Dynamic Aspects of Protein Adsorption onto Titanium Surfaces

France, Faculte´ de Chimie, ULP Strasbourg, 1 Rue Blaise Pascal, 67008 ... Recherches sur les Macromole´cules, Institut Charles Sadron, CNRS-ULP, 6 ...
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Dynamic Aspects of Protein Adsorption onto Titanium Surfaces: Mechanism of Desorption into Buffer and Release in the Presence of Proteins in the Bulk V. Ball,†,‡ A. Bentaleb,† J. Hemmerle,† J.-C. Voegel,*,† and P. Schaaf §,| INSERM U-424, Centre de Recherches Odontologiques, 1 Place de l’Hoˆ pital, 67000 Strasbourg, France, Faculte´ de Chimie, ULP Strasbourg, 1 Rue Blaise Pascal, 67008 Strasbourg, France, Centre de Recherches sur les Macromole´ cules, Institut Charles Sadron, CNRS-ULP, 6 Rue Boussingault, 67083 Strasbourg Ce´ dex, France, and Ecole Europe´ enne des Hautes Etudes des Industries Chimiques de Strasbourg, 1 Rue Blaise Pascal, BP 296F, 67008 Strasbourg Ce´ dex, France Received September 5, 1995. In Final Form: December 4, 1995X The release process for adsorbed IgG molecules on titanium particle surfaces in the presence of pure buffer or IgG molecules in solution was investigated by means of radiolabeling techniques. This study is part of a general investigation of the basic mechanisms underlying exchange processes of adsorbed proteins by macromolecules in the bulk and in particular of the kinetic laws describing such processes. This study shows the existence, in the investigated time scale (from 1 to 43 h), of two different populations of adsorbed IgG molecules: a population called type I which is releasable from the surface and a type II population which is irreversibly adsorbed on the surface. As in previous studies, we confirm that for the population of type I, the kinetics of the release mechanism is enhanced by the presence of IgG molecules in solution. This is a strong indication that it constitutes an exchange process. Moreover, we show that this release process is of order 1 with respect to both adsorbed and bulk molecules. Finally, we show that the percentage of molecules of type I (reversibly adsorbed on the surface at the adsorption time scale investigated) seems to increase linearly with the bulk concentration of proteins during the release experiments, in the investigated bulk concentration range. This unexpected result constitutes a new observation and we do not posses a satisfactory explanation for it. A similar increase was also reported for exchange experiments performed with polyacrylamide onto aluminosilicated glass beads by Pefferkorn et al. (Pefferkorn, E.; Carroy, A.; Varoqui, R. J. Polym. Sci., Polym. Phys. Ed. 1985, 23, 1997).

Introduction Adsorption of macromolecules on solid surfaces shows specific properties due to the large number of links that these molecules can establish with the surface: partial irreversibility; slow change of the conformations of the adsorbed macromolecules;1,2 release of the adsorbed molecules through exchange reactions.3,4 Despite the importance of this exchange phenomenon in the behavior of a layer of adsorbed macromolecules, very little is known about the mechanism from a microscopic and kinetic point of view. It has been demonstrated that if a surface is brought into contact with plasma (or a mixture of different macromolecules), small molecules are the first to reach the surface and adsorb on it. Due to their smaller diffusion coefficient, molecules of larger molecular weight reach the surface at a later stage. However these molecules usually have a higher affinity for the surface and thus gradually replace the adsorbed molecules of smaller molecular weight on the surface. This is known as the * Author to whom correspondence should be addressed: e-mail [email protected]. † INSERM U-424, Centre de Recherches Odontologiques. ‡ Faculte ´ de Chimie, ULP Strasbourg. § Centre de Recherches sur les Macromole ´ cules, Institut Charles Sadron. | Ecole Europe ´ enne des Hautes Etudes des Industries Chimiques de Strasbourg. X Abstract published in Advance ACS Abstracts, March 1, 1996. (1) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87. (2) Soderquist, M. E.; Walton, A. G. J. Colloid Interface Sci. 1980, 75, 386. (3) Jennissen, H. P. Adv. Enzyme Reg. 1981, 19, 377. (4) Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers. Vol. 2 Protein Adsorption; Plenum Publishing Co.: New York, 1985; pp 1-85.

Vroman effect in the case of biopolymers.5-7 The replacement of the molecules from the surface is usually referred to as an exchange process. Exchange processes of both biopolymers and synthetic polymers adsorbed on surfaces by macromolecules from the solution have long been demonstrated.8-18 More precisely, it has been shown that if a surface covered by adsorbed macromolecules is brought into contact with a pure solvent, little or no desorption is detected. For proteins, this is particularly true when they are adsorbed on hydrophobic surfaces.19-21 On the other hand, if a coated surface is brought in contact with a solution of macromolecules, molecules from the solution further (5) Vroman, L.; Adams, A. L. J. Biomed. Mater. Res. 1969, 3, 43. (6) Wojciekowsky, P.; Ten Hove, P.; Brash, J. L. J. Colloid Interface Sci. 1986, 111, 455. (7) Wojciekowsky, P.; Brash, J. L. J. Biomater. Sci. Polym. Ed. 1991, 2, 203. (8) Brash, J. L.; Uniyal. S.; Samak, Q. Trans.sAm. Soc. Artif. Intern. Organs 1974, 20, 69. (9) Brash, J. L.; Samak, Q. M. J. Colloid Interface Sci. 1978, 65, 495. (10) Brash, J. L.; Uniyal, S.; Pusineri, C.; Schmitt, A. J. Colloid Interface Sci. 1983, 95, 28. (11) Voegel, J. C.; De Baillou, N.; Schmitt, A. Colloids Surf. 1985, 16, 289. (12) Pefferkorn, E.; Carroy, A.; Varoqui, R. J. Polym. Sci., Polym. Phys. Ed. 1985, 23, 1997. (13) Jennissen, H. P. J. Colloid Interface Sci. 1986, 111, 570. (14) Elwing, H.; Askendal, A.; Lundstro¨m, I. Prog. Colloid Polym. Sci. 1987, 74, 103. (15) Mura, M. J.; Behr, S.; Voegel, J. C. J. Biomed. Mater. Res. 1989, 23, 1411. (16) Schmidt, C. F.; Zimmermann, R. M.; Gaub, H. E. Biophys. J. 1990, 57, 577. (17) Lundstro¨m, I.; Elwing, H. J. Colloid Interface Sci. 1990, 136, 68. (18) Feng, L.; Andrade, J. D. Biomaterials 1994, 15, 323. (19) Mac Ritchie, F. J. Colloid Interface Sci. 1972, 38, 484. (20) Lee, S. H.; Ruckenstein, E. J. Colloid Interface Sci. 1988, 125, 365. (21) Malmsten, M.; Lassen, B. J. Colloid Interface Sci. 1994, 166, 490.

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adsorb on the layer by displacing preadsorbed species. This phenomenon is called an exchange reaction. Very little is known about the molecular aspects and the mechanism of this process which makes macromolecular adsorption different from the adsorption process of small molecules. On the basis of experiments of Pefferkorn et al.12 who studied the homogeneous exchange process of low polydispersity polyacrylamide adsorbed onto aluminosilicated glass beads, the suggestion has been made that the exchange reaction could be modeled by a firstorder kinetic law with respect to both molecules in solution and adsorbed molecules. It has been proposed that this rate equation is related to a rate-limiting step due to the diffusion of macromolecules from the bulk though a repulsive barrier formed by the preadsorbed species.22 Such a simple law implies that only one characteristic time governs the whole exchange process. However, in some cases the observed kinetics are more difficult to analyze since a whole spectrum of characteristic time scales is necessary to describe the behavior.23 Moreover Granick’s group noted that in decreasing the temperature of the medium, the process behaves in a nonexponential way and the characteristic time scale of the exchange process followed a power law with the molecular mass of the preadsorbed polymer24,25 indicating a diffusion-limited process. In the case of proteins, we have found that the same simple first-order rate equation used by Pefferkorn et al.12 was able to describe the homogeneous exchange reaction of human immmunoglobulins adsorbed onto sulfated latex particles26 as well as the heterogeneous exchange process whereby IgG molecules adsorbed on a hydrophilic silica surface were replaced by human fibrinogen molecules from solution.27 The present paper constitutes an aspect of a more general study of the basic mechanisms underlying the release processes of proteins adsorbed on solid surfaces and in particular the kinetic aspects of the exchange (turnover) process. While such an extensive study has been widely undertaken by Granick’s group for synthetic polymers,24,25 very few studies are reported for proteins. From two previous studies performed in our group, at ambiant temperature,26,27 the following seem to emerge: (i) Exchange processes of adsorbed proteins can be modeled, as in the case of synthetic polymers, by a chemical reaction of first order with respect to proteins in solution. (ii) This reaction seems also to be of order 1 with respect to the adsorbed molecules, but the arguments supporting this statement remain weak. (iii) In the study of the heterogeneous exchange process whereby IgG molecules adsorbed on a hydrophilic silica tube under laminar flow conditions were replaced by human fibrinogen molecules from the bulk, different kinds of adsorbed molecules had to be introduced: (a) molecules which can be exchanged or easily desorbed from the surface, (b) molecules which desorb slowly, and finally (c) molecules which are irreversibly adsorbed on the surface over a time scale of a few days. Even if oversimplified, this classification allowed a description of all the experimental data reported in the study of ref 27. (22) De Gennes, P. G. C. R. Acad. Sci., Se´ r. II 1985, 301, 1399. (23) Haouam., A. The`se de doctorat d'Universite´, Universite´ Louis Pasteur, Strasbourg, 1988. (24) Johnson, H. E.; Douglas, J. F.; Granick, S. Phys. Rev. Lett. 1993, 70, 3267. (25) Douglas, J. F.; Johnson,H. E.; Granick, S. Science 1993, 262, 2010. (26) Ball, V.; Huetz, Ph.; Elaissari, A.; Cazenave, J. P.; Voegel, J. C.; Schaaf, P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7330. (27) Huetz, Ph.; Ball, V.; Voegel, J. C.; Schaaf, P. Langmuir 1995, 11, 3145.

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The aim of the present work is to investigate the release process of adsorbed molecules from an almost pure titanium surface which constitutes, with its oxides, a widely used range of biomaterials.28 The adsorption properties of these surfaces are fairly well known,29-31 but the dynamic aspects of this adsorption have not yet been investigated to our knowledge. As a model protein, we used human immunoglobulins (IgG molecules) which play an important role in the immune response of the organism.32 Here, the kinetics of desorption and of release of the adsorbed molecules in presence of IgGs in the bulk will be analyzed. It will be determined whether the kinetic laws previously observed26,27 as well as the existence of different kinds of adsorbed molecules on the surface are still valid. Moreover, the knowledge of the dynamic aspects of the adsorption process, involving desorption and exchange phenomena, is necessary to understand the biocompatibility of such materials as titanium since they induce constant modifications in the adsorbed layer including the replacement of denaturated proteins by native ones. Materials and Methods Adsorbent. Experiments were performed with titanium particles (Weber Me´taux, Paris, France) stored under vacuum (10-3 mmHg) in the presence of CaCl2 in order to avoid the growth of oxide and/or hydration layer. The polydisperse particles were sieved, the fraction between 38 and 53 µm was washed twice in analytical grade ethanol and oven-dried for 2 h at 120 °C before use. Scanning electron micrographs (JEOL JSM 35C) showed that particles were irregularly shaped. The specific surface area, lower than 1 m2‚g-1, could not be estimated with the BET technique. A lower limit surface area of 360 cm2‚g-1 could be calculated by assuming the particles as perfect spheres having a diameter of 38 µm and a specific mass of 4.39 g‚cm-3. Small angle X-ray scattering data were collected using a Siemens Kristalloflex D500 diffractometer equipped with a Se monochromator working at 40 kV and using Co KR1 radiation (λ ) 0.17890 nm). The following JCPDS files were used for identification of the species present at the surface of the titanium particles: 44-1294 for pure titanium, 21-1276 for TiO2 rutile, and 21-1272 for TiO2 anatase. The diffraction pattern obtained was in good agreement with pure titanium (Figure 1a), although some TiO2 form should be present at the surface. But it could not be observed in the X-ray spectrum because of the large scatter in the data indicating the low cristallinity of the particles. This point was confirmed by applying a procedure to convert TiO2 from anatase to the rutile form.33 Diluted nitric acid (pH ) 1.0) was added to the powder while stiring. The powder was then washed with distilled water (Milli.R04 Millipore, Bedford, MA) until pH 5.5 was attained in the supernatant. The powder was then boiled in distilled water for 7 h and heated in air at 200 °C for 16 h. The diffraction pattern for the treated powder was virtually identical to the diagram of the untreated one (Figure 1b), confirming the absence of any detectable amount of titanium oxide. However it should be noted again that a small amount of titanium oxide must be present at the surface due to spontaneous oxidation in air even at very low pressures (ref 34 and references therein). Thus, the powder was stored under vacuum to avoid the growth of the passivating oxide layer which could significantly modify the surface properties of the particles with time. X-ray photoelectron spectroscopy analysis using the 1486.6 eV radiation of Al KR indicated traces of carbon, sodium and (28) Kasemo, B. J. Prosthet. Dent. 1983, 49, 832. (29) Ivarsson, B. A.; Hegg, P. O.; Lundsto¨m, I.; Jo¨nsson, U. Colloids Surf. 1985, 13, 169. (30) Ellingsen, J. E. Biomaterials 1991, 12, 593. (31) Walivaara, B.; Arronsson, B. O.; Rodahl, M.; Lausmaa, J.; Tengvall, P. Biomaterials 1994, 15, 827. (32) Roit, I. Immunologie; Pradel: Paris, 1990. (33) Browne, M.; Gregson, P. J. Biomaterials 1994, 15, 894. (34) Effah, E. A. B.; Bianco, P. D.; Ducheyne, P. J. Biomed. Mater. Res. 1995, 29, 73.

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Figure 1. Small angle X-ray scattering of untreated (a) and treated particles (b). See Materials and Methods for the experimental conditions. The solid vertical lines correspond to the simulated reflections for pure titanium. iron, as often observed for titanium.31 Contact angle measurements were realized by the deposition of a 10 µL water droplet onto ethanol and water cleaned titanium wafers (having the same diffraction pattern as titanium particles). The mean contact angle for the droplets (θ ) 10 ( 3°) was estimated from enlarged photographs, demonstration that the surface was hydrophilic. Proteins. Lyophilized polyclonal human immunoglobulins, denoted IgG, were kindly provided by the Centre de Transfusion Sanguine of Strasbourg (France). Purified according the method of McKinney,35 their purity was higher than 95% as controlled by SDS/Page with Coomassie blue staining. The IgGs were dissolved in PBS buffer using 50 mM NaH2PO4‚H2O and 50 mM Na2HPO4‚7H2O in the presence of 150 mM NaCl, the pH being adjusted to 7.50 (Mettler Electrode DG111-SC, Greifensee Switzerland) by mixing given volumes of both solutions. All chemicals were of analytical grade (Sigma Chemicals, St. Louis, MO). Buffer solutions were prepared with deionized water (Milli.RO4, Millipore, Bedford, MA). The buffer solutions were filtered through a membrane (Millex, Millipore, Bedford, MA) with pore size 0.22 µm, degassed, and stored at 4 °C under vacuum in a double-necked bottle until use. After dissolution, the IgG solution was again filtered through Millex membranes and the concentration determined by measuring absorbance at 280 nm using a spectrophotometer (Beckman DU 640, Fullerton CA). The extinction coefficient  was taken equal to 1.38 cm2‚mg-1.36 The solutions were then rapidly frozen in liquid nitrogen and stored at -70 °C for less than 1 month before use. A control experiment was performed using quasi-elastic light scattering with a protein solution at 2.8 × 10-2% (w/w) after centrifugation at 5000 rpm during 3 h. The light beam was produced by an Ar laser, λ ) 488 nm and 5 W (Spectra, Physics Stabilite 2016). A hydrodynamic radius of 5.4 nm was obtained by the determination of the autocorrelation function g2(t) and by using a cumulant method analysis.37 This hydrodynamic radius corresponds to a (35) McKinney, M. M.; Parkinson, A. J. Immunol. Methods 1987, 96, 271. (36) Schulze, H. E.; Heremans, J. F., Eds. In Molecular Biology of Human Proteins with Special Reference to Plasma Proteins; Elsevier: Amsterdam, 1966; pp 173-235. (37) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814.

Ball et al. diffusion coefficient of 4.0 × 10-7 cm2‚s-1 at 20 °C. This diffusion coefficient is in good agreement with the value obtained from ultracentrifugation measurements.38 Moreover, the polydispersity factor obtained with the cumulant method was rather low (of the order of 0.1, where a single exponential profile corresponds to a polydispersity equal to zero), which probably indicates that there are few IgM molecules (IgG pentamers32) in solution. Proteins were labeled according to a modification of the McFarlane method39,40 using iodine monochloride as oxydizing agent and Na125I for labeling (IMS30, Amersham France, Les Ulis, France). Free iodine not involved in the protein labeling process was eliminated with the aid of a column filled with an ionic exchange resin (AG1, Bio-Rad Lab, Hercules, CA). The conductivity (conductometer consort, K 610 Turnhout, B.2300 Belgium) of a 3.0 × 10-2% (w/w) IgG solution was similar to the conductivity of the pure buffer solution (20.3 ( 1.0) mS, indicating low contribution of the salts (essentially NaCl) from lyophilized proteins. Protein desorption and exchange experiments lasted generally over 40 h. It was thus necessary to check the stability of the bond between iodine and IgG molecules. For this, we filled a dialysis membrane, with a 3500 g/mol molecular weight cutoff (Spectrapore Medical Industries Inc., Los Angeles CA), with 20 mL of 1.5 × 10-2% (w/w) IgG solution and followed the increase of radioactivity in 300 ( 20 mL of PBS buffer. Less than 6% of the initial activity was found in the outer medium after 40 h. A comparable value was found previously for albumin labeled by the same technique.9 Adsorption Procedure. The adsorption experiments were performed in polycarbonate Eppendorf tubes (V ) 2 mL). The amounts of IgG molecules adsorbed by the walls of the tube, after 4 h of contact with a labeled IgG solution of 3.0 × 10-2% (w/w), was too small to be detected within the experimental precision. In all experiments the surface to volume ratio was maintained constant by adding 1.00 ( 0.02 g of titanium particles to 1.80 ( 0.02 g of solution containing various amounts of proteins. Both the particles and the solution were precisely weighed. During the adsorption experiments, lasting from 1 to 21 h at a temperature of 25 ( 2 °C, the tubes were gently rotated end over end at 33.5 ( 1.0 rpm. At the end of the adsorption, the rotation of the tube was stopped in order to allow the powder to settle, which was achieved in a few seconds due to the high density of the powder. Three precisely weighed samples of 200 µL were then taken and their activity was measured by γ counting (Minaxi γ, United Technologies Packard, Downers Grove, IL). This activity was compared to that of the initial added protein solution. This allowed a precise estimate of the amount of adsorbed proteins (depletion method). No important ionic releases from the titanium powder seemed to occur as verified by the constant conductivity values for the supernatant solution before and after protein adsorption. First of all, we checked the influence of radiolabeling on the adsorption properties of the proteins, as is suggested for each different protein and surface.41 Most of the labeled tyrosine residues are disubstituted42,43 with roughly one molecule in 1000 effectively labeled. We estimated the effect of the label on the adsorption properties of the IgGs using the method of Bale et al.44 and Schmitt et al.45 adsorbing various proportions of labeled immunoglobulins in solution at a given constant IgG concentration of (4.20 ( 0.06) × 10-2% (w/w) and contact time of 1.25 h. The sorbent consisted of untreated titanium particles. The amount of fixed labeled IgG molecules versus radioactive dilution (Figure 2) varied linearly and intercepts at the origin. We may thus assume that the labeled molecules possess the same adsorption properties as the unlabeled ones. In another series of experiments, we tried to establish the incubation time necessary to reach adsorption steady state for initial low and high bulk concentrations respectively of (6.4 ( 0.2) × 10-3% and (38) Cohn, E. J.; et al. J. Am. Chem. Soc. 1950, 72, 465. (39) McFarlane, A. S. Nature 1958, 182, 53. (40) McFarlane, A. S. Biochem. J. 1965, 62, 135. (41) Grant, W. H.; Smith, L. E.; Stromberg, R. R. J. Biomed. Mater. Res. Symp. 1977, 8, 33. (42) Li, C. H. J. Am. Chem. Soc. 1942, 64, 1147. (43) Li, C. H. J. Am. Chem. Soc. 1945, 67, 1065. (44) Bale, M. D.; Mosher, D. F.; Wolfarht, L.; Sutton, R. C. J. Colloid Interface Sci. 1988, 125, 516. (45) Schmitt, A.; Varoqui, R.; Uniyal, S.; Brash, J. L.; Pusineri, C. J. Colloid Interface Sci. 1983, 92, 25.

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Figure 2. Influence of the 125I label onto the adsorption process. Adsorption was performed for 1.25 h at a constant initial concentration of (4.20 ( 0.06) × 10-2% (w/w) by varying the fraction of radioactive molecules. A Student’s test showed that the straight line intercepts statistically at the origin. The dotted lines represent the limit of the 95% confidence interval.

Figure 3. Adsorption kinetics performed on untreated particles at an initial concentration C*IgG ) 6.22 × 10-3% (w/w) (O) and at 3.48 × 10-2% (w/w) (0), and on particles treated with the “nitric acid method” (1) at an initial concentration of 6.59 × 10-3% (w/w). See Materials and Methods section for the detail of the chemical treatment procedure. (3.48 ( 0.1) × 10-2% (w/w). The adsorption kinetics (Figure 3) of IgG molecules on both treated and untreated surfaces indicated that the adsorbed amount remained unchanged after 2 to 3 h of contact with the sorbent. The adsorption isotherm was thus determined after 4.00 ( 0.08 h of contact of the proteins with the sorbent surface (Figure 4). Some experiments were also performed by determining the final bulk concentration according to an adaptation of the Lowry coloring method.46,47 The experimental data again confirmed the absence (within the experimental error) of influence of labeling on the adsorption process. There was no observable difference in either adsorbed amount at steady state and in the adsorption kinetics between treated and untreated samples, as expected from the similarity in their X-ray diffraction pattern. We notice also the small initial isotherm slope, demonstrating a weak affinity of IgG molecules for this sorbent, and the attainment of the adsorption plateau for a relatively high bulk concentration of about 7.0 × 10-2%. Desorption Experiments against Pure Buffer after Intermediate Residence Time of Adsorbed Protein. These experiments will be called experiments of type A. For the desorption experiments, which were realized in the presence of a pure buffer solution, an experimental procedure had to be developped in which the protein excess could be rapidly removed (46) Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 73, 265. (47) Duinhoven, S. Ph.D. Thesis, Agricultural University, Wageningen, The Netherlands, 1992.

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Figure 4. Adsorption isotherm after 4 h of incubation with labeled IgGs at different initial concentrations. The horizontal axis corresponds to bulk concentrations at the end of the adsorption experiment. (O) and (1) represent experiments performed with untreated and treated titanium particles, respectively. (0) Data obtained by using untreated particles and measuring the final bulk concentration of unlabeled molecules with the Lowry coloring method. Error bars were calculated from the 95% confidence interval in the calibration curve for the Lowry method (data not shown).

Figure 5. Time at which step number i begins in experiments of type A (full line and b) and in those of type B (dashed line and O). See text for further explanations. and then the protein concentration in solution maintained as low as possible. So, after 4 h of protein adsorption, we stopped the stirring and allowed the powder to settle. Supernatant solution (1.4 ( 0.1 mL) was then withdrawn and the adsorbed amount determined as previously described. The Eppendorf tube was then again filled with a precisely weighed amount of PBS buffer, the powder was redispersed, and gentle rotation again started for about 1 min. A full cycle consists of powder sedimentation, then solution withdrawal, buffer addition, powder redispersion, and contact with pure buffer. Three such cycles were performed quickly, in succesion, taking a total of 21 ( 3 min, in order to remove the initial protein solution. Additional cycles were realized by contact of variable duration with the buffer; during this step, the tube was gently rotated so that the solution remained as homogeneous as possible. The duration of a cycle was chosen so that the protein concentration in the tube remained lower than 2 × 10-3% (w/w). The duration of cycle 4 was thus equal to 1.5 h, 1 h for cycle 5, 3 h for cycles 6 and 7, 6 h for cycle 8, and 3 h for cycles 9 and 10 (Figure 5). In each cycle, the dilution induced by the addition of a pure buffer solution was precisely calculated after PBS addition (by weight) and the desorbed amount estimated by comparison of this initial activity with the measured bulk activity at the beginning of the subsequent cycle.

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Ball et al.

Desorption and Release Experiments in the Presence of Unlabeled Proteins after Long Interfacial Residence Time (Type B Experiments). Release experiments in the presence of unlabeled IgG molecules in solution were realized for a constant adsorbed protein amount of 100 ( 20 mg‚g-1. Labeled IgG molecules were adsorbed during 4 h at (27.2 ( 2) × 10-2% (w/w). After the precise evaluation of the adsorbed amount, the first five cycles were performed exactly as for the desorption experiments with pure buffer. The duration of cycle 6, also performed in the presence of pure buffer, was equal to 15 h (overnight), rendering possible some protein readsorption. The contact time between the adsorbed IgG molecules and the titanium surface was thus equal to at least 18 h. The experiments were then pursued in cycle 7 by replacing 1.4 mL of supernatant by an unlabeled IgG solution of concentration between 0 and 12.6 × 10-1% (w/w). By precisely weighing and determining activities in solution, immediately before and after unlabeled protein addition one could estimate precisely the labeled IgG concentration in solution at the beginning of an experiment. The duration of cycle 7 was 2 h and 1.5 h for each of the subsequent cycles until cycle 18. Cycle 18 was performed over 6 h and cycle 19 over 2 h. The start of cycle 7 correponds to the time t ) 0 of type B experiments (see Figure 5). During these release experiments, all protein solutions were kept at 4 °C to minimize thermal denaturation and brought to ambient temperature a half hour before use.

Results and Discussion Desorption Experiments in the Presence of Buffer Solution for Type A Experiments. Type A desorption experiments were realized as described previously (see Materials and Methods) after an initial adsorption time of 4 h, varying the concentration of the labeled IgG molecules during the adsorption process. Thus the influence of the surface coverage on desorption kinetics after a constant adsorption time was first investigated. The beginning of cycle 1 was taken as the time t ) 0 for the desorption. The data analysis began at cycle 5; in this and all subsequent cycles, the bulk concentration of labeled IgG molecules was always lower than 2 × 10-3% (w/w). The amount of proteins remaining on the surface was fit by a single exponential decay as a function of the desorption time, according to eq 1 for the time interval lasting between 2 and 21 h (Figure 6a).

Γ*IgG(t) ) {Γ*IgG(t) - Γ*IgG(∞)} e-kdt + Γ*IgG(∞)

Figure 6. (a) Type A desorption kinetics performed after 4 h of adsorption at an initial bulk concentration of 2.7 × 10-2% (w/w). The full line represents an exponential fit of these data from cycle 5 on. (b) ln[Γ*IgG(t) - Γ*IgG(∞)] versus time plot. Γ*IgG(∞) was chosen to be equal 42 µg‚g -1 as suggested by Figure 5a. The full line corresponds to a linear regression through the data (O) and the dotted lines represent the limit of the 95% confidence interval. Table 1. Desorption Rate Constants kd for Type A Experiments for Different Adsorbed Amounts

(1)

Γ*IgG(t) represents the amount of labeled IgG molecules remaining at the sorbent surface at time t, Γ*IgG(0) and Γ*IgG(∞) are the labeled IgG amounts present at t ) 0 and t ) ∞, respectively, and kd is the rate constant for desorption. The validity of the curve fit (LevenbergMarquardt method) by a single exponential (eq 1) is strengthened by the linearity of ln(Γ*IgG(t) - Γ*IgG(∞)) versus time (Figure 6b). The variation of kd with the adsorbed amounts after the rapid 2 h desorption period do not show any interfacial concentration dependence (see Table 1). A mean value for kd of (4.3 ( 0.6) × 10-5 s-1 could thus be obtained. This desorption kinetic law implies that our system can be described by the existence of two kinds of adsorbed molecules as found in previous studies: 8,9,11 molecules of type I that are desorbable and whose amount is given by Γ*IgG(0) and Γ*IgG(∞) at time t ) 0 and molecules of type II which are irreversibly adsorbed on the surface at the time scale of the experiment. The amount of these molecules is Γ*IgG(∞). Kurrat et al.48 analyzed the desorption of human serum albumin adsorbed onto a TiO2/SiO2 waveguide and found that the desorption rate decreased with the surface coverage in (48) Kurrat, R.; Ramsden, J. J.; Prenosil, J. E. J. Chem. Soc., Faraday Trans. 1994, 90, 587.

a

Γ*IgG (µg‚g-1)

kd ( σa (in 105 s-1)

76.8 99.3 100.8 117.9 211.4

4.9 ( 0.9 4.3 ( 0.4 3.5 ( 0.8 4.6 ( 0.5 4.2 ( 0.8

σ, standard deviation obtained from the fits.

the course of the kinetic experiment. The apparent contradiction with our experimental findings could be due to the significant ionic strength differences in the solutions used in the two studies but it should be emphasized that IgG molecules may have a different adsorption behavior than albumin molecules. The fact that desorption experiments can be fitted by eq 1 strongly indicates that for the explored concentration range, lateral interactions between adsorbed molecules of type I should be very weak. Thus, adsorbed IgG molecules behave as almost independent molecules. This looks like Langmuir behavior although several basic assumptions of the Langmuir adsorption model49 as surface homogeneity and total reversibility against dilution appear not to be fullfilled. For other models50 which assume irreversible adsorption and absence of surface (49) Langmuir, I. J. Am. Chem. Soc. 1916, 38, 2221.

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Figure 7. Example of a protein release experiment performed in presence of an unlabeled protein solution of 2.23 × 10-2% (w/w) in cycle 7 and 2.15 × 10-2% (w/w) in subsequent cycles (+). (O) Protein desorption by PBS buffer in the six initial cycles.

diffusion, a limit surface coverage of about 0.5 (depending on the geometry of adsorbing objects) is generaly found. Thus, for an IgG molecule having an ellipsoidal shape of dimensions 23.5 × 4.0 × 4.0 nm3,51 limit adsorption plateau values of about 0.34 and 0.06 µg‚cm-2 are expected for “side-on” and “end-on” adsorptions, respectively. From the plateau value of the adsorption isotherm (250 µg‚g-1), one can thus estimate that the specific surface area of the titanium powder lies betweem 740 and 4200 cm2‚g-1. These values are compatible with the upper limit defined by the detection limit of the BET apparatus (104 cm2‚g-1) and the lower limit of 360 cm2‚g-1 given by the assumption of nonporous spheres with diameter 38 µm and a density of 4.39 g‚cm-3. Desorption and Protein Release Experiments in the Presence of Nonlabeled Protein Molecules in the Bulk. A series of experiments was also performed in the presence of unlabeled proteins in solution (compared to experiments of type A with PBS buffer alone) after long contact time of the adsorbed protein layer with the bulk solution (at least 18 h for the lastest adsorbed proteins). These experiments are of type B (see Materials and Methods section). For the experiment performed with PBS buffer, the adsorbed amount of labeled proteins remaining on the surface could again be fit with a single exponential decay function as described by eq 1. The rate constant kd is equal to 2.5 × 10-5 s-1 for the type B experiment in comparison with the mean value of 4.3 × 10-5 s-1 for the type A experiments. The significant decrease of the value of kd (for the same surface coverage) can be attributed to structural modifications and stronger interfacial anchorings of the adsorbed molecules after a long residence time at the sorbent surface. These observations can be compared with those of Soderquist and Walton with the same proteins adsorbed onto poly(amino) acid complexes2 in which the characteristic time for desorption increases with longer interfacial residence times. A typical kinetic curve performed at a constant bulk concentration of 2.1 × 10-2% (w/w) of labeled IgG is represented in Figure 7. In Figure 8, we represent the amount of labeled adsorbed proteins that are released as a function of time for different bulk concentrations of unlabeled IgG molecules in the bulk. The curve in Figure (50) Viot, P.; Tarjus, J.; Ricci, S. M.; Talbot, J. J. Chem. Phys. 1992, 97, 5212. (51) Quash, G. A.; Rodwell, J. D. In Covalently Modified Antigens and Antibodies in Diagnosis and Therapy; Marcel Dekker, Inc.: New York, Basel, 1989; p 156.

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Figure 8. IgG releases for protein solutions at different concentrations in cycle 8 and the following cycles: [* ) 0%, O ) 1.44 × 10-2% (w/w), 0 ) 2.07 × 10-2% (w/w), b ) 2.15 × 10-2% (w/w), 2 ) 4.82 × 10-2% (w/w), 4 ) 6.15 × 10-2% (w/w), 1 ) 9.68 × 10-2% (w/w), × ) 1.03 × 10-1% (w/w)].

7 seems discontinuous between steps 6 and 7 since the duration of step 6 was 15 h during which, due to desorption, the concentration of proteins in solution increased slightly leading to a new equilibrium point. However, this should not affect our experimental results since most of the adsorbed proteins were still more than 18 h in contact with the surface at the beginning of step 7. For these release experiments, the time t ) 0 is taken at the beginning of step 7. In order to analyze the kinetic release data of adsorbed proteins, after step 7, we postulate the following kinetic law:

d{Γ*IgG(t) - Γ*IgG(∞)} ) dt -K1(CIgG){Γ*IgG(t) - Γ*IgG(∞)}β (2) in which Γ*IgG(t) represents the amount of labeled IgG molecules that can be released at time t through this process and K1 is the rate constant of the release process. This “constant” is however a function of the concentration of IgG molecules present in the solution which is in contact with the surface and contains information on the partial reaction order R with respect to bulk molecules. β is the partial reaction order with respect to adsorbed species. When no proteins are present in solution (CIgG ) 0), eq 2 reduces to pure desorption and K1(CIgG) ) kd. Moreover, we have already shown that for type A experiments, β ) 1. In eq 2, K1 is the rate constant whose variation with CIgG should account for the protein releases in the presence of molecules in solution. The determination of β and the dependence of K1 with CIgG should furnish useful information about the rate limiting step of the IgG release process. Let us assume that β is an integer (β ) 1, 2, ...). We then obtain the following integrated forms of eq 2:

for b ) 1, one obtains Γ*IgG(t) ) {Γ*IgG(0) - Γ*IgG(∞)}e-K1t + Γ*IgG(∞) (3) and for b ) 2 1 1 ) K1t (4) Γ*IgG(t) - Γ*IgG(∞) Γ*IgG(t) - Γ*IgG(0) If eq 3 is valid, then ln[Γ*IgG(t) - Γ*IgG(∞)] should vary linearly with t, which is in fact observed (Figure 9). The

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Figure 9. ln[G*IgG(t) - G*IgG(∞)] versus time plot (O) for the data of Figure 7. The full line is a linear regression trough of the data and the dotted lines represent the limit of the 95% confidence interval.

Figure 10. K1 versus bulk concentrations used from cycle 8 for the release experiments shown in Figure 8. The full line is a linear regression through the data. The dotted lines represent the limit of the 95% confidence interval.

validity of eq 4 was tested by plotting the left-hand member in eq 4 versus time. The obtained curve (data not shown) was far from the expected linear dependence for β ) 2. Thus we considered β ) 1 in the following analyses. This result indicates that the release process of adsorbed IgG molecules seems to be a first-order process with respect to the adsorbed molecules. From eq 3, ∆Γ*(t), the amount that has left the surface at time t, can be expressed by

∆Γ*(t) ) {Γ*IgG(0) - Γ*IgG(∞)}(1 - e-K1t)

(5)

The experimental curves were then fit by the aid of eq 5, the two fitting parameters being K1 and ∆Γ*∞ ) [Γ*IgG(0) - Γ*IgG(∞)]. These parameters are plotted as a function of the IgG concentration respectively in Figures 10 and 11. From Figure 10, one observes that K1 is a linear function of CIgG, with a value of K1, for CIgG ) 0 corresponding statistically to kd as expected from the previous analysis of eq 2. One can thus write:

K1 ) kcCIgG + kd

(6)

and one can conclude that partial reaction order R with respect to bulk species must be equal to 1.

Figure 11. ∆Γ*∞ versus CIgG plot: (O) calculated values from the fits according to eq 5; (×) the last experimental points for the three kinetics (Figure 8) achieved until cycle 19.

This clearly indicates that the release process is cooperative in the presence of protein molecules in the bulk of the solution since the characteristic time K1-1 decreases with CIgG. This process seems thus to correspond to an exchange reaction since it is described by the same rate equations as those obtained in our previous studies.26,27 To put this hypothesis on a firm ground, we should also determine the amount of labeled proteins that are adsorbed through this mechanism onto the surface covered with unlabeled species. However, due to the small surface of the particles, this was not possible. We can thus only assume, with a high degree of confidence, that this release process corresponds to true exchange. Moreover, the process is of first order against bulk molecules as found for the same protein system onto sulfated latexes26 and for human fibrinogen exchanging preadsorbed IgGs onto a silica tube under laminar flow conditions.27 The characteristic time for the process τ is of the order of 6 h for the highest bulk concentration in the explored experimental range. From eq 6 and from the slope of the curve of Figure 10 one obtains kc ) 9.53 × 103 L‚h-1‚mol-1, a value which should be compared with the value of 5.8 × 103 L‚h-1‚mol-1 obtained for the same proteins exchanging on latex particles.26 Both values are low in comparison to the one obtained for fibrinogen exchanging with labeled IgG molecules under laminar flow conditions: kc ) 3.84 × 105 L‚h-1‚mol-1.27 Further studies are needed to investigate the influence of hydrodynamic conditions and in particular shear stresses, onto cooperative desorption or exchange processes. Moreover, the variation of ∆Γ*∞ (Figure 11), the amount released at infinite time, seems also to be linear in CIgG for the explored concentration domain. Such an observation was never clearly described previously but seems also to appear in the exchange kinetics of synthetic polymers adsorbed onto modified silica beads (Figure 6 in ref 12) and for phosphorylase b adsorbed onto butylagarose gels (Figure 4a in ref 13). The fact that ∆Γ*∞ increases with the bulk concentration could be explained in the following way: the anchoring of the adsorbed protein varies (increases) with the adsorption time. This effect leads, for example, to a decrease of the desorption rate constant kd with the contact time of the proteins and the surface as has been observed (see preceeding paragraph). But the change in the surface anchoring also modifies the ability of adsorbed proteins to be exchanged by molecules from the solution. However, adsorbed molecules are more rapidly released from the surface through an exchange process at higher bulk concentration than at lower ones. Thus, at high bulk concentrations, the number of adsorbed

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amount of exchangeable molecules, ∆Γ*(t)0) is known. This, however is very difficult since the amount released at infinite time is a function of CIgG and thus is not directly the image of ∆Γ*(t)0). But if our previous explanation of the varition of ∆Γ*∞ with CIgG is true, ∆Γ*(t)0) should be independent of the IgG bulk concentration and one can thus estimate that this quantity is greater than 40 µg‚g-1 (see Figure 11) and less than 70 µg‚g-1 the remaining adsorbed amount at the beginning of the type B kinetic experiments. One then obtains a value for kc between 7.47 × 103 and 1.31 × 104 L‚mol-1‚h-1 which is reasonably close to the value obtained by plotting the rate constants of the release kinetics against the IgG bulk concentrations in Figure 9. Even if this calculation suffers from the lack in precision of the estimation of ∆Γ*∞, it provides some self-consistency to our model.

Figure 12. Initial release rates of labeled IgGs versus CIgG, concentration of unlabeled proteins. Rates are calculated from the amounts released in cycle 7. The full line is a linear regression through the data. The dotted lines represent the limit of the 95% confidence interval.

molecules that will stay on the surface for a time that is long enough so that they become irreversibly adsorbed, is smaller than at low bulk concentrations. This behavior is indeed observed. This qualitative explanation cannot however predict the particular dependence of ∆Γ*∞ on CIgG. Note that a similar observation has been made52 by Meinders and Busscher who analyzed the desorption of latex particles under flow conditions. Under these conditions it is even surprising that eq 3 describes so well the release of molecules. Further investigations are necessary to evaluate precisely the surface denaturation and the desorption probability reduction as a function of time. A previous study of the kinetics of homogeneous IgG replacement on colloidal latex particles26 used only the initial slopes determination in the evaluation of the rate equation. This method should also fairly apply here in the 2 h time interval of cycle 7 for type B experiments because K1-1 is systematically larger than 6 h. We have verified that for this time interval the involved unlabeled protein amounts induce a bulk concentration change of less than 14%. For this time interval, degenerency conditions (i.e., very little variations in bulk concentration) versus bulk molecules are thus at first sight fullfilled. The initial desorption rates increased linearly with IgG concentration (Figure 12) demonstrating again that the process could be described by a first-order reaction against the species in solution. Thus, provided that one measures the released amounts at sufficiently short times, it is possible to characterize the exchange process by measuring “initial slopes” as a function of bulk molecules excesses. With the value of the slope of the straight line in Figure 12, and by means of eq 2 and eq 6 (with β ) 1 and with t close to zero), one can estimate kc provided that the total (52) Meinders, J. M.; Busscher, H. J. Langmuir 1995, 11, 327.

Conclusion These results thus indicate, as in previous studies, the presence of two kinds of adsorbed molecules: (i) molecules of type I which are desorbable and/or exchangeable and (ii) molecules of type II which are, over the time scales investigated, irreversibly adsorbed on the surface. We have shown that the molecules of type I are released from the surface in the presence of molecules in the bulk according to a process which is of first order with respect to both molecules in solution and those adsorbed. We believe strongly that this reaction corresponds to a true exchange process but it could also correspond to “readsorption inhibition” as was previously suggested by Jennissen.3 This “exchange” hypothesis seems however meaningful since the same kinetic law has already been observed or strongly suggested by previous studies on adsorbed proteins and it could thus constitute a general law. In addition, such a bimolecular exchange process has also been observed for synthetic polymers at ambient temperature. (At lower temperatures the rate limiting step is probably due to diffusion of the desorbing polymer through the adsorbed layer.25) However, even if this model accounts for our experimental observations, it is certainly oversimplified. Indeed, we have also observed in this study that the amounts of proteins of types I and II change with contact time on the surface and at constant contact time between the adsorbed proteins and the surface this amount is a function of the protein concentration in solution. This observation has been qualitatively explained by an interplay between an increasing anchoring process of adsorbed molecules on the surface, the time scale of this process being long or of the order of the explored experimental times, and the exchange process. A detailed investigation of this latter hypothesis will constitute our next goal. Acknowledgment. The authors are indebted to Dr. E. K. Mann for improvement of the English and critical reading of the manuscript and to Dr. Y. Haikel for providing the titanium powder and for the fruitful discussions we had with him. LA950735V