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Conformation Change of Albumin Adsorbed on Polycarbonate Membranes as Revealed by ToF-SIMS M. Henry,† C. Dupont-Gillain,‡ and P. Bertrand*,† Unite´ de Physico-Chimie et de Physique des Mate´ riaux, Universite´ catholique de Louvain, 1 Croix du sud, 1348 Louvain-la-Neuve, Belgium, and Unite´ de Chimie des Interfaces, Universite´ catholique de Louvain, 2/18 Croix du Sud, 1348 Louvain-La-Neuve, Belgium Received January 17, 2003. In Final Form: May 21, 2003 The adsorption of proteins at biomaterial surfaces depends on the properties of the substrate and can cause changes in protein conformation. Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) was used in this study to characterize human serum albumin (HSA) adsorption on two different polycarbonate surfaces: a native membrane and a hydrophilic treated one. The amount adsorbed as a function of HSA concentration in solution was compared for the two substrates. The treated membrane was found to have a lower affinity for albumin than the native one. Principal component analysis was used to reveal changes in albumin conformation as a function of albumin concentration in solution and to compare the conformations adopted on the two substrates. The albumin conformation was different on the two substrates, and in every case, the protein lost its native structure. A correlation was found between the amount adsorbed on the hydrophilic surface and the albumin conformation on this surface.
Introduction Protein adsorption at interfaces is the first event happening when a foreign material is implanted in a living environment. Once adsorbed, a protein either keeps its native structure or undergoes changes in concentration, orientation, or conformation.1 These properties of adsorbed protein layers determine the subsequent cellular interactions, which can significantly impact the performance of biomaterials in a biological environment. Conformational changes in adsorbed proteins can make the biomaterial bioreactive or can induce the recognition of the protein as a foreign body for the system. As a result, the organism releases biological cascades such as coagulation and complement activation.2 The extent of the protein rearrangement is affected by the nature of the surface. The interactions governing protein adsorption are the redistribution of charged groups, the changes in state of hydration, and the structural rearrangements in protein molecules.3 On hydrophilic surfaces, adsorption of proteins is expected to be governed by electrostatical interactions and is favored by structural rearrangements.3 Interaction between protein and hydrophobic surface is mainly based on a dehydration mechanism.4 Time-of-flight secondary ion mass spectroscopy (ToFSIMS) has already proved its efficiency in studying protein adsorption.5 Its very high surface sensitivity permits one to probe very low levels of protein adsorption,6 which would * Corresponding author. Phone: +32-10-473581. Fax: +32-10473452. E-mail:
[email protected]. † Unite ´ de Physico-Chimie et de Physique des Materiaux. ‡ Unite ´ de Chimie des Interfaces. (1) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28. (2) Brash, J. L.; Horbett, T. A. In Proteins at Interfaces II: Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series, Vol. 602; American Chemical Society: Washington, DC, 1995; pp 1-50. (3) Norde, W. Cells Mater. 1995, 5, 97. (4) Schmidt, R. Comportement des mate´ riaux dans les syste` mes biologiques: Applications en me´ decine et biotechnologie; Presses Polytechniques et Universitaires Romandes: Lausanne, Switzerland, 1999; Chapter 7. (5) Chilkoti, A. In ToF-SIMS: Surface Analysis by Mass Spectrometry; Vickerman, J. C., Briggs, D., Eds.; IM Publications: Huddersfield, U.K., 2001; Chapter 23.
be very interesting in studying the efficiency of proteinresistant surfaces. Previous research work allows us to identify the secondary ion (SI) mass spectra peaks, which are characteristic for each amino acid.7-10 In addition to identifying the adsorbed protein type, static ToF-SIMS can be used to study the conformation, the orientation, and the degree of denaturation of an adsorbed protein film.11,12 This information is obtained in ToF-SIMS by looking at the modifications in the nature and/or in the intensity of the amino acid SI fragments detected at the uppermost surface of the adsorbed protein. The treatment of the secondary ion peaks with principal component analysis (PCA) has proved its efficiency in studying conformational changes in adsorbed protein films.13 In particular, if only one type of protein is adsorbed on solid surfaces, PCA can differentiate samples based on protein conformation.13 This study uses ToF-SIMS to characterize albumin adsorption on two different polycarbonate (PC) surfaces: a native PC membrane and a treated PCOx/PVP membrane. These surfaces were chosen during the first step of the European project aiming at the development of a bioartificial pancreas.14 Polycarbonate is a good biomaterial due to its known biocompatibility properties.15 A surface (6) Kingshott, P.; McArthur, S.; Thissen, H.; Castner, D. G.; Griesser, H. J. Biomaterials 2002, 23, 4775. (7) Castner, D. G. Characterization of Proteins at Surface and Interface in Probing and Imaging of Cells and Molecules, Oral Communication, University of Washington, Seattle, WA. (8) Bartiaux, S. Undergraduate Thesis, Unite de Chimie des Interfaces, Faculte des Sciences Agronomiques, Universite Catholique de Louvain, Louvain-La-Neuve, Belgium, 1995. (9) Mantus, D. S.; Ratner, B. D.; Carlson, B. A.; Moulder, J. F. Anal. Chem. 1993, 65, 1431. (10) Lhoest, J.-B. Ph.D. Thesis, Unite de Physico-Chimie et de Physique des Materiaux, Applied Science Faculty, Universite Catholique de Louvain, Louvain-La-Neuve, Belgium, 1997. (11) Tidwell, C. D.; Castner, D. G.; Golledge, S. L.; Ratner, B. D.; Meyer, K.; Hagenhoff, B.; Benninghoven, A. Surf. Interface Anal. 2001, 31, 724. (12) Lhoest, J.-B.; Detrait, E.; van den Bosch de Aguilar; Bertrand, P. J. Biomed. Mater. Res. 1998, 41, 95. (13) Xia, N.; May, C. J.; McArthur, S. L.; Castner, D. G. Langmuir 2002, 18, 4090. (14) BARP Bio-artificial Pancreas, European Commission, Craft Action of the Biomed Programme, References: BMH4-CT98-9516.
10.1021/la034081z CCC: $25.00 © 2003 American Chemical Society Published on Web 06/21/2003
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treatment was realized to make the surface more hydrophilic and thus to increase its glucose and insulin permeability.16 First, the intensities of characteristic amino acid SI peaks were measured on the two membranes as a function of the albumin concentration in solution and this permitted the detection of differences in the adsorbed protein amount. Then PCA was performed on these SI fragments to detect protein conformational changes with the hydrophilicity of the surface and with the albumin concentration in solution. Materials and Methods Substrate. The polymer substrate used for protein adsorption was a bisphenol A PC membrane with 15 µm thickness and 30 nm diameter pores with a density of 2 × 109 pores/cm2 (Lexan). Some PC membranes were treated at the CTTM (Centre de Transfert de Technologie du Mans, France) in order to make them more hydrophilic. The first treatment step was a surface activation by cold plasma in an argon atmosphere (50 W) for 10 min, which induces the creation of polar sites (CdO, -C-OH, -COOH). The activated surface was then dipped for a few seconds in an aqueous solution of a hydrophilic polymer, poly(N-vinylpyrrolidone) (PVP). The polymer concentration was 1 wt %. After dipping, the film was rinsed in water and dried in atmosphere. The contact angle with water decreased from 47° to 20° after surface treatment.16 Protein. Human serum albumin (Sigma, St. Louis, MO) is the smallest blood protein (MW ) 65 kDa17). It consists of a 585 amino acid chain containing a high percentage of cysteine and charged amino acids18 (aspartate, glutamate, lysine). Native human serum albumin (HSA) is a heart-shaped molecule that can be approximated to an equilateral triangle with sides of 8 nm and a thickness of 3 nm.18 The isoelectric point corresponds to a pH of 4.8.17 In a physiological solution (pH ) 7), HSA is consequently negatively charged. Albumin Adsorption. The adsorption was performed using 2 mL solutions with a range of HSA concentrations varying from 0 to 2 mg/mL in physiological conditions (T ) 37 °C, pH ) 7.2). The buffer used to prepare solutions was phosphate-buffered saline (PBS). It was adjusted at pH 7.2 and consists of NaCl 137 mM, KCl 2.68 mM, KH2PO4 6.44 mM, and Na2HPO4‚2H2O 8 mM. The samples were deposited in the wells of a tissue-culture plate (Falcon 3043 from Becton Dickinson, Franklin Lakes, NJ). After 6 h of incubation, each sample (square of 1 cm2) was rinsed by five successive dilutions. First, 2 mL of ultrapure water was added and 3 mL of liquid was removed from the well. Then the following operation was repeated four times: 3 mL of ultrapure water was added and 3 mL of liquid was removed again. Afterward, the samples were flushed under nitrogen flow and stored in Petri dishes before analysis. Ultrapure water was prepared with a Milli-Q system (resistivity g 18 mΩ). ToF-SIMS Analysis. Positive ToF-SIMS spectra were recorded with a Phi-Evans TFS-4000MMI (TRIFT 1) spectrometer. A pulsed 15 keV gallium ion beam (800 pA direct current, 8 kHz pulsing frequency, and 2 ns pulse width) was rastered over a 120 µm × 120 µm area for an acquisition time of 300 s. The total fluence per spectrum was about 1.5 × 1012 ions/cm2, which ensured static analysis conditions. The secondary ions were accelerated to 3 keV and focused by two lenses before undergoing a 270° deflection in three hemispherical electrostatic analyzers. A 7 keV postacceleration was applied at the detector entry in order to increase the detection efficiency of high-mass ions. The charge compensation was performed using a pulsed electron beam (24 eV) and a nonmagnetic stainless steel grid placed on each sample. A piece of aluminum paper was added behind each sample to fit the grid exactly and to improve charge compensation. For (15) Kessler, L.; Legeay, G.; Coudreuse, A.; Bertrand, P.; Poleunis, C.; Vanden Eynde, X.; Mandes, K.; Belcourt, A.; Pinget, Submitted. (16) Legeay, G.; Bertrand, P.; Belcourt, A.; Kessler, L. Patent FR2820057, 2002. (17) Horbett, T. A.; Ratner, B. D.; Schakenraad, J. M.; Schoen, F. J. In Biomaterials Science: An Introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: San Diego, 1996; Chapter 3. (18) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153.
Figure 1. Positive ion ToF-SIMS spectra of the native PC membrane before (a) and after (b) HSA adsorption from a 500 µg/mL protein solution.
Figure 2. Positive ion ToF-SIMS spectra of the treated PC membrane (PCOx/PVP) before (a) and after (b) HSA adsorption from a 2 mg/mL protein solution. each sample, three spectra were recorded at different areas in order to check the reproducibility. Moreover, two sample series were realized. The total intensity (Itotal) of a spectrum as used in the results section corresponds to the total SI peak intensity from which the hydrogen and the contaminant SI peaks were subtracted. Principal Component Analysis. A brief description of the PCA principles applied to SIMS data can be found in ref 19. PCA was performed only on the HSA peaks in order to determine the variation in the relative intensities of the different amino acid fragments, which can provide information about conformation evolution as a function of the experimental conditions. For each spectrum, the peak intensities were normalized to the sum of all the HSA peak intensities in order to eliminate differences in total secondary ion yield from spectrum to spectrum. Before PCA, the data set was also mean-centered. This operation centers the data set at the origin so that the variance in the data set is due only to differences in sample variances instead of differences in sample means.20 PCA was performed using the Multion Software in development (Biophy Research, Fuveau, France).
Results Figures 1 and 2 show positive ToF-SIMS spectra of the PC membrane and the treated PCOx/PVP membrane, respectively. Only positive mode spectra are presented because there are few albumin characteristic peaks in negative mode. Moreover, charging problems are encountered in this mode. Figures 1a and 2a concern surfaces (19) Vanden Eynde, X.; Bertrand, P. Surf. Interface Anal. 1997, 25, 878. (20) Wagner, M. S.; Castner, D. G. Langmuir 2001, 17, 4649.
Conformation of Albumin on Polycarbonate Membranes
Langmuir, Vol. 19, No. 15, 2003 6273 Table 1. Principal Positive Albumin Peaks Detected on the Two Substratesa albumin secondary ion fragments molecular structure H3N+ H4N+
CH2N+ CH4N+ CH5N+ C2H4N+ CH3N2+ CH2NO+ C2H6N+ CHS+
CH3S+ C3H6N+ a
m/z
molecular structure
17.03 18.04 28.03 30.05 31.06 42.06 43.05 44.03 44.08 45.08 47.10 56.09
C2H4NO+ C3H8N+ C2H6NO+ C2H5S+ C4H6N+ C4H8N+ C4H10N+ C2H7N3+ C3H8NO+ C5H6N+ C4H6NO+ C5H10N+
m/z 58.06 58.11 60.08 61.13 68.10 70.11 72.13 73.10 74.11 80.11 84.10 84.14
molecular structure C5H12N+
C3H6NO2+ C4H4NO2+ C4H5NO2+ C4H10N3+ C4H8NO2+ C5H8N3+ C6H10NO+ C8H10N+ C9H8N+
m/z 86.16 88.09 98.12 99.13 100.14 102.11 110.14 112.15 120.17 130.16
The PCA analysis was realized on these peaks.
Figure 3. Comparison of the intensity ratio between the C4H10N+ (m/z ) 72) peak and the sum of the C4H10N+ and the C9H10O+ (m/z ) 135) peaks as a function of albumin concentration for the PC membrane and the PCOX/PVP membrane.
before albumin adsorption, and Figures 1b and 2b, surfaces after 6 h albumin adsorption at a concentration corresponding to the saturation (500 µg/mL for the PC membrane and 2 mg/mL for the treated PC membrane). The effect of the treatment is observable in Figure 2a. The intensity of characteristic PC peaks decreases (C9H7+ (m/z ) 115), C9H11O+ (m/z ) 135), C13H9+ (m/z ) 165)), whereas some PVP peaks appear in the spectra (C6H10NO+ (m/z ) 112), C7H10NO+ (m/z ) 124), C8H12NO+ (m/z ) 138)).21 After albumin adsorption, a lot of albumin peaks appear in the untreated PC mass spectrum (Figure 1b), but the substrate peaks remain present. These albumin peaks are mainly C4H8N+ (m/z ) 70), C4H10N+ (m/z ) 72), C5H10N+ (m/z ) 84), and C8H10N+ (m/z ) 120). In the case of the treated membrane, the effect of protein adsorption is more difficult to detect (Figure 2b) due to the fact that nitrogen-containing fragments are already present before adsorption. Nevertheless, an intensity increase is observed for many albumin-related peaks such as C4H8N+ (m/z ) 70) or C4H10N+ (m/z ) 72). Despite the low analysis depth, membrane characteristic peaks are still detected after albumin adsorption on the two substrates. This is certainly due to a noncontinuous coverage of the protein film. To evaluate the protein adsorption on each substrate and to make a comparison between the two substrates, the evolution of the intensity ratio of a characteristic protein peak (C4H10N+) over the sum of a characteristic substrate (PC) peak (C9H11O+) and the protein peak was followed as a function of the albumin concentration in the solution (see Figure 3). In the case of the native surface, the curves show a rapid initial increase directly followed by a saturation. A slower increase up to the saturation follows the rapid initial increase in the case of the treated surface. There is a little but statistically significant difference between the two saturation values. A lower saturation value (I72/[I72 + I135] ) 0.852) is found at the plateau for the treated surface as compared to the untreated surface (I72/[I72 + I135] ) 0.907). On the treated polycarbonate, the plateau is reached for a higher protein concentration value (1 mg/mL) as compared to the more hydrophobic surface where the plateau is reached at 5 µg/mL. (21) The Static SIMS Library; Vickerman, J. C., Briggs, D., Henderson, A., Eds.: Surface Spectra: Manchester, U.K., 1997.
Figure 4. PC1 score plot from PCA of positive ion spectra of HSA adsorbed on the native PC membrane (open squares) or on the treated PC membrane (black diamonds) as a function of albumin concentration in solutions.
Albumin Conformation on PC Surfaces. This study was conducted to detect the importance of substrate composition and hydrophilicity on albumin conformation. The effect of albumin concentration in solution was also checked. Only the positive albumin peaks were used; these are detailed in Table 1. Figure 4 presents the first principal component (PC1) scores for the positive ion spectra of the PC membrane and the treated PCOx/PVP membrane for several albumin concentrations. PC1 collects about 81% of the data variance and permits the differentiation of the two types of substrate. The PC1 score is negative for hydrophilic samples (black diamonds), but it increases when the albumin concentration increases. The PC1 score is positive and rather constant in the case of albumin adsorbed on the more hydrophobic surface (open squares). Therefore, there are differences in albumin SIMS signals when adsorbed on the native PC membrane or on the treated PCOx/PVP membrane. A difference is also observed as a function of the albumin concentration in solution for the treated surface. These albumin evolutions can be characterized by looking at the PC1 loadings. Figure 5 presents the PC1 loading plot. This figure has to be analyzed in parallel with Figure 4 showing the PC1 scores and with Table 2, which presents the SIs corresponding to the different PC1 loadings. For the treated membrane, PC1 scores are negative and they increase when albumin concentration in solution increases. The
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Figure 5. PC1 loading plot from PCA of positive ion spectra of HSA adsorbed on native and treated PC membranes as a function of SI mass.
SI mass peaks with negative PC1 loadings are thus characteristic of the amino acids present at the albumin uppermost surface for the treated surface; they correspond to arginine, lysine, histidine, glutamic acid, asparagine, and phenylalanine. Most of these amino acids have positively charged and polar side chains. For the treated membrane, the increase of the PC1 score with the protein concentration in solution means that the albumin SIMS signal changes with the protein concentration and other peaks corresponding to other amino acids are more intensively detected. These peaks correspond to zero PC1 scores. The following amino acids were found to have zero PC1 loading fragments: arginine and glutamic acid; some amino acids containing sulfur (methionine and cysteine); threonine, tryptophan, aspartic acid, and glutamine. Most of these amino acids have polar side chains. For the native PC membrane, PC1 scores are positive and they do not evolve as a function of albumin concentration in solution. The SI mass peaks with positive loadings are thus characteristic of amino acids present at the albumin uppermost surface for the PC surface. They correspond to leucine, alanine, valine, proline, serine, lysine, and isoleucine. These amino acids are neutral and apolar.
Table 2. Properties of the Amino Acids Detected on the Native Membrane (Positive PC1 Loadings) and on the More Hydrophilic Membrane (Negative and Zero PC1 Loadings) PC1 loadings
characteristic secondary ion
positive
CH4N+ N+
C2H6 C2H4N+ C4H8N+ H4N+ CH2N+ N+
H3 C4H10N+ C5H10N+ C2H6NO+ C5H12N+ negative C6H10NO+
Discussion Albumin Adsorption Curves. The affinity between a protein and a surface is determined from the initial slope of its adsorption isotherm.3 The slope of the albumin adsorption curve is seen to be steeper for the PC membrane than for the treated one (see Figure 3). A previous study22 showed that the albumin adsorption is markedly influenced by the properties of the sorbent surface. It was found that albumin had a higher affinity for hydrophobic siliconized glass compared to hydrophilic clean glass. The authors attributed this affinity dissimilarity to the important contribution of dehydration in the adsorption process on the more hydrophobic surfaces. The treated polycarbonate (PCOx/PVP) membrane actually shows a lower affinity for albumin. The treatment applied to the poly(22) Van Dulm, P.; Norde, W. J. Colloid Interface Sci. 1983, 91, 248.
zero
associated amino acids leucine and a lot of other amino acids alanine alanine proline every amino acid alanine (and a lot of other amino acids) every amino acid valine lysine serine leucine, isoleucine arginine
C3H6N+
lysine
C4H6N+
lysine
CH3N2+
arginine
C4H4NO2+ C5H8N3+
asparagine histidine
C3H8N+
glutamic acid
C8H10N+ CH2NO+ C2H5S+ C3H8NO+ C4H10N3+
phenylalanine asparagine methionine threonine arginine
C9H8N+ C4H8NO2+
tryptophan glutamic acid
CH3S+
cysteine
C3H6NO2+
aspartic acid
C4H6NO+ C2H7N3+
glutamine arginine
CHS+
cysteine
amino acid side chain properties (ref 17) neutral-apolar neutral-apolar neutral-apolar neutral-apolar neutral-apolar neutral-apolar positively chargedpolar neutral-polar neutral-apolar positively chargedpolar positively chargedpolar positively chargedpolar positively chargedpolar neutral-polar positively chargedpolar negatively chargedpolar neutral-apolar neutral-polar neutral-apolar neutral-polar positively chargedpolar neutral-apolar negatively chargedpolar negatively chargedpolar negatively chargedpolar neutral-polar positively chargedpolar negatively chargedpolar
Conformation of Albumin on Polycarbonate Membranes
carbonate surface is consequently efficient to reduce drastically protein adsorption from solutions at low albumin concentration on the polycarbonate surface. Robinson and Williams23 already showed that on silica particles, a PVP coating significantly inhibits bovine serum albumin adsorption but does not completely prevent it. This could be useful for the development of new proteinresistant biomaterial surfaces. Up to now, the most-used surface treatment to reduce protein adsorption is the grafting of poly(ethylene oxide) (PEO) on the biomaterial surface. In this case, the mechanism of protein adsorption resistance appears to be related to the high hydrophilicity, high flexibility, and thus high excluded volume of the PEO chains.2 However, with the plasma/PVP treatment, there is only a small difference in the plateau reached at high concentration for both surfaces. Beyond 1 mg/mL, the adsorbed amount is nearly the same for the native and the more hydrophilic surface. Albumin Conformation on the PC Surface. PCA analysis of the amino acid fragments revealed an evolution of PC1 scores as a function of the albumin concentration in solution (Figure 4). In this figure, the two kinds of surface can be differentiated by their PC1 scores. Indeed, PC1 scores reveal only differences in the amino acid composition at the protein outer surface. These considered molecular fragments can only be emitted from the uppermost surface.24 The applied data normalization process has ruled out the difference in the adsorbed amount. We can consequently conclude that albumin adopts different conformations when it is adsorbed on treated compared to native PC surfaces. For a better understanding of these conformational variations with surface hydrophobicity, PC1 loadings were analyzed (see Table 2 and Figure 5). On native polycarbonate (positive scores in Figure 4), amino acids with apolar and neutral side chains are mainly detected. High surface concentrations of amino acids with apolar side chains have been observed in an earlier study of protein adsorption on silicon substrates by ToF-SIMS.13 The authors attributed this important presence of apolar groups to the dehydration of the samples during the drying step. We can conclude that under our ultrahigh vacuum (UHV) analysis conditions, the albumin molecule loses its native structure in these adsorption conditions on this native surface. Indeed, with its biological structure, only polar amino acid fragments would be detected because in a biological aqueous medium, the apolar residues are preferentially located inside the protein to avoid contact with water while ionized and polar residues are on the outside of the protein in contact with the aqueous phase.17 On treated polycarbonate membranes, the nature of the amino acids detected by ToF-SIMS depended on the albumin concentration in solution. For adsorption at low albumin concentration (negative scores in Figure 4), amino acids with polar and positively charged side chains were mainly detected. Protein adsorption on more hydrophilic surfaces is known to be governed by electrostatical interactions.25 This adsorption mechanism is favored by protein structural rearrangements. In the experimental adsorption conditions (pH ) 7.2), albumin is negatively charged.17 ToF-SIMS shows that the treated polycarbonate surface, on the other hand, bears NH+ positive charges on its surface. Our results support the theoretical predictions that electrostatical interactions are predominant in the (23) Robinson, S.; Williams, P. A. Langmuir 2002, 18, 8743. (24) Delcorte, A.; Bertrand, P.; Wischerhoff, E.; Laschewsky, A. Langmuir 1997, 13, 5125. (25) Norde, W.; Lyklema, J. J. Biomater. Sci., Polym. Ed. 1991, 2, 183.
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Figure 6. PC1 score plot from PCA of positive ion spectra of HSA adsorbed on the native PC membrane (open squares) or on the treated PC membrane (black diamonds) as a function of the ratio between the albumin SI peak intensity and the total SI peak intensity. The PC1 scores represent HSA conformation, and the intensity ratio represents the HSA adsorbed amount.
albumin adsorption mechanism on a more hydrophilic surface. Indeed, in such conditions ToF-SIMS detects mainly positively charged fragments at the outer surface of the adsorbed protein, allowing the negatively charged fragments to be located at the protein-substrate interface in order to interact with the positively charged substrate surface. But this is seen only for adsorption from a weakly concentrated albumin solution. At higher albumin concentrations, the detected amino acids are no longer mainly the positively charged ones. Most of these amino acids have neutral polar side chains; some amino acids with negatively or positively charged side chains are also detected. Seeing that amino acids with very different properties are detected by ToF-SIMS on the external surface, one cannot predict the mechanism responsible for protein adsorption at high albumin concentration. The PC1 score plot for the treated PC substrate (Figure 4) presents the same behavior as the adsorption isotherm curve (Figure 3). When comparing the two substrates, the difference between the two plateaus is however larger in the case of the PC1 score plot. This behavior similarity between Figures 3 and 4 shows that albumin conformation on the treated polycarbonate is related to its adsorbed amount. Figure 6 highlights the relation between the albumin adsorbed amount (represented by the ratio of Ialb and Itotal) and the albumin conformation at the surface (represented by the PC1 scores). Ialb corresponds to the sum of the albumin SI peak intensity of Table 1. Itotal is defined in Materials and Methods. In the case of the native surface, there is no relation between the adsorbed amount and the surface conformation. In the case of the treated surface, there is a proportional relation between the two variables. In other words, whatever the adsorbed amount, the albumin molecules will adopt the same conformation on the native PC membrane. In contrast, on the treated PC membrane, a distinct conformation of the albumin molecules corresponds to each level of adsorbed amount. This might be related to the higher surface affinity of HSA for the native compared to the treated PC membrane. On the native membrane, even at low albumin concentration in solution, the affinity for the surface is such that a monolayer of molecules adopting a given conformation is quickly formed. On the treated membrane, at low
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albumin concentration in solution, molecules will adsorb slowly which allows conformation changes to take place. When the concentration in solution is raised, arrival of albumin at the surface is proceeding faster and different conformations may be reached, depending on the surface occupancy. The contribution of the UHV environment during the ToF-SIMS analyses on the protein orientation/conformation after adsorption cannot been ruled out, and more work is needed to elucidate this possible effect. However, this effect cannot be dominant, otherwise it would not have been possible to detect any structural changes by ToF-SIMS analyses. The albumin conformation changes should be checked with other techniques. Fourier transform infrared spectroscopy seems to be one of the most powerful methods to investigate protein conformation.26 It permits mainly detection of changes in secondary structure. Previous studies using atomic force microscopy have permitted the detection of conformation and orientation changes with the time of contact of the surface with the protein solution.27 These methods provide nevertheless other kinds of information. Only ToF-SIMS allows the determination of amino acids implicated in conformational changes. Conclusion ToF-SIMS combined with PCA data analysis was used to detect changes in albumin adsorbed amount and conformation at the surface of two different polycarbonate membranes: a native polycarbonate membrane and a treated polycarbonate membrane (PCOx/PVP). From our results, one can conclude the following: (i) The more hydrophilic membrane (PCOx/PVP) exhibits a lower affinity for albumin than the native one. The (26) Chittur, K. K. Biomaterials 1998, 19, 357. (27) Dupont-Gillain, Ch. C.; Fauroux, C. M.; Gardner, D. C. J.; Leggett, G. J. J. Biomed. Mater. Res., in press.
Henry et al.
applied treatment can consequently be very interesting in developing in vivo biomaterials with reduced adsorbed proteins, allowing the preservation of membrane permeability properties. (ii) On the native surface, once adsorbed, albumin loses its biologically active structure, which can affect its functionality. (iii) PCA allowed us to differentiate the albumin SI peaks in relation with the two different substrates. The protein adopts a different conformation if it is adsorbed on hydrophobic or on hydrophilic material. On the more hydrophobic surface, the conformation of adsorbed albumin does not depend on albumin concentration in solution and would be determined by the dehydration happening during the drying step. In this case, the adsorbed amount reaches saturation at very low protein solution concentration (5 µg/mL). On the more hydrophilic surface, the conformation of adsorbed albumin evolves as a function of albumin concentration in solution. In this case, the adsorbed amount increases with the protein concentration in solution up to saturation at high concentration (1000 µg/mL). The adsorption mechanism at low albumin concentration is thought to be dominated by the electrostatical interactions between the membrane surface and the protein. (iv) The conformation adopted by albumin on the treated polycarbonate membrane depends directly on the amount adsorbed on these membranes, whereas the conformation is independent of the adsorbed amount for the native membrane. Acknowledgment. The authors acknowledge Dr. Etienne Ferrain (UCL) for providing the PC membranes and Dr. Gilbert Legeay (CTTM, Le Mans) for providing the plasma surface treatments. C. Dupont-Gillain is a postdoctoral researcher of the Belgian National Foundation for Scientific Research (FNRS). LA034081Z
