2230
Langmuir 1991, 7, 2230-2235
Kinetics of Fibrinogen Adsorption from Solutions Flowing through Polymer Hollow Fiberst Feng Yan and Philippe D6jardin* Institut Charles Sadron (CRM-EAHP), CNRS-ULP, 6 rue Boussingault, 67083 Strasbourg Cedex, France Received May 3,1990. I n Final Form: April 10, 1991 We present results on kinetics (25 min) of fibrinogen adsorption under flow conditions (370 s-1 shear rate) within a bundle of charged polyacrylonitrile fibers in the concentration range 0.021-0.80 mg/mL. Successive well-separated kinetic regimes are demonstrated. We estimate the intrinsic kinetic constant in the first regime to be 2.35 X 10-4 cm s-1. Values for the other regimes vary between 0.7 X 106 and 1.0 X 1P6 cm 8-l for the second and about 0.3 X 10-6 cm s-1 for the third observed only at high bulk concentrations. High adsorbancessuggest the existenceof a multilayer structure and/or a rough surface. These experiments are compared to simulations of an adsorption process describing formation of a multilayer structure.
Introduction In many cases, transport phenomena have to be taken into account to propose coherent models for protein adsorption in natural vessels or on foreign materials. Coupling between diffusion and interfacial reaction occurs under static conditions.'-5 This is due to the fact that protein molecules are relatively large with diffusion coefficientstypically (2-6) X cm2s-l. In some dynamic experiments coupling between diffusion, convection and interfacial reaction occurs and mathematical models should include both modes of transport.- Experimental literature on protein adsorption often concerns powders of high specific area packed inside a chromatography column.lO In such techniques, transport phenomena are complicated and difficult to model, one of the difficulties being the distribution of values for the shear rate which exists through the column. In order to obtain known and reproducible hydrodynamic conditions at the interface, different experimental systems have been chosen. The simplest use tubes or slits.6~9However, very sensitive techniques are required to detect the presence of the adsorbed material, such as labeling with fluorescent probes for optical techniques6 or with radioactive isotopes for radioactivity measurements.11-13 The total internal reflection fluorescence (TIRF) technique used by Robertson and co-workerssand by Andrade et a1.14gives continuous recording of the adsorbance versus t Part of this work was presented at the 4th Symposium on Protein Purification Technologies, March 14-16, 1990,Clermont-Ferrand, France. (1)Ward, A. F. H.; Tordai, L. J. Chem. Phys. 1946,63,485. ( 2 ) Miller, R. Colloid Polym. Sei. 1981,259, 375. (3) Mysels, K. L.; Frisch, H. L. J . Colloid Interface Sci. 1984,99,136. (4)Varoqui, R.;Pefferkorn, E. J . Colloid Interface Sci. 1986,109,520. ( 5 )Schaaf, P.; DBjardin, Ph. Colloids Surf. 1987,24, 239. (6)Lok, B. K.; Cheng, Y.-L.; Robertson, C. R. J. Colloid Interface Sci.
time and therefore provides experimental data allowing precise analysis of the kinetic process. Conversely, most experiments performed with radiolabeled molecules give discontinuous measurements. After the adsorption solution is displaced by buffer a t different times, the activities of pieces of tubes are determined.16 However, it seems to be possible using radiolabeling techniques's to obtain the same kind of results as those derived from TIRF. Lack of precision in the first tentatives could be attributed to an inefficient photomultiplier, while solution-surface exchange was measured with good accuracy.17 Continuous recording of radioactivity may likewise be applied to columns of beads.18Jg We present here an improved experimental system as compared to the first model,ls which is applied to a study of the adsorption of fibrinogen within a bundle of polyacrylonitrile hollow fibers. The increased number of fibers enables greater precision of the measurements. Since the hollow fibers examined in this work are currently used in hemodialysis, the results are relevant to our understanding of the role of fibrinogen in surfaceinduced coagulation, especially as the data are obtained under shear rate conditions close to those appearing in clinical applications. The subject is also of interest in light of the previous work of Chuang and co-workers,20 who found a high interfacial concentration of fibrinogen on polyacrylonitrile membrane disks, largely above the expected value for an adsorbed monolayer. A kinetic study should add useful information complementary of these results.
Materials and Methods Hollow Fibers. Modules containing bundles of hemodialysis hollow fibers (48 fibers per bundle) were kindly provided by Hospal Company (Meyzieu, France). Each fiber has an internal diameter of 270 pm and a wall thickness of 50 pm. The main
constituent is a random copolymer of acrylonitrile and sodium
1983,91,87. (7)Lbv6que, M. A. Ann. Mines 1928,13,256. (8)Andrade, J. D.; Hlady, V. Adu. Polym. Sci. 1986,79,1. (9)DBjardin, Ph. J. Colloid Interface Sci. 1989,133, 418. (10)Grainaer,D. W.:Okano. T.: Kim. S. W. J. Colloid Interface Sci. 1989,132,xi. (11)Uniyal, S.;Brash, J. L. Thromb. Hoemostasis 1982,285, 47. (12)Slack, S.M.; Horbett, T. A. J . Colloid Interface Sci. 1988,124, toe
(15)Brash, J.L.; Uniyal, S. J. Polym. Sci., Polym. Symp. 1979,66,377. (16)Voegel, J. C.; de Baillou, N.; Sturm, J.; Schmitt,A. Colloids Surf. 1984., 10. ~ -9. - , (17)Brash,J. L.; Uniyal,S.;Pueineri,C.;Schmitt,A.J . Colloidlnterface Sci. 1983,95,28. (18)de Baillou, N.; Voegel, J. C.; Schmitt, A. Colloids Surf. 1986,16,
(13)Young, B. R.; Pitt, W. G.; Cooper, S6L. J. Colloid Interface Sei. 1988,124,28;125,246. (14) Hlady, V.; Reinecke, D. R.; Andrade, J. D. J. Colloid Interface Sei. 1986,11 1, 555.
(19) Voegel, J. C.; de Baillou, N.; Schmitt, A. Colloids Surf. 1986,16, 289. (20)Chuang,H. Y. K.; Sharpton,T. R.; Mohammad, S. F. Int. J.Artif. Organs 1983,199,6.
OJJ.
0743-7463f 91/2407-2230$02.50/0
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0 1991 American Chemical Society
Langmuir, Vol. 7, No. 10, 1991 2231
Fibrinogen Adsorption
J
I
Figure 1. Experimental system. Microcomputer (3) controls two syringe pumps (1, 2) and acquires data from a balance (7) and a multichannel analyzer (6) connected to a NaI detector (5) in front of the module under study (4). 12Oooo
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.
80000
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9
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labeling and fibronectin content low. Concentrated Tyrode's buffer (160 g of NaCl, 4 g of KCl, 20 g of NaHCOs, 1.16 g of NaH2PO~H20,completed to 1 Liter with deionized water (Super-Q Millipore)) was filtered through 0.45-pm Millex filters before storing at +4 OC. Tyrode's buffer used for fibrinogen solutions was prepared just before experiments by dilution 1:20 in water and adjusted to pH 7.35 with 1M HCl. A few milliliters of fibrinogen (C = 2 mg/mL) was labeled by the iodogen technique13 using Nal%I,extensively dialyzed against Tyrode's buffer, and 0.125-0.5-mL aliquota of the radiolabeled fibrinogen solution were stored at -18 O C . Quick-thawed (37 O C ) aliquota were then diluted with filtered buffer just before starting the experiments. Determination of Fibrinogen Adsorbance and Apparent Kinetic Constants. We performed a calibration in situ by estimation of the radioactivity change due to filling or emptying of the tube (radius R)with a solution of known concentration cb. Schematic representation of an experimental curve is shown in Figure 2. Given Ah the activity rise while filling the tube, which is equal to the activity drop Ah! when rinsing starta, and AH the residual activity after rinsing (Figure 2), we have I' = 0.5RCb( AH/Ah)
(1)
and the apparent adsorption constant
AH 600
800
1000
1200
Time (seconds)
Figure 2. Schematic representation of radioactivity recording. The rise, Ah, and the drop, Ah', are directly proportional to the known solution concentration, thus allowing determination of the amount of protein adsorbed from AH. The slope dI'/dt is measured in the same way via dH,/dt. methallylsulfonate, known as AN69. The copolymer bears a negative charge of 600 mequiv/kg. Assuming on the surface the average charge spacing existing in the bulk polymer, we calculate acharge density u = 0.57 electron/nm2. This is a minimum value since ESCA studies reveal an enrichment in sulfonate groups near the surface. We note also that membrane swelling in an aqueous medium leads to a lower limit u = 0.25 electron/nm2. Fibers without pores were provided by the manufacturer, who measured a contact angle of 22O inside the transparent fibers. Therefore the model of an impermeable hydrophilic tube may be considered as a good approximation of the system. The dialysate compartment, outside the fibers, was flushed continuously with a buffer solution thermostated at 37 OC, while buffer or a fibrinogensolution flowed through the blood compartment, inside the fibers. Experimental System. The experimental system is schematically represented in Figure 1. A microcomputer controls two syringe pumps, one containing Tyrode's buffer pH 7.35, the other a solution of fibrinogen at concentration cb in the same buffer. Flow rate was 2 mL/min, corresponding to a shear rate of 370 s-l. We performed 25-min alternate flows of solution and buffer. This period appeared to be too short to reach a stable value of adsorbance, while during rinsing with buffer a continuous slow decrease of the interfacial concentration I' was observed. Diversion toward a waste vessel is possiblejust before the module to be examined, to eliminate air bubbles. The syringes are connected via Teflon tubing to the 20 cm long hollow fibers module, of which the middle portion is positioned in front of a 5 cm diameter NaI detector. Data are visualized on a screen and stored in a microcomputer, each experimental point corresponding to the integrated radioactivity over 10s. At the capillary exit the liquid is collected on a balance, from which the readings also appear graphically on the screen. Thus any disturbances in the flow line are instantaneously visualized. Preparation of Fibrinogen Solutions. Human fibrinogen purified by the solvent/detergent technique was obtained from the Centre RBgional de Transfusion Sanguine (Strasbourg, France). The coagulability was 98% before and 95% after
Hence this procedure does not require independent measurement of the specific activity. Ratios of lengths on the graph lead directly to I' and dI'/dt when R is known. For small diameters, however, complications may arise from the fact that when the process is partially controlled by the transport of solute toward the interface, the activity rise Ah does not correspond to a tube entirely filled with a solution of concentration cb, since concentration depletion occurs in the Nernst layer near the wall. The importance of the correction depends on the thickness of the depleted layer as compared to the tube radius and on the magnitude of the depletion, which is related to the intrinsic adsorption constant k,. For instance, if we consider the extreme case of complete control of the kinetic process by transport to the interface, as in the L W q u e model for a slit, the ratio r of the Nernst layer thickness XN to the half width of the slit w/2 is given by
r = (3.72/~)(Dz/y)'/~
(3)
With D = 2.8 X 10-' cm2 s-lF1 z = 10 cm, y = 370 s-l, and w = 0.0135 cm, we calculate r = 0.25, which is not negligible. Therefore, we computed flow simulations for the cylindrical geometry of a tube. The real amount of solute in the volume under initial steady-state conditions, as compared to an assumed full tube, was determined for different kinetic constants .k It appears that the right-hand side of eqs 1 and 2 should be multiplied by a correction factor fc, with 0.70 < fe < 1, to provide a correct interpretation of our experiments. This represents a limitation to quick interpretation of the data. One way to avoid this disadvantage is to consider, instead of the activity rise Ah, the activity drop Ah' when starting the riming step (Figure 2). As we know that for most proteins desorption occurs slowly if at all, this drop should correspond to the amount of solute present in the tube volume. If, in addition, the experiment is performed until a stable value of activity is reached, then we do not expect a depleted layer near the wall and the activity drop will correspond to a full tube. In fact, to compare Ah and Ah' provides a good control of correct calibration. For example, the scale chosen for Figure 3 is not well adapted for Figure 3b, where cb = 0.037 mg/mL. Such a representation could lead to neglect of the first adsorption regime by including it in the filling step. This can be avoided by looking at the activity drop at the start of rinsing. Since it is sometimes tricky or impossible to distinguish between tube filling and the beginningof adsorption, whereasdesorption occurs much more slowly than adsorption, calibration from the activity drop would appear to be more reliable. It is also assumed that desorption occurs slowly relative to complete rinsing of the tube.
Yan and Dhjardin
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Time (seconds) Time (seconds) Figure 3. Experimental adsorbance of fibrin0 en 88 a function of time from a solution of concentration c b (mg/mL): (a) c b = 0.021; (b) c b = 0.037;(C) c b 0.12; (d) c b = 0.21; (e7 c b 0.38; (0 c b 0.80.
Results and Discussion Qualitative Analysis. Figure 3 represents the experimental recordings of activity versus time from c b = 0.021 to 0.80 mg/mL. Full lines show the fits of the activity A versus time t data (i) by a single exponential A = A,, - (Ali,,,- A,) exp[-(t - t0)/7,1 (4) for the adsorption process and (ii) by a linear function for the desorption process. A b and A0 are respectively the calculated activities at infinite time and time to. It can be seen from the dashed lines which represent the initial slopes that most experimental curves, have quasi-linear parts. Statistical analysis included in the software (SigmaPlot, RJA, Germany) confirmed that using eq 4 to fit the adsorption data was too complex a function, as revealed by dependency parameters close to 1for the characteristic time T* and the activity at infinite time A b . Therefore we do not report these parameters. From the two experiments at c b = 0.037and 0.80mg/ mL, the existence of different kinetic regimes is clearly demonstrated. We note, however, that the activity plateau at the end of adsorption for cb = 0.80 mg/mL is an artifact
due to an accidental interruption of the flow. From assumed continuity of the phenomena with concentration, we suppose that the first regime observed at low concentration still exists at higher concentration but cannot be analyzed, as separation between tube filling and the start of adsorption here becomes impossible, given the present state of the technique. Nevertheless, even with an improved method, analysis of the data would not be easy at high concentration because of the concomitance of the two phenomena. This problem has been studied by Lok et al.,21 who showed that establishment of a steady-state profile while adsorption occurs can lead to an initial adsorption rate smaller than that predicted by LBvt3que's law, especiallyat high concentrations. Moreover it should be kept in mind that, contrary to TIRF, the method described here detects the simultaneous variation of the amount of solute on the surface and in the volume. We will see later that simulations (Figure 6) reveal that solute variations in the volume contribute to the total variation, depending on the bulk concentration. Indeed, we tried to (21)
Lok, B. K.;Cheng, Y.-L.; Robertson, C . R.J. Colloid Interface
Sci. 1989, 91, 104.
Langmuir, Vol. 7, No. 10,1991 2233
Fibrinogen Adsorption
Table I. Amounts of Fibrinogen I'j Adsorbed from Solutions of Concentration cb in the Three Successive Regimes Observed, Each Regime Lasting Atj, with Apparent Kinetic Constant kj loSki, cm/s rj, pg/cm2 (Ati, min)
cb, mg/mL
rl (Ah) 0.29 (3.6) 0.31 (2.4) 0.39 (1.7) 0.35 (1.2) 0.21 (-) ?
0.021 0.037 0.120 0.210
0.380 0.800 5
.
,
1
,
.
,
.
,
r2 (At21 0.22 (21.4) 0.42 (22.6) 1.10 (23.3) 1.96 (23.8) 1.86 (11.9) 1.68 (6.3)
,
r3 (At31
1.25 (13.1) 2.23 (18.7)
kl 5.5 6.2 i2.71 i2.71 ? ?
k2
0.88 1.00 0.66 0.65 0.68 0.64
k3
Cri
[0.37] 0.28
0.50 0.73 1.50 2.31 3.32 3.91
I
*
i
Bulk Concentration (mg/mL)
Figure4. Adsorbance of fibrinogenversus solution concentration after a 25-min adsorption period: ( 0 )rl; (+) = rl + r2+ r3(when r3# 0).
rl+ r2; (*) rbu
increase the number of experimental points in this region by choosing a shorter countingtime but were not successful because of the increased dispersion of the data. In any case, it seems reasonable to conclude qualitatively that above cb = 0.021 mg/mL, at shear rate 370 s-l, at least two successive kinetic regimes exist within 25 min. For a quantitative interpretation, we need to look first at the calibration procedure. QuantitativeAnalysis. The rinsing activity drop Ah' (Figure 2) was used for calibration. It is useful to verify if the tube is almost filled with solute at concentration cb when rinsing begins. Let us consider that the first regime observed at low concentration is the fastest regime, say the L6vi3que regime. Simulation gives the lowest correction factor fc = 0.70. Now the ratio of the initial slopes for the two successive regimes is approximately 6. This means that the concentration near the wall (see eq 8below) would be at least about 6/,th of the bulk concentration c b at the beginning of the second linear regime, thus leading to a negligible correction factor, fc 1, in eqs 1 and 2. Therefore using Ah' for calibration instead of Ah allows us to apply eqs 1and 2 for the determination of protein adsorbance and kinetic constants. We arbitrarily defined the intercepts of the initial slopes as points separating the successive regimes, except for c b = 0.38 mg/mL where the separation between the second and third regimes was roughly estimated from the curvature change. The first adsorbanceslope was determined from the first experimental points above the activity level corresponding to the total [background+ calibration Ah'], represented by a dotted line on Figure 3. This total was the zero level for calculation of the adsorbances. Table I summarizes the quantitative parameters associated with the three regimes of the adsorption process, each occurring over a time interval Ati. We define the experimental kinetic constant ki of the regime i as ki = C,.,-'(dI'/dt),,i (5) Fibrinogen Adsorbance. In accordance with the previous results of Chuang,20 who measured in batch
Figure 5. Scanning electron micrograph of membrane AN69. Bar scale is 1 pm.
experiments adsorbance as high as 12 pg/cm2, we found large amounts of adsorbed fibrinogen (Table I). Figure 4 shows the variation of I'i with solution concentration. The amount adsorbed in the first regime varies between 0.2 and 0.4 pg/cm2and lies between the two extremevalues for closed-packed side-on (0.14-0.21 pg/cm2) and end-on (1.0 pg/cm2) adsorption models, within the schematic representation of the fibrinogen molecule by a parallelepiped of dimensions 45 X 9 X 6 nm3. It suggests that at low solution concentration most molecules can adopt a side-on conformation, even if some of them were possibly adsorbed first in the end-on orientation. When the solution concentration increases, the rate of arrival of molecules at the surface leads to rapid surface occupation, reducing the possibilities of end-on/side-on transformation and increasing the interfacial concentration attained in the first regime. If such an increase is observed until c b = 0.12-0.21 mg/mL,an apparent decrease occurs at higher concentrations,which could be due to the arbitrarymethod used to separate the two successive regimes. A limit of about 1.7-2 pg/cm2 is found for the second regime, while for the third the existence of only two data points does not permit determination of a possible third limit. The amounts of protein adsorbed suggest the formation of an intricate multilayer structure. A contribution to an apparent high adsorbance could originate from the roughness of the surface. However, this factor could not be solely responsible for the succession of well-separated quasi-linear regimes, and as shown in Figure 5 the membrane surface examined by scanning electron microscopy does not exhibit significant roughness; using atomic force microscopy in an aqueous medium should help in the future to quantify the roughness under more reliable conditions than with electron microscopy. We note that for a similar experimental system,n say the same (22) Schmitt, A.; Varoqui,R.;Uniyal, S.;Brash, J. L.;Pwineri, C.J. Colloid Interface Sei. 1983,92, 25.
Yan and D&jardin
2234 Langmuir, Vol. 7, No. 10,1991 polymer coated onto a glass tube, an interfacial concentration slightly higher than 1 pg/cm2 was observed for solution concentrations above 0.5 mg/mL. Therefore, it could be argued that manufacture of the fibers leads to a special structure on their inner wall which differs from the state obtained by contact with an organic solution followed by solvent evaporation. Stretching of the polymer material probably induces an orientational order and/or some roughness which favors accumulation of fibrinogen on the surface. The existence of multilayer5 has already been proposed by Silberberg23 for albumin on glass, given the large hydrodynamic thicknesses observed. Adsorption Kinetic Constants. If the reaction were entirely controlled by diffusion to the interface in the Nernst layer, the LbvvBque model' would lead, for zero bulk concentration near the interface, to
k,
= C
