Surface Science 605 (2011) 2017–2024

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Surface Science

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Adsorption of Candida rugosa lipase at water-polymer interface: The caseof poly(DL)lactide

Gihan Kamel a,⁎, Federico Bordi b, Laura Chronopoulou c, Stefano Lupi a, Cleofe Palocci c,Simona Sennato b, Pedro V. Verdes d

a Dipartimento di Fisica, Sapienza Università di Roma, Piazzale A. Moro 2, 00185 Roma, Italyb Dipartimento di Fisica and CNR-IPCF, Sapienza Università di Roma, Piazzale A. Moro 2, 00185, Roma, Italyc Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italyd Soft Matter and Molecular Biophysics Group, Department of Applied Physics, Campus Vida, Faculty of Physics, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain

⁎ Corresponding author at:Dipartimentodi Fisica, SapieAldo Moro, 2-00185 Rome, Italy. Tel.: +39 06 4991 3506

E-mail address: gihan_kamel@yahoo.com (G. Kamel

0039-6028/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.susc.2011.07.021

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 March 2011Accepted 27 July 2011Available online 7 August 2011

Keywords:Protein adsorptionNanostructured polymersLangmuir films

Insights into the interactions between biological macromolecules and polymeric surfaces are of great interestbecause of potential uses in developing biotechnologies. In this study we focused on the adsorption of a modellipolytic enzyme, Candida rugosa lipase (CRL), on poly-(D,L)-lactic acid (PDLLA) polymer with the aim to gaindeeper insights into the interactions between the enzyme and the carrier. Such studies are of particular relevancein order to establish the optimal conditions for enzyme immobilization and its applications. We employed twodifferent approaches; by analyzing the influence of adsorbed CRL molecules on the thermodynamic behavior ofLangmuir films of PDLLA deposited at air–water interface, we gained interesting information on the molecularinteractions between the protein and the polymer. Successively, by a systematic analysis of the adsorption of CRLonPDLLAnanoparticles,we showed that the adsorption of amodel lipase, CRL, onPDLLA is described in terms of aLangmuir-type adsorption behavior. In thismodel, onlymonomolecular adsorption takes place (i.e. only a singlelayer of the protein adsorbs on the support) and the interactions between adsorbed molecules and surface areshort ranged. Moreover, both the adsorption and desorption are activated processes, and the heat of adsorption(the difference between the activation energy for adsorption and desorption) is independent from the surfacecoverage of the adsorbing species. Finally, we obtained an estimate of the number of molecules of the proteinadsorbedper surface unit on the particles, a parameter of a practical relevance for applications in biocatalysis, anda semi-quantitative estimate of the energies (heat of adsorption) involved in the adsorption process.

nzaUniversità diRoma, Piazzale; fax: +39 06 44 63 158.).

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Polylactic acid (PLA) is one of the most commonly used polymersfor biotechnological applications [1,2]. In fact, it possesses features likenon-toxicity, biodegradability and biocompatibility that make it anideal material for a wide number of biotechnological applications [3].

Moreover, the biodegradation of PLA leads to pharmacologicallyinactive substances, which are absorbed by the body or removed bymetabolism [4]. PLA is derived from renewable sources, such as cornstarch and sugar cane, and its use in the preparation of bioplastics,usefulness for producing loose-fill packaging, compost bags, foodpackaging, and disposable tableware, qualify it as a sustainablealternative to petrochemical-derived products [5,6]. PLA monolayerisothermshavebeen recently studied [7]. They showa feature that lookssimilar to the well-known monolayer main transition of fatty acids or

phospholipids, with analogous temperature dependence, although PLAhas a completely different molecular structure. The dependence of themain transition on the length of the tails of the molecules is mirrored bythe dependence on the length of the polymer chain (or molecularweight). Materials of different molecular weight are miscible in mono-layers and themain transition like feature is shifted in proportion to theconcentration. This is similar to the behavior of mixtures of biologicallyrelevant lipids and suggests the possible use of polymers as membranematerials for artificial applications because polymers should be able toprovide muchmore durable membranes than natural or small moleculefilms can afford.

All cellular properties are strongly dependent from the specificfunctional features provided to the subcellular space by interfaces.Lipases are soluble enzymes comprisinga categoryof themost frequentlystudied interfacial enzymes [8,9],mainly acting at the oil–water interface(surface of oil droplets) andnot at the level of cellularmembranes, on thecontrary to some phospholipases. Having a catalytic action which isstrictly dependent upon the presence of a water–lipid interface, lipasesare an example of the importance of studying protein adsorption andprotein–surface interactions [10–14]. However most of the published

2018 G. Kamel et al. / Surface Science 605 (2011) 2017–2024

studies on lipase–monolayer interface interactions concern the in-teractions with lipid films that is with the substrate. Moreover, lipaseimmobilization for biocatalytic applications is particularly important andrepresents a very active research area [15–17]. When lipases are used asimmobilized enzymes, their substrate may also be solubilized in organicsolvent, thus increasing dramatically the number of their potentialapplications [18–20]. Candida rugosa lipase (CRL) is one of the mostimportant industrial enzymes, thanks to its ability to produce chiralchemicalswithhighenantiomericpurity [21,22].Having theGRAS status,its multiple applications of commercial interest cover a broad range ofindustrial fields, from food, to chemical synthesis, to skin care [23].

It is well established that a hydrophobic interface can improvelipase stability. Moreover, recent studies have demonstrated that theinteraction of lipolytic enzymes with nanostructured materials canenhance the activity of the adsorbed protein [24]. The mechanismslikely to occur during the adsorption of lipase onto monomolecularfilms at the air–water interface have been studied to some extent[25–27]. Further investigations of protein interactions with syntheticinterfaces may inform the design of protein-based biosensors and,since adsorbed proteinsmediate the interaction of cells with a surface,complement our understanding of cell–surface interactions.

Adsorption of proteins at an interface can be investigated by usinga number of methods. The Langmuir balance is a useful technique formodel studies of molecular interactions at interfaces because surfacepressure-area isotherms reflect the intermolecular forces operating intwo-dimensional arrays of macromolecules, and also provide infor-mation about their organization and conformational changes [28–31].When lipases are used as immobilized enzymes, their substrate isusually solubilized in organic solvents. Therefore, a double interest instudying lipase adsorption arises: one can study adsorption in relationwith the mode of action of the enzyme on its natural substrate, but alsoin relation with enzyme immobilization for other purposes. In thiscontext, the adsorption of an enzymatic protein, candida rugosa lipase(CRL), on poly-D,L-lactic acid (PDLLA)-based polymeric surfaces, wasinvestigated. Qualitative information on the interactions between theprotein and the polymeric surface as well as on their mutualorganization was obtained. CRL adsorption isotherms on PDLLAnanoparticles were also studied and analyzed to gain quantitativethermodynamic information and an estimate of the heat of adsorption.

2. Experimental section

2.1. Materials

The polylactide employed in this study, a copolymer of poly-D andL-lactic acid, PDLLA, (MW 75–120 kDa), and the lipase from Candidarugosa (CRL), type VII (MW 57 kDa) were purchased from Sigma-Aldrich (St. Louis MO). Bradford Reagent and bovine serum albumin(BSA), chloroform, dimethylformamide and other solvents, analyticalgrade, were also purchased from Sigma. Na2HPO4 and KH2PO4 werepurchased from Carlo Erba Analyticals (Milano, Italy). All thechemicals were used without further treatment. Water used in allexperiments and cleaning procedures was purified using a Milli-Qsystem (Billerica, MA), to a specific resistance of 18 MΩ cm−1.Phosphate buffered saline (PBS, pH 7.6, I 0.1)was used. Protein sampleswere centrifuged at 14000 rpm for 3 min at 4 °C. Following thecentrifugation, a UV/Visible spectrophotometer Pharmacia BiotechUltrospec 4000 (Stockholm, SE) was used to determine the proteinconcentration by the Bradford method using bovine serum albumin(BSA) as a standard.

2.2. Methods

2.2.1. Thermodynamic measurements on Langmuir filmsSurface measurements were carried out using a thermostated KSV

Langmuir Mini-trough system (KSV LTD, Finland), placed on an anti-

vibration table and enclosed in a Plexiglas box to avoid impurities anddust deposition. In this system, compression is achieved with thesymmetric movement of two opposing barriers. In all experiments thecompression rate was 20 cm2/min. A rectangular trough with a totalarea of 246.4 cm2 was used. Trough and barriers were thoroughlycleanedbefore eachmeasurementwith appropriate solvents, and rinsedwith ultrapure water. Prior to film deposition, the surface was cleanedrepeatedly by slowly sweeping the barriers and vacuum aspirating thesurface in between, until no change in surface pressure was detectablecomparing the closed and open positions. Due to the rather small effectof the presence of PDLLA on the surface pressure, the cleaning processwas a critical step of the measurement procedure.

PDLLA was dissolved in chloroform. A known amount of thesolution was carefully spread with a micro-syringe onto the clean air–water interface. All the measurements were performed at 25±0.2 °C.The solvent was allowed to evaporate for about 10 min before startingthe compression. The desired subphase temperaturewas controlled by awater circulatingbath (C25,Haake,Karlsruhe,Germany). Surfacepressuremeasurements were carried out by the Wilhelmy method, using aroughened platinumplate, to an accuracy of 1 mN/m. Due to unavoidableexperimental uncertainties, the maximum difference between every tworepeated curves is contained in a band of ~±2.5 mN/m on the average.

Surface potential measurements were performed using thenoncontact vibrating plate capacitor method, originally introducedby Kelvin and improved by Yamins and Zisman [32,33]. We used acomputer-controlled device (SPOT1, KSV LTD, Finland), with a 17 mmdiameter active electrode, placed at less than 3 mm above the air–water interface, and a stainless steel reference electrode immersed inthe subphase [34].

Surface pressure and surface potential were measured simulta-neously during film compression; at the beginning of each experiment,the surface potential of the aqueous phasewasmeasured, and this valuewas assumed as a reference. Before starting the compression, thesolvent was allowed to evaporate for about 10 min for a goodstabilization of the initial potential value. The probe parameter settingwas adjusted in order to reduce the stray capacitance effect, due to thesmall variation of the electrode distance from the interface in differentmeasurements, to a negligible extent. The reproducibility of surfacepotential measurements was within 5 mV.

The interaction of CRL with PDLLA films was studied by comparingthe thermodynamic behavior of PDLLA films spread on pure PBSsubphase and on CRL-containing subphase. In a different series ofexperiments, changes in the surface parameters of PDLLA induced bythe CRL were investigated by monitoring the effect of injectingdifferent concentrations of the protein beneath the compressed filmsat different target pressures of compression (5, 10, and 15 mN/m).More in detail, a fixed volume of CRL buffered solution (at theconcentration required to obtain the final desired one in the wholesubphase volume) was carefully injected in the subphase by a syringeending in a thin Teflon tube placed in the subphase well below thefilm, thus avoiding any perturbation of the film [28]. The increase insurface pressure upon CRL injection wasmonitored until it reached anequilibrium value. All experiments were repeated at least three times.

2.2.2. Adsorption on PDLLA-nanoparticlesPDLLA nanoparticles were prepared by using a recently patented

methodology [35]. In brief, the commercially available polymer wasdissolved in dimethylformamide. The obtained solution (5 mg/ml) wasthen transferred in a dialysis bag and dialyzed against water, which is anon-solvent for the polymer (solvent/non solvent ratio 1:20). After5 days at 4 °C the precipitate was recovered, rinsed several times inwater, centrifuged and freeze-dried. The size and morphology of theobtained nanoparticles (average diameter 220±5 nm) were charac-terized by scanning electronmicroscopy and dynamic light scattering asdescribed elsewhere [24]. Adsorption experiments of CRL onto PDLLAnanoparticles were performed in Pyrex tubes containing a known

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amount of the particles dispersed in 2 ml of a phosphate buffer solution(PBS 0.1 M, pH 7.6) of lipase (50 mg/ml), under magnetic stirring(600 rpm) and at controlled temperature. After following the timecourse of the adsorption for 6 h, the samples were filtered throughcellulose nitrate filters (Whatman, pore diameter 0.025 μm) to recoverthe particles. The amount of immobilized enzyme was obtained bystandard Bradford assay of the initial lipase solutions, the supernatants,and the washing solutions after immobilization. Protein concentrationwas determined spectrophotometrically by measuring the adsorptionpeak at 595 nm.

3. Results and discussion

3.1. PDLLA Langmuir films

Typical surface pressure, π, vs. meanmolecular area, A, isotherms ofPDLLA monolayers at air–water interface are shown in Fig. 1. PDLLAfilms were deposited on pure PBS subphase (solid line) and on CRL-containingsubphase (at a concentration of 0.12 mg/ml, dashed line). Forcomparison, the typical “isotherm” obtained by simply sweeping thebarriers on a subphase containing CRL (at a concentration of0.12 mg/ml), but without spreading any film on its surface, is alsoshown (dotted line). The presence of a variable amount of protein at theinterface that adsorbs from the bulk is clearly apparent. The isotherm ofPDLLA on pure PBS subphase is qualitatively similar to those reported inliterature for PLLA and PDLA homopolymers [7,36,37]. However, thesmoothed step that is observed for PLLA and PDLA in the meanmolecular area (Mma) range between 20 and 30 Å2/molecule, resem-bling the feature that in fatty acids monolayers would be typical of themain phase transition, is absent here. It should be noted that the areahere refers to the lactic acidmonomers. On the contrary, the breakdown(where themonolayer breaks down and its fragments slide one over theother beginning to form amultilayer) appears to occur at similar valuesof area per molecule and surface pressure (~18 Å2/molecule and~10 mN/m; 17–18 Å2/molecule and same pressure for PLLA [7]).Increasing compression beyond this point, pressure of the PDLLA filmincreases rather gradually and no plateau is observed, again as in thehomopolymers isotherms.

By using atomic force microscopy techniques, the main-transition-like step that appears in the isotherms of lactide homopolymers hasbeen recently shown to be due to a genuine disorder/order or liquidexpanded-to-condensed (LE/LC) phase transition [38], possiblyfavored by the helix conformation that the homopolymers tend toassume at the interface under the effect of compression [36]. Both the

Fig. 1. π-A isotherms of PDLLA deposited on a pure PBS subphase (solid line) and on aCRL-containing subphase (dashed line). The isotherm obtained by compressing theinterface of thepure CRL subphase,without anyfilmdeposition is also shown (dotted line).

absence of an evident first-order transition in the low pressurezone, and the continuous rise of the pressure beyond the breakdown(observed here for the PDLLA copolymer) are consistent withthose findings. Actually, the intrinsic disorder that characterizes thecopolymer, where stretches of all-D or all-lthat can organize in helices,are separated by coiled regions where the D and l monomers are atrandom, reflects in a much lower ordering of the monolayer and agreater plasticity of the whole structure.

The presence of CRL in the subphase (Fig. 1; dashed line) notablyincreases disorder and plasticity of the PDLLA film. Consequently, eventhe breakdown is completely absent. In addition, this isotherm,compared to that of the PDLLA film spread on pure PBS, is characterizedby higher values of pressure in the whole compression range. Thisfinding suggests that, upon compression, the protein that initially is atthe air–water interface is not completely squeezed out into thesubphase, but at least partially remains at the interface, even at thehighest compression, forming a CRL-PDLLA mixed film.

That the protein itself is surface active and tends to stay at the air–water interface, is apparent from the isotherm obtained by justsweeping the barriers over the pure CRL subphase without depositingany PDLLA film on the surface (Fig. 1; dotted line), where a notable riseof the pressure is observed upon compression. In this case, CRLmolecules were not deposited on the surface but spontaneouslyadsorbed at the interface from the bulk subphase (Gibbs isotherm).Since the number of adsorbed molecules is not known and varies inprinciple upon compression, the variable shown on the horizontal axisshould simply be the area between the barriers. However, in order toplot the curve for the pure CRL-isotherm, and to make a comparisonwith the PDLLA isothermspossible,we chose to give this area in termsofafictitious “area permonomer”, as if the sameamount of PDLLAused forthe other isotherms in the figure had been deposited at the interface.

Another and more subtle effect suggests a rather strong affinity ofCRL for the PDLLA film. As usual, the measured pressure is set to zeroafter cleaning the surface and immediately before film deposition.Withthis procedure, on a pure PBS subphase, when the proper amount ofPDLLA is spread, andwith the barrier in the initial position, the pressureremains close to zero (Fig. 1; solid line). On the contrary, when the sameamount of PDLLA, after having zeroed the pressure, is spread on a CRL-containing subphase, a vertical (i.e. with motionless barriers) rapidincrease of the pressure is observed. Such a sudden increase is clearlydue to the interaction of PDLLAwith CRL at the interface, and possibly tothe recruitment of further CRL molecules from the bulk to the interfacedue to this interaction.

Inorder to gain qualitative informationon themechanical propertiesof the PDLLA-CRL mixed film we calculated the static elasticity, ε=−A(∂π/∂A)T, from the isotherms shown in Fig. 1.WhenPDLLA is spreadon apure PBS subphase (Fig. 2; solid line), ε increases steadily as the film iscompressed. Initially, the polymer segments do not overlap and themolecular arrangements of the polymer, due to its strongly hydrophiliccharacter, lie mostly in the subphase [38]. However, upon compression,the hydrophobic methyl groups direct themselves to the air/waterinterface producing a progressivelymore closely packedmonolayer of acohesive structure that finally breaks down at 18 Å2/molecule. Again,there is no trace here for the net LE/LC phase transition which is clearlyvisible in the homopolymer isotherms, confirming the intrinsic disorderof the film formed by the DL copolymer. Intrinsic disorder and plasticityis also pointed out by the small, but significantly different from zero andgradually increasing value of the elasticity after the breakdown, asexpected when the structure does not collapse uniformly. As thecompression continues, ε increases rapidly, which corresponds to theformation of multilayers.

The rather sharp peak shown by the isotherm in the low area regioncorresponding to pure CRL (Fig. 2; dotted line) could be indicative of anorder–disorder transition or of an experimental artifact, however thedetailed analysis of the behavior of Gibbs isotherms of pure CRL wasbeyond the scope of the presentwork andwedid not investigate further

Fig. 2. ε-A isotherms of PDLLA deposited on a pure PBS subphase (solid line) and on aCRL-containing subphase (dashed line). Also shown the isotherm of pure CRL subphase(dotted line).

Fig. 3. Surface potential ΔV (a) and dipole moment μn (b) as a function of the meanmolecular area of PDLLA deposited on a pure PBS subphase (solid line) and on aCRL-containing subphase (dashed line) and also for the pure CRL subphase (dottedlines). The arrows point to the scale quantifying each curve.

2020 G. Kamel et al. / Surface Science 605 (2011) 2017–2024

this effect. What concerns here is that after a first steady rise of theelasticity (suggesting a cohesive structure) followed by an abruptdecrease (suggesting the collapse of that structure), the static elasticityremains stable, suggesting that in this whole compression range theprotein is only partially squeezed out of the interface into the bulksubphase.

Examining the elasticity pattern of the PDLLA-CRL mixed film(Fig. 2; dashed line), it is clear that it behaves in a much different waythan pure PDLLA or CRL films. The mixed film seems to exhibit newcharacteristics owing to the mutual interactions between materials.After a sharp peak similar but higher than that observed in the CRLisotherm, and probably sharing the same origin (i.e., the coordinationof protein molecules), now, favored by the polymer presence, anextended region is observed where static elasticity remains stable.This region is followed by a rapid increase of the elasticity and finallyby a collapse. This behavior is consistent with the hypothesis of amixed film that is compressible down to ~10 Å2/molecule (of PDLLA),where the structure becomes much more cohesive. Notably, whenPDLLA is deposited on a pure PBS subphase, at this same value ofarea per molecule, the formation of multilayer begins (Fig. 1). Thetransition zone around 15 Å2/molecule and 10 Å2/molecule, wherethe slope changes more gradually, probably corresponds to theprotein being partially expelled from the interface.

The above described scenario, characterized by a rather strongaffinity of CRL for the PDLLA film, was supported by surface potentialmeasurements. Surface potential (ΔV) and dipolemoment (μn) curvesof PDLLA monolayers are shown in Fig. 3. The measured surfacepotential, ΔV, is defined as the difference between the measuredpotential of monolayer-covered and monolayer-free subphase, whichwas taken as reference. This potential can be related to the normalcomponent of the dipole moment μn of themolecules forming the filmthrough the Helmholtz equation:

ΔV = μn = Aε0 εrð Þ + φ0 ð1Þ

where εr and ε0 are the “effective” dielectric constant within the layerand the permittivity of free space, respectively; μn is the normalcomponent of themolecular dipole moment; A is the area occupied byeach molecule, or monomer; and φ0 is the double-layer contribution,that can be neglected since CRL, the only charged species, is onlyweakly charged at the pH of the measurement (pH 7.6).

The dipole moment μn results from the contribution of differentcomponents: the polar groups of adsorbed molecules bathed by thesurface; the reorientation of the few layers of water moleculesinduced by the presence of the film; the hydrophobic part of adsorbed

moleculeswhich extend from the surface to the air. However, a simplesurface potential measurement cannot discriminate between thesedifferent contributions. The quantity shown in Fig. 3, ΔVA (Eq. (1)),determined experimentally, should hence be regarded as an overalleffective dipole that takes into account all these effects. Nevertheless,from the comparison of the curves in Fig. 3, (panel b), interestinginformation can be obtained.

As far as PDLLA on pure PBS (solid line) is concerned, during thecompression that precedes the collapse at approximately 18 Å2/molecule,the potential ΔV increases steadily. However, in the same area range,the dipole moment per monomer μn is almost constant; suggesting thatthe potential increase may be just due to the increasing density of thepolymer on the surface when the area is reduced. Nonetheless, thedensity increase does not affect the orientation or the ordering ofmolecules.

After the collapse, the effective dipole per molecule decreases withits value almost halved (from ~0.34 to ~0.13V Å2/molecule), consis-tently with the formation of a double layer. The final slight decrease ofthe dipole moment is probably due to a change in the arrangement ofthe superimposed layers induced by compression.

2021G. Kamel et al. / Surface Science 605 (2011) 2017–2024

Dipole moment behavior of PDLLA on a CRL-containing subphaseis shown in Fig. 3 (panel b, dashed line). In fact, after an extendedrange of area variation where the dipole remains almost constant(from ~20 down to ~11 Å2/molecule), it decreases upon furthercompression. The decrease of the dipole moment per moleculewhich is observed upon compression, above ~15 Å2/molecule and~12 Å2/molecule for “pure CRL” and “mixed film”, respectively, afteran extended zonewhere its value remains constant, may be attributedto a decrease of the density of molecules at the surface. However, itcan be also due to a conformational/positional rearrangement thatmodifies the dipole moment or its vertical component. This decreaseof the effective dipole values occurs, in the presence of PDLLA, at evenlower values of the surface area (although, due to the presence of thepolymer, the surface area available to the protein is smaller) and iscomparatively reduced, suggesting, again, the existence of attractiveinteractions between the PDLLA and the protein, that stabilize themixed film and reduce the expulsion of the protein from the interface.

3.2. Injection of the protein into the subphase

Fig. 4 shows the CRL injection into the PBS subphase beneath thestabilized PDLLA films compressed to a target pressure. The increaseof the film pressure, Δπ, is measured upon CRL injection at the chosentarget pressure of the PDLLA film after its relaxation.

In Fig. 5, the increase of film pressure, Δπ, measured after CRLinjection, is shown for three different target pressures of the PDLLA film(5, 10, and 15mN/m), as a function of the nominal concentration ofprotein in the subphase, i.e. the concentration that would be reached inthe subphase if theproteinwashomogeneously distributed in thewholevolume. The reproducibility in themeasuredΔπ (Figs. 4 and 5) is better

a

bT=25OC

T=25OC

Fig. 4. CRL injection into the subphase beneath the PDLLA film compressed at 25 °C to atarget pressure (a) 5 mN/m, (b) 15 mN/m. Surface pressure increment, Δπ, induced byCRL injection is shown in dotted line.

than ±0.5 mN/m away from the “coexistence zone” (above or below~10mN/m) and rather erratic in this zone (~10 mN/m, filled circles inFig. 5). Represented points of this erratic behavior at ~10 mN/mare onlyan example of a series of measurements. Other series measured at thesamezoneare completely different yet still erratic. That is, themeasuredΔπ is non-reproducible and the uncertainties are very large that anyfurther analysis is meaningless. Notably, while for π=5 and 15 mN/m,Δπ increases smoothly towards a plateau with increasing proteinconcentration, the observed behavior for π=10mN/m is erratic andpoorly reproducible. This behavior is consistent with the fact that at thispressure (corresponding to the plateau in the isotherm: see Fig. 1, solidline) the PDLLAmonolayer has already collapsed but a complete bilayerhas not formed yet, so that the interface is now an inhomogeneousmixture of monolayer and multilayer fragments that slowly reorganizeunder the combined effect of pressure and interactionswith the protein.Inotherwords, theprotein adsorbs “regularly” causing a larger variationof the pressure of the film when this is a monolayer, than when it is abilayer, however this is probably due only to the greater rigidity (seeFig. 2) of the bilayer rather than to a different interaction of the proteinwith thefilm surface.When thefilm is at the collapse, thefilm instabilitycannot be decoupled from the effect of the adsorption, and the pressurevariation is apparently erratic. A final comment is in order. In all theexperiments at 15 mN/m, after reaching the target pressure, a largevariation of the measured pressure is observed. This relaxation isprobably due to the rearrangement of the film fragments after thecollapse, as suggested also by the large hysteresis that appearswhen thefilms are compressed above the collapse and decompressed again (datanot shown, but see [36]). The presence of such a large relaxation couldraise doubts about the pressure that has to be considered, if the targetoneor thepressuremeasured at the injection.However,while the targetpressure characterizes the physical state that has been reached by thefilm upon compression (at 15 mN/m the formation of an at least partialbilayer), by measuring the pressure during the relaxation one onlymonitors the mechanical rearrangement of the different zones (monoand bilayers), to be sure that the protein is injectedwhen the pressure isstabilized, but this pressure is not indicative of the physical state of thefilm fragments, that is assumed to remain substantially unaltered afterthe compression.

When the layers at the surface are homogeneous, a regularincrease of Δπ with CRL concentration is observed, and both data setat π=5 and 15 mN/m fit reasonably with the expression:

Δπ = A 1� e�C =C0� �

ð2Þ

where C is CRL concentration and A and C0 are constants.

Fig. 5. Pressure increase of the PDLLA film vs protein concentration in the subphasemeasured after CRL injection, at different initial pressures of the PDLLA film: (■) 5 mN/m;(●) 10 mN/m; (▲) 15 mN/m.

Fig. 6. Linear fitting of the pressure increase the of PDLLA films vs protein concentrationupon CRL injection in the subphase, at different initial pressures of the PDLLA film:π=5mN/m (■) and π=15 mN/m (▲).

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Assuming, as a first approximation, that the surface pressureincrease is proportional to the number of lipase molecules that adsorbon the PDLLA film, the qualitative exponential behavior of thecurves shown in Fig. 5, well satisfied for target surface pressures 5and 15 mN/m, suggests that the adsorption could be described asLangmuir-type, following the equation:

LE =LM

CC� e−ΔHads =KBT

1 + CC� e−ΔHads =KBT

ð3Þ

Here, LE is the mass of substance adsorbed per unit area of theadsorbing surface when C is its concentration in the bulk solution, andLM the mass of substance required to saturate the adsorption sites ofthe unit surface area. ΔHads=Ea−Ed(EabEd) is the heat of adsorption,i.e., the difference between the energies needed for adsorption, Ea, anddesorption Ed, C* is a constant with dimensions of a concentration,and finally, KB is Boltzman constant (1.380×10−23 J/K). Within thismodel, the adsorption rate is simply written as the product betweenthe rate at which the adsorbing molecules collide with the surface(assumed proportional to their concentration in the bathing solution)and the probability (1-LE/LM) of striking a vacant site, with anactivation term e−Ea/K

BT. Similarly, the desorption rate is given by the

product between the already covered surface fraction (LE/LM), and thecorresponding activation term e−Ed/K

BT. At equilibrium the two rates

must be equal. By simple algebra, Eq. (3) can be put in a linear form

Fig. 7. Adsorption isotherms of CRL on PDLLA nanoparticles at different temperatures: (●) 10

that allows an easier check of the hypothesis that this model fits theexperimental data:

CLE

=C�

LMeΔHads =KBT +

CLM

ð4Þ

Assuming again the proportionality between Δπ and LE, a plot ofC/Δπ vs. C should give a straight line of slope 1/Δπ* and interceptC�Δπ� eΔHads =KBT , where 1/Δπ* is the pressure increment reached for themaximum lipase adsorption. Actually, both data set, plotted in thisway (Fig. 6), show a well-defined linear behavior so the hypothesis ofa Langmuir-type adsorption of CRL on PDLLA seems to be confirmed,where forπ=5and 15mN/m, the slopes are 0.073±0.002 and 0.089±0.003 m/mN, and intercepts are (7.3±0.7)×10−4 and (9.9±1.5)×10−4 mg m/ml mN, respectively (the regression coefficients R,are 0.997 and 0.998).

From the slopes, the maximum pressure increments, Δπ*, can becalculated, which would be expected when the PDLLA films werecompletely “saturated” by the adsorbed lipase. At the two targetpressures, 5 and 15 mN/m, these increments are 14.3 and 11.1 mN/m,respectively. The smaller value of the increment corresponding to thehigher target pressure could indicate both that the protein interactdifferentlywith amonolayer (π=5mN/m)or a bilayer (π=15mN/m),or that the multilayered structure, being more “resistant” and compact,is less prone to the “expansion” induced by the protein adsorption.

Interestingly, the products of the inverse slope and the intercept,that is in terms of Eq. (4), the quantities C*eΔHads/KBT, (0.0104 and0.0109 mg/ml for π=5 and 15 mN/m, respectively) are coincidentwithin the errors (~4.5%) for the two target pressures. This should beexpected for a Langmuir-type adsorption, since within this model boththe heat of adsorption,ΔHads, and the parameter C*, depend only on thelocal interaction of the adsorbing substancewith the surface, and hence,in our case, are expected to be independent from the mono- ormultilayered structure of the film. This simple qualitative finding seemsto exclude that lipase interaction with a monolayer or a multilayershould be different.

Such a qualitative result, the identification of the model, is aprerequisite to use the model for quantitative predictions (for example,the adsorption enthalpy). However, due to the unknown proportionalityfactor between themeasured pressure incrementΔπ and themass of theenzyme adsorbed per unit area, LE amore, quantitative analysis does notappear feasible. Conversely, such quantitative results can be obtained byusing a slightly different system. In fact, as reported in the followingsection, the thermodynamic study of the adsorption of CRL on PDLLAnano-particles, analyzed in terms of Langmuir adsorption isotherms,

°C; (▪) 20 °C; (▲) 25 °C; (▼) 30 °C; (◂) 40 °C and (▸) 50 °C. (Uncertainties within ~5%).

2023G. Kamel et al. / Surface Science 605 (2011) 2017–2024

results in a quantitative determination of the surface coverage of theparticle by the protein and of the adsorption energy.

Fig. 9. Logarithm of the (intercept/slope) ratio as a function of the inverse absolutetemperature.

3.3. CRL adsorption on PDLLA-nanoparticles

In order to gain quantitative data on CRL adsorption on PDLLAsurfaces and to further explore the hypothesis of a Langmuir-typeadsorption, we studied the adsorption of CRL on PDLLA nanoparticleswith a diameter of 220±5 nm suspended in an aqueous solution(PBS, pH=7.6) at different temperatures (Fig. 7). In Fig. 8, the iso-therms are shown in their linearized form.

All curves have a similar profile which corresponds well to theLangmuir-type isotherm. In the low protein concentration range,protein loading increases by increasing concentration for all theinvestigated temperatures. At higher temperature, protein loadinggrows faster with concentration.

Under the premises of Eq. (4) and based on the linear fit parametersshown in Fig. 8, we estimated that the mass of protein required for thecomplete coverage of PDLLA surface (LM) is ~66.1 mg per 100 mg ofPDLLA, taking the average of LM values at the investigated temperatures(in agreement with the Langmuir model assumptions LM is largelyindependent of the temperature, see inset of Fig. 8).

The average diameter of PDLLA nanoparticles as obtained from DLSmeasurements is 220±5 nm intensity average, (150±5 nm, if numberaverage is used). Assuming for the density of PDLLA nanoparticles thevalue of 1.26 g/cm3 [39] and the value of 57.780 Da for the molecularweight of the protein, an average area per protein molecule in theadsorbed layer is calculated to be of ≈3.14 nm2 (4.6 nm2 using thenumber averagevalueof the radius)withanerror of 15%. This qualitativeestimate appears reasonable since it compares very well with a“molecular diameter” of 5.2 nm of the CRL that can be deduced fromits X-ray structure, suggesting a rather tight packing of the protein in theadsorbed layer [40].

As shown in Fig. 9, and according to Eq. (4), the heat of adsorptionΔHads, can be calculated from the equation: ln(a/b)=ΔHads/KB.1/T+lnc* by plotting the natural logarithm of the (intercept/slope) ratio, ln(a/b), as a function of the inverse absolute temperature. Linear fitshown in Fig. 9, gives ΔHads=(2±1)103KB. This energy correspondsapproximately to 5–6KBT at room temperature. This value appearsreasonable, since the adsorption of a large protein such as CRLprobably involves many contact points, each with interaction energyof a fraction of KBT. Conversely, the total energy is built up by all thecontacts and results in several KBT. Based on this, CRL adsorption onPDLLA nanoparticles is practically irreversible.

Fig. 8. Linear fitting of the adsorption isotherms of CRL on PDLLA nanoparticles at differen(Uncertainties within ~5%). LM dependence on temperature, T, is shown in the inset.

To better understand the interactions between CRL and PDLLA NPs,the kinetics of the adsorption phenomenon was investigated. Byincreasing the time of contact, the enzyme loading increasesaccordingly, until a plateau is reached after approximately 4.5 h,which resulted to be the optimal contact time. Desorption studies onCRL-PDLLA nanobioconjugates proved a good stability of the immo-bilization in PBS, in which, after a 24 h suspension, the amount ofdesorbed protein was approximately 10% (data not shown, see [24]).

4. Conclusions

Advances in biotechnology have made proteins more and moreavailable, including variants genetically modified for specific tasks, thushugely expanding their practical use in the conversion of chemicals andmaterials. Several proteins have been shown to be able to self-assembleupon adsorption on polymeric surfaces, thus opening the possibility ofbioactive surface patterning at the molecular level, particularlyattractive for applications that include novel diagnostic or therapeuticagents on the single cell scale. The influence of CRL presence on thethermodynamic behavior of PDLLA films deposited at air–waterinterface was investigated providing qualitative information on themolecular interactions between the protein and the polymer films. Byusing the Langmuir model, we showed that the adsorbed lipase forms arather compact layer on PDLLA surface, and obtained an estimate of theaverage area occupied in this layer by a single protein, that compares

t temperatures: (●) 10 °C; (▪) 20 °C; (▲) 25 °C; (▼) 30 °C; (◂) 40 °C and (▸) 50 °C.

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reasonably with X-ray structural data reported for CRL in literature. Anestimate of the heat of adsorption was also obtained.

Acknowledgments

P. V. Verdes acknowledges financial support from Ministerio deCiencia e Innovación, Programa Nacional de Movilidad de RecursosHumanos del Plan Nacional de I-D+i 2008–2011.

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