‑D) shown in Figure 2C leads to very close results whatever the Voigt (viscoelastic) or Sauerbrey (rigid) analysis model; this is consistent with the observed quasi-superimposition of the normalized resonance frequencies.47 Unexpectedly, it comes out that PhV induces a massive decrease of the film in hydrodynamic thickness and wet mass. Figure 2D, which reports the mean stepby-step evolutions of these parameters (according to the Voigt model) over the 10 buildups performed, confirms the general

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Figure 4. 3D and 2D AFM images representing the topographies of (A) a PEI/(ChS/PLL)6 film and (B) a PEI/(ChS/PLL)6/PhV film immersed in Tris buffer. Mean roughness values Ra over three separate images are accompanied by standard errors.

Figure 5. (A, B) Respective optical micrographs of dry PEI/(ChS/ PLL)25 and PEI/(ChS/PLL)25/PhV films built up on top of quartz substrates. Surfaces were scratched with a needle and then dried prior to observation. (C, D) 3D and 2D AFM images showing the topographies of PEI/(ChS/PLL)25 and PEI/(ChS/PLL)25/PhV films after drying. Mean roughness value Ra over three separate images is accompanied by the standard error.

character of this behavior. Based on the fact that QCM-D detects adsorbed material together with bound water, the concomitant increase in resonance frequencies and decrease in dissipation factors observed can be explained by (i) a desorption or partial destruction of the LbL deposit and/or (ii) a densification of the LbL deposit accompanied by a release of water (upon cross-linking, for instance).44,48 In order to decide between these two effects, we monitored in situ the buildups of three similar PEI/(ChS/PLL)6/ PhV architectures by means of the OWLS technique, which is sensitive to adsorbed material only, not to bound water. Figure 3A shows the continuous evolution of raw OWLS data (effective indices NTE and NTM) for a typical buildup. Again, the sharp decrease of NTE and NTM upon contact of the PEI/(ChS/ PLL)6 architecture with PhV indicates the strong influence of the protein onto the film structure. Figure 3B
D reporting the mean step-by-step evolution of optical parameters (refractive index 14373

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Table 1. Atomic Composition Derived from XPS Measurements for a PEI/(ChS/PLL)6 Film (PLL#6) and a PEI/(ChS/ PLL)6/PhV Film (PhV) Built Up onto Glass Substrates PLL#6

PhV

C 1s

55.6%

31.9%

O 1s

28.4%

36.1%

N 1s

8.9%

8.1%

S 2p

2.3%

0.9%a

P 2p Si 2p


4.3%

2.0% 16.6%

Cl 2p

0.4%

1.1%

Na 1s

0.1%

1.6%

Al 2p




0.9%b

K 2p




0.4%b

Zn 2p 3/2




0.2%b

a

Uncertainty in this value is large because of the enhanced detection of silicon, whose Si 2s transition generates a wide characteristic peak called “plasmon” overlaying the S 2p signal in the range 160
185 eV and disabling a reliable automatic quantification of the latter. Manual estimation of the peak area still allowed consideration of the S 2p signal for atomic percentages calculation. b Small amounts of these elements are assumed to originate from the commercial source of PhV.

Figure 6. (A) Respective ATR-FTIR spectra of ChS, PLL, and PhV deposits. Each deposit was obtained by evaporation of a solution of the polymer or protein in pure water atop the ZnSe crystal. (B) ATR-FTIR spectra of a typical PEI/(ChS/PLL)6 film built up without intermediate drying, before and after treatment with PhV. (C) Mean step-by-step evolution of ATR-FTIR absorbances at (b) 1644 cm
1 (ChS and PLL), (9) 1053 cm
1 (ChS), and (2) 1246 cm
1 (ChS) over the buildup of three PEI/(ChS/PLL)6 films. Bars represent standard errors. Open symbols (O, 0, 4) at the PLL#6 step report mean absorbances at the same wavenumbers over four dry PEI/(ChS/PLL)6 films built up without intermediate drying. Open symbols at the PhV step report the absorbances at the same wavenumbers for two dry PEI/(ChS/PLL)6 films after treatment with PhV.

nOWLS, optical thickness dOWLS, and optical mass QOWLS) for the three films confirms the extensive film destruction induced by PhV, bringing the optical parameters back roughly to the values measured at the PLL#1 or ChS#2 step. The inset in Figure 3D shows an optical micrograph of one of the films, after it was scratched with a needle and dried with argon. Continuity of the scratch boundaries shows that the film was not totally destroyed upon interaction with PhV. Figure 4 compares the AFM topographies imaged in Tris buffer of a PEI/(ChS/PLL)6 film and a PEI/(ChS/PLL)6/PhV film. Treatment with PhV provoked a dramatic change of morphology, consisting of the disappearance of the material islets and a strong decrease in roughness Ra from ∼40 nm down to ∼5 nm, consistently with extensive film destruction. The destructive effect of PhV on ChS/PLL architectures was also verified on a PEI/(ChS/PLL)25 film, as illustrated in Figure 5 showing optical and AFM images obtained before and after treatment with PhV. Additional ATR-FTIR measurements were performed on two PEI/(ChS/PLL)6 films built up atop the ZnSe spectrometer

substrate and then dried and analyzed. Then, the films were rehydrated with Tris buffer, treated with PhV, and again dried and analyzed. Figure 6A shows the reference ATR-FTIR spectra of a PLL deposit, a ChS deposit, and a PhV deposit, and Figure 6B shows the spectra obtained for one of both PEI/(ChS/PLL)6 films before and after treatment with PhV (similar spectra were obtained for the second film). In Figure 6C, we supplement the mean step-by-step evolution of absorbances measured over three distinct buildups (discussed in a previous work)49 with the data concerning PhV-treated films. Again, the absorbance decrease observed upon treatment with PhV indicates an important film destruction. The presence of a significant amount of PhV in the final deposit is revealed by the specific peak relative to the dianionic phosphoryl groups of PhV at 976 cm
1.50 Thus, the decrease in the absorbance relative to amide bands accompanying the release of ChS and PLL must be partially compensated by adsorbed PhV and certainly does not account exactly for the film destruction. The presence of immobilized PhV in the final deposit constitutes a key information for interpreting the structure and for considering possible biological applications of these systems. We further inspected this point by characterizing PhVtreated films with two complementary techniques, X-ray photoelectron spectroscopy (XPS) and confocal Raman microspectroscopy (MCRam). A PEI/(ChS/PLL)6 film and a PEI/(ChS/PLL)6/PhV film were built up onto glass substrates and analyzed by XPS to detect the possible presence of phosphorus associated with PhV, which was the only possible source of this element in the deposit. Table 1 reports the derived atomic surface compositions for both architectures. The presence of PhV on the PhV-treated film is clearly confirmed by the phosphorus signal, representing 2% of the detected elements. XPS analysis raises two additional remarks. (i) The detected silicon necessarily originates from the glass support. The analysis was carried out under vacuum conditions, thus with highly dehydrated deposits, over a depth of about 10 nm. It is not unexpected that the films were reduced below 10 nm in thickness under such conditions and 14374

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Table 2. Structural Parameters of PEI/(ChS/PLL)6 (PLL#6) and PEI/(ChS/PLL)6/PhV (PhV) Films Derived from OWLS and QCM-D Measurements and Variation Thereof upon PhV Treatmenta PLL#6 146 ( 11

51 ( 7


65 ( 10%

16.07 ( 1.22

5.63 ( 0.75


65 ( 10%

nOWLS

1.423 ( 0.009

1.471 ( 0.028

Figure 8. Quartz slides covered with (left) a PEI/(ChS/PLL)6, (middle) a PEI/(ChS/PLL)6/ChS, and (right) a PEI/(ChS/PLL)6/PhV architecture, after immersion for 24 h at 22 °C in a 200-mL equimolar supersaturated calcium phosphate solution prepared at 7.5 mM.

thus allowed the detection of Si. The significantly higher amount of Si detected for the PhV-treated film compared to the untreated film is consistent with a reduced thickness due to partial film destruction. (ii) The S 2p signal related to ChS is clearly lowered for the PhV-treated film compared to the untreated film, consistently again with partial film destruction. The MCRam technique was not adapted for characterizing PEI/(ChS/PLL)6 and PEI/(ChS/PLL)6/PhV films because concerned amounts of material were below the detection limit. Therefore we characterized a PEI/(ChS/PLL)25 film and a PEI/(ChS/PLL)25/PhV film built up atop quartz substrates. Figure 7 shows the corresponding Raman signatures over the range 1200
1750 cm
1, as well as the reference signature of a PhV deposit obtained by evaporation of a pure protein solution. The Raman signature of the PhV-treated film contains specific components of the PhV and the ChS/PLL system signatures, within the form of separate bands in domain “1” and shoulders in domains “2” and “3”. This confirms that (i) film destruction was not complete and (ii) PhV molecules were immobilized onto the surface. In order to preliminary verify that immobilized PhV molecules could influence the heterogeneous nucleation of CaP, quartz slides were covered with a PEI/(ChS/PLL)6, a PEI/(ChS/ PLL)6/ChS, or a PEI/(ChS/PLL)6/PhV film and then immersed together for 24 h at 22 °C into an equimolar supersaturated calcium phosphate solution prepared at 7.5 mM.34 Promisingly, as illustrated by Figure 8, CaP deposition was by far promoted by the residual PEI/(ChS/PLL)6/PhV architecture compared to the native PEI/(ChS/PLL)6/ChS and PEI/(ChS/ PLL)6 architectures. We note also that the ChS-ending film exerted a promotive effect compared to the PLL-ending film. In the discussion below, we will propose an explanation for the destructive effect of PhV on ChS/PLL films. For that purpose, we rely upon the analysis of the structure of PhVtreated ChS/PLL films and of the buildup profile of the PhV/ PLL system.

dOWLS (nm) Q OWLS,wet (μg cm
2) Q OWLS (μg cm
2) %H2Odense zone a

variation (%)

Q QCM‑D (μg cm
2)

dQCM‑D (nm)

Figure 7. Raman signatures in the range 1200
1750 cm
1 of a PEI/(ChS/PLL)25 film, a PEI/(ChS/PLL)25/PhV film, and a pure PhV deposit. Signatures of pure PhV and the PEI/(ChS/PLL)25 film were normalized with regard to the 1250 and 1340 cm
1 bands of the PEI/(ChS/PLL)25/PhV film, respectively, in order to facilitate the comparison of the spectra.

PhV

91 ( 11

14 ( 3


85 ( 19%

10.02 ( 1.18

1.49 ( 0.29


85 ( 19%

4.32 ( 0.12

0.92 ( 0.06


79 ( 6%

57 ( 7%

38 ( 8%

The parameters QOWLS,wet and %H2Odense zone are defined in the text.

’ DISCUSSION Structure of the PEI/(ChS/PLL)6/PhV Films. Table 2 gathers the optical (OWLS) and hydrodynamic (QCM-D) parameters of PEI/(ChS/PLL)6 films before and after treatment with PhV. For both architectures, one first notes significantly lower values for dOWLS compared to dQCM‑D. Concerning the PEI/(ChS/ PLL)6 films, this discrepancy has already been discussed in a previous work.49 Briefly, it was attributed to the presence of a diffuse outer layer atop the LbL architecture. Because this zone is close to the adjacent buffer medium in term of refractive index, it remains undetectable by optical techniques. Therefore, the optical thickness describes only the inner, dense zone of high refractive index, while the hydrodynamic thickness describes the whole hydrated film extent.4 The outer diffuse layer of PEI/ (ChS/PLL)6 films is assumed to be comprised of the islets of material observed in Figure 4A. From the difference between the values of dOWLS (∼15 nm) and dQCM‑D (∼50 nm) measured after treatment with PhV, we conclude that the PEI/(ChS/ PLL)6/PhV architectures must also be comprised of an inner dense zone and an outer diffuse zone. From OWLS data, we can derive two distinct mass values: (i) Q OWLS, standing for the “dry” mass of the inner dense layer, given by45

Q OWLS ¼

Δn  dOWLS dn=dC

ð1Þ

with Δn = nOWLS
nbuffer and dn/dC = 0.18 mL g
1 and (ii) QOWLS,wet standing for the hydrated mass of the inner dense layer, given by49 Q OWLS, wet ¼ FLbL  dOWLS

ð2Þ

with FLbL = 1.1 g cm
3. The difference between Q OWLS,wet and Q OWLS gives access to the hydration of the dense zone, %H2Odense zone. The overall hydrated mass, Q QCM‑D, is related to dQCM‑D through the same equation as eq 2.44 Despite the fact that the decreases in optical thickness (
85 ( 19%) and dry mass (
79 ( 6%) measured by OWLS cannot be strictly compared with the decreases in hydrodynamic thickness and hydrated mass (
65 ( 10%) measured by QCM-D because they do not refer to the same film extents, both techniques indicate roughly similar massive destruction rates. The decrease of the dense zone hydration %H2Odense zone, together with the increase in refractive index nOWLS (from ∼1.42 up to ∼1.47), indicates that the PhV treatment further densifies the inner zone of residual films compared to that of pristine films. 14375

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Langmuir The low roughness of PEI/(ChS/PLL)6/PhV films, Ra = 4.5 ( 0.7 nm, determined from AFM measurements (Figure 4) is not compatible with a diffuse layer formed by islets of material, as was assumed for PEI/(ChS/PLL)6 films.49 Given the maximal chain lengths of ChS (20
30 kDa) and PLL (40
60 kDa), which are equal to 40
60 nm51 and 115
170 nm,52,53 respectively, the diffuse layer could rather be comprised of chains of one of these polyelectrolytes extending into the adjacent solution.4,54 Considering the structural features of PhV, immobilized proteins might also be involved in the formation of the hydrated diffuse layer: PhV molecules are ∼30 nm in length, flexible, and subject to strong intra- and intermolecular electrostatic repulsions between their numerous phosphate groups, in particular along large blocks of up to 15 contiguous phosphoserine residues.24,55 Moreover, a glycosylated site of PhV bears an oligosaccharide comprised of 13 saccharide units likely strengthening both the hydrophilic character and the steric repulsion between neighboring immobilized PhV chains.56 Such stress-free immobilization of the PhV molecules must preserve the availability of phosphoryl moieties, thus explaining the remarkable promotive effect of PEI/(ChS/PLL)6/PhV on the heterogeneous nucleation of CaP. Buildup of the PEI/(PhV/PLL)7 Architectures. The QCM-D profiles obtained during the buildup of a PEI/(PhV/PLL)7 deposit can help to explain the destructive effect of PhV on ChS/PLL films. From Figure 1B describing the typical cyclical pattern of QCM-D parameters from PhV#4 to PLL#5 steps of the construction, it comes out that PhV (respectively PLL) causes sharp increases (respectively decreases) of both the dissipation factors and the hydrated mass (from Figure 1C, the evolution patterns of the hydrated mass and the frequencies are similar, except the sign). The mass loss caused by PLL may be due to PhV desorption and/or to a release of water molecules trapped within the deposit. A simple desorption of PhV molecules, within the form of released PhV/PLL complexes, should reduce the QCM-D parameters back to values intermediate to those measured respectively after the PEI and PhV#1. This is verified for resonance frequencies (and thus, for mass), but not for dissipation factors, which reach values lower than that measured after the initial PEI deposition (Figure 1A), indicating deposit stiffening upon PLL injections. It is worth noting that the different normalized overtones are clearly separated following PhV injections, which reveals, together with increased dissipation factors, a nonrigid character of the deposit.47 On the contrary, all overtones become superimposed after PLL injections, which is the mark of a film stiffening. Concomitant mass loss and deposit stiffening upon PLL injections are consistent with a release of water, while PhV desorption is rather unlikely to occur since Grohmann et al. detected an uptake of material upon PLL injections.41 Consequently, the overall film deswelling is not surprising considering that polycationic PLL chains may crosslink the highly hydrated polyanionic diffuse layer suggested by the high dissipation factors and separated overtones evolutions following contact with PhV (Figure 1A, B). This diffuse layer is likely to be formed by loops and tails of PhV chains extending into the contacting solution. The osmotic shock caused by the replacement of monovalent counterions by polycations might also contribute to the release of water.57 Subsequent PhV adsorption step brings the QCM-D parameters back to values similar to those measured at the end of the preceding PhV adsorption step. This corresponds, in particular, to a wet mass uptake, whereas Grohmann et al. detected a dry mass loss upon contact with the protein.41 PhV thus necessarily

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causes the desorption of the PLL molecules immobilized during the intermediate step and the uptake of water. In addition, QCMD data indicate that the possible PhV desorption provoked by the previous PLL injection step must be exactly compensated by newly adsorbed PhV molecules. These observations are fully consistent with the existence of the hydrated diffuse PhV layer assumed above, and with its further cross-linking by PLL: PhVinduced PLL desorption next cancels the cross-linking and restores the hydrated diffuse layer. From a kinetic point of view, the rapidity of both PLL adsorptions and desorptions indicates that these processes are controlled by the transport of chains in solution toward or from the surface.58 The almost complete displacement of PLL by PhV is necessarily due to higher stability of the PhV/PLL complexes in solution than in the immobilized state. To explain this behavior, Grohmann et al.41 referred to the phase diagram of polyelectrolyte complexes in solution proposed by Kovacevic et al.,59 establishing that complexes are dissolved when one of both polyelectrolytes is in large excess, the exact threshold depending on ionic strength. This effect is often extended to the case of LbL films in order to explain the partial disassembly sometimes occurring upon contact with a solution of one of both polyelectrolytes, or when a zone of the film is enriched in one of the polyelectrolytes by diffusion.49 It is assumed that the film disassembly occurs through the conversion from immobilized into water-soluble polyelectrolyte complexes, driven by an important gain in entropy. Here, the instability of immobilized PhV/PLL complexes may be enhanced by the fact that PLL chains have to compete with underlying PEI chains of the precursor layer for creating interactions with the protein. Highly charged PEI chains may interact strongly with the PhV molecules, limiting the number and size of free loops and tails of the latter available to maximize interactions within PhV/PLL complexes to the same extent as solubilized PhV chains do. Additionally, it seems that the PhV/PEI complexes forming the film are more stable than the solubilized PhV/PLL complexes; otherwise, adsorbed PhV should be totally displaced upon interaction with PLL, as is PLL upon interaction with PhV. PhV-Induced Disassembly of PEI/(ChS/PLL)6 Films. The high stability of solubilized PhV/PLL complexes discussed above is likely to govern the disassembly of PEI/(ChS/PLL)6 films. PhV must destabilize weaker ChS/PLL complexes constituting the films, causing them to dissolve in favor of more stable watersoluble PhV/PLL complexes consequently released into the solution, along with the ChS chains. Preferential interactions of PLL with PhV rather than with ChS can be attributed to the numerous dianionic phosphoryl moieties of the protein at physiological pH, as well as to its flexibility, promoting its diffusion within the films and allowing the optimization of interactions with PLL. Similarly, Ball et al. explained the partial dissolution of hyaluronic acid (HA)/PLL and HA/PAH films upon contact with multivalent Fe(CN)64
ferricyanide anions through preferred interactions of the latter with PLL.16 In addition, the release of the displaced ChS chains might generate an osmotic pressure within the films causing them to swell and thus contributing to their destruction. Mjahed et al. have proposed a similar explanation for the formation of holes within HA/PLL films following a decrease in ionic strength.60 The persistence of a dense deposit despite massive destruction of the ChS/PLL films can be due to a better resistance to destructive competitive interactions with PhV, of the dense, more cohesive and stable internal zone, compared to the external diffuse zone. 14376

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Langmuir

Figure 9. Schematic drawing of the dissolution mechanism of ChS/ PLL films upon contact with PhV. For details, see the main text. (A) ChS/PLL film is put in contact with PhV. (B) PhV chains diffuse within the film. (C) Due to stronger interactions of PLL with PhV than with ChS, ChS/PLL complexes are replaced by PhV/PLL complexes. Released ChS chains provoke osmotic pressure. (D) Osmotic pressure and higher stability of PhV/PLL complexes in solution than in the film lead to dissolution of the LbL architecture.

The immobilization of PhV chains into the residual deposit is then likely to occur by means of free fragments of polycationic PEI and PLL chains composing it. A sketch of the proposed dissolution mechanism is shown in Figure 9. Several studies already described competitions between similarly charged polyelectrolytes within LbL architectures, as is the case here between PhV and ChS. First examples were reported by Boulmedais et al.61 and Jomaa et al.62 for PGA/PLL and PMA/ PDADMAC systems, respectively, contacting PSS solutions. In both cases, the polyanions constituting the films (PGA or PMA) were totally replaced by PSS chains. In the same manner, Ball et al. observed that competitive interactions of polycationic PAH and PLL toward polyanionic HA, and of polyanionic β-1,3glycan sulfate (GlyS) and alginate (Alg) toward PLL, led respectively to the quantitative replacement of PLL by PAH within HA/PAH films16 and of Alg by GlyS within Alg/PLL films.63 In the latter study, the exchange process was governed by the chemical nature of the polyanions, with sulfate moieties generating stronger interactions with polycations than carboxylate moieties, thus allowing the formation of particularly stable LbL films.64,65 Within this context, our results suggest that phosphorylated polyanions can interact with polycations even more strongly than sulfated polyanions, since PhV destroyed the ChS/ PLL complexes. One can cite also the work of Kharlampieva et al. describing the exchange of polymers incorporated within LbL films through hydrogen bonds, by charged polymers in solution,66 and the work of H€ubsch et al. describing the exchange of Fe(CN)64
ferricyanide anions incorporated within PGA/PAH films by diffusion, by PGA chains in solution.67 In the latter case, ferricyanide ions were not displaced by HA chains, whose persistence length is too large, and hence flexibility is too low, to allow the diffusion process underlying the exchange mechanism. Finally, in the only example involving proteins, No€el et al. reported the replacement of PLL in pectin/PLL architectures by globular proteins described as weakly charged polyampholytes.68

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From these various studies, it is established that, provided that an incorporated compound retains sufficient mobility within a LbL film, it is susceptible to undergo a dynamic exchange process with an outer polyelectrolyte generating stronger interactions within the film. However, to our knowledge, the disassembly of LbL films upon contact with a competitive polyelectrolyte, a fortiori with a protein, has never been reported. Targeted dissolution of LbL systems upon contact with proteins specifically related to given pathologies, similar to the PhV-induced disassembly of ChS/PLL films reported here, might provide an original complementary route for controlled drug release. In order to further explore this approach, the next steps should consist of (i) study of the influence of the protein concentration on dissolution kinetics and (ii) rationalization of the structural features of eligible LbL systems and proteins. In particular, “scaling up” the two-component phase diagram approach developed by Kovacevic et al.59 toward the establishment of three-component phase diagram of polyelectrolytes and proteins mixtures would help to predict favorable conditions for protein-triggered LbL film disassembly. Highly phosphorylated and/or flexible proteins similar to PhV (e.g., fetuin and casein) and LbL systems comprised of highly hydrated carboxylated or sulfated polysaccharides (e.g., dermatan sulfate, heparin, hyaluronic acid, carboxymethylcellulose) should next be explored.

’ CONCLUSION In summary, the experimental data reported here concerning the immobilization of the model phosphoprotein phosvitin by means of the LbL method highlight unexpected behaviors: (i) in spite of its polyanionic character and flexibility, it was hardly possible to use the protein as a constitutive component of LbL films, and (ii) in disagreement with the general rule, the protein did not adsorb onto preformed biomimetic ChS/PLL films; instead, PhV triggered instantaneous massive film disassembly. Insofar as we know, this is the first instance of a destructive exchange process between LbL films and a third polyelectrolyte, a fortiori a protein, moreover in physiological conditions. We assume that this phenomenon may open a route toward the design of protein-responsive films and capsules for unique biosensing and drug delivery applications. Notwithstanding the rather reluctant behavior of PhV in terms of LbL buildup, proteins were still immobilized onto surfaces and exerted promotive influence on the heterogeneous nucleation of calcium phosphate. More detailed investigations on this point will be reported in a forthcoming article. ’ AUTHOR INFORMATION Corresponding Author

*Phone (+33) (0)2 32 39 90 87; fax (+33) (0)2 32 39 90 80; e-mail [email protected].

’ ACKNOWLEDGMENT vreux Agglomeration” The authors acknowledge the “Grand E and the “Conseil General de l’Eure” for partial financial support of La2B (Laboratoire de Biophysique et Biomateriaux). The authors thank Dr. Bernard Senger from Inserm U977 in Strasbourg for access to the computer codes used for processing the experimental OWLS data and Dr. Arnaud Ponche from Institut de 14377

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Langmuir Science des Materiaux de Mulhouse (IS2M - LRC 7228) for XPS analyses.

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