Surface Properties of Polystyrene Nanoparticles Coated with Dextrans

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Surface Properties of Polystyrene Nanoparticles Coated with Dextrans and Dextran-PEO Copolymers. Effect of Polymer Architecture on Protein Adsorption A. De Sousa Delgado, M. Le´onard,* and E. Dellacherie Laboratoire de Chimie-Physique Macromole´ culaire, UMR CNRS-INPL 7568, Groupe ENSIC, BP 451, 54001 Nancy Cedex, France Received December 5, 2000. In Final Form: March 1, 2001

Hydrophobically substituted dextran (dextran phenoxy, DexP) and dextran phenoxy-poly(ethylene oxide) copolymers (DexP-PEO) have been used to modify the surface of polystyrene latex particles. To avoid polymer desorption in the presence of hydrophobic species such as proteins, the adsorbed layer was stabilized by chemical cross-linking and then characterized in terms of adsorbed amount, thickness, and stability. The interfacial concentration in anchoring phenoxy groups and the PEO grafting density were both varied, and their effects on nonspecific bovine serum albumin (BSA) adsorption were examined. It was found that the most important parameter in preventing BSA adsorption is the number of interactions between the adsorbed dextran and the surface, even in the presence of DexP-PEO layers with high grafting ratios of PEO chains. We also examined the ability of dextran layers to bind specifically concanavalin A (Con A) as the Con A molecule exhibits a good specific affinity for glucose-containing carbohydrates. Flocculation of DexP-modified particles by Con A was observed in the course of the experiments. All of these results are discussed in relation to the importance of polymer architecture and surface-protein interactions in protein rejection by dextran and dextran-PEO coatings.

Introduction Polymeric particles with controlled surface compositions have been increasingly used as tools in studies of colloidal models and in a variety of applications, including adhesive technology and coating, drug delivery systems, medical diagnostic tests, separation media, etc. One challenge of applications in relation to biomaterials is to create nanoparticles that resist protein adsorption while maintaining suspension stability. This can be achieved by immobilizing hydrophilic and neutral polymers on the particle surface, either by physical adsorption1-7 or by covalent coupling.8-12 The ability of surface-bound poly(ethylene oxide) (PEO) or naturally occurring polysaccharides to resist protein adsorption in relation to biomaterials has attracted considerable attention in the past two decades. In

particular, the mobility of PEO chains has been shown to contribute to elastic repulsive forces and, thus, to play an important role in the ability of PEO layers to reduce nonspecific protein adsorption. Extensive reviews on the adsorption of biomolecules onto PEO-grafted surfaces have been reported that address the advantages of PEO chains in terms of immunogenicity, antigenicity, flexibility, and protein-repelling properties.5,8,10-15 However, the conventional explanation of steric repulsion resulting from the view that the grafted PEO chains form a brush16-19 on the surface has recently been revisited for a few reasons.20-24 In most cases, the grafting density and the PEO chain length are far from the values required to reach the brush regime. Furthermore, this theory cannot account for the inhibition of protein adsorption by very low molecular weight PEO25,26 or by more rigid polymers such as dextran or cellulose derivatives.2,8,27-31

* To whom correspondence should be addressed. (1) Schroe¨n, C. G. P. H.; Cohen Stuart, M. A.; Van der Voort Maarschalk, K.; Van der Padt, A.; Van't Riet, K. Langmuir 1995, 11, 3068. (2) Khamlichi, S.; Serres, A.; Muller, D.; Jozefonvicz, J.; Brash., J. L. Colloid Surf. B 1995, 4, 165. (3) Malmsten, M.; Van Alstine, J. M. J. Colloid Interface Sci. 1996, 177, 502. (4) Freij-Larsson, C.; Nylander, T.; Jannasch, P.; Wessle´n, B. Biomaterials 1996, 17, 2199. (5) Shar, J. A.; Obey, T. M.; Cosgrove, T. Colloid Surf. A 1998, 136, 21. (6) Duro, R.; Souto, C.; Gomez-Amoza, J. L.; Martinez-Pacheco, R.; Concheiro, A. Drug Dev. Ind. Pharm. 1999, 25, 817. (7) Cassidy, O. E.; Rowley, G.; Fletcher, I. W.; Davies, S. F.; Briggs, D. Int J. Pharm. 1999, 182, 199. (8) O ¨ sterberg, E.; Bergstro¨m, K.; Holmberg, K.; Schuman, T. P.; Riggs, J. A.; Burns, N. L.; Van Alstine, J. M.; Harris, J. M. J. Biomed. Mater. Res. 1995, 29, 741. (9) Beeskow, T.; Kroner, K. H.; Anspach., F. B. J. Colloid Interface Sci. 1997, 196, 278. (10) Holmberg, K.; Tiberg, F.; Malmsten, M.; Brink, C. Colloid Surf. A 1997, 123-124, 297. (11) Malmsten, M.; Emoto, K.; Van Alstine, J. M. J. Colloid Interface Sci. 1998, 202, 507. (12) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1999, 31, 5059.

(13) Malmsten, M.; Muller, D. J. Biomed. Mater. Sci., Polym. Ed. 1999, 10, 1075. (14) Kidane, A.; Lantz, G. C.; Jo, S.; Park, K. J. Biomater. Sci., Polym. Ed. 1999, 10, 1089. (15) Kenausis, G. L.; Vo¨ro¨s, J.; Elbert, D. L.; Huang, N.; Hofer, R.; Ruiz-Taylor, L.; Textor, M. A.; Hubbell, J.; Spencer, N. D. J. Phys. Chem. B 2000, 104, 3298. (16) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159. (17) Harris, J. M. In Poly(ethylene glycol) Chemistry; Plenum Press; New York, 1992. (18) Torchilin, V. P.; Papisov, M. I. J. Liposome Res. 1994, 4, 725. (19) Torchilin, V. P. J. Microencapsulation 1998, 15, 1. (20) Szleifer, I. Biophys. J. 1997, 72, 595. (21) Szleifer, I. Biomaterials 1997, 2, 337. (22) MacPherson, T.; Kidane, A.; Szleifer, I.; Park, K. Langmuir 1998, 14, 176. (23) Halperin, A. Langmuir 1999, 15, 2525. (24) Leckband, D.; Sheth, S.; Halperin, A. J. Biomater. Sci., Polym. Ed. 1999, 10, 1125. (25) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (26) Feldman, K.; Ha¨hner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134. (27) Fournier, C.; Le´onard, M.; Dellacherie, E. Int. J. Biochromatogr. 1997, 2, 235.

10.1021/la001701c CCC: $20.00 © 2001 American Chemical Society Published on Web 06/12/2001

Protein Adsorption on Dex and Dex-PEO-PS Nanoparticles

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Figure 1. Adsorption of amphiphilic dextran (DexP) with (a) high and (b) low amounts of grafted phenoxy groups and (c) of graft DexP-PEO copolymer at the hydrophobic surface.

In a previous paper,32 we focused on the adsorption mechanism of surface-active dextrans (dextran phenoxy, DexP) and dextran-PEO copolymers (PEO-substituted DexP, DexP-PEO) on polystyrene (PS) nanoparticles and on the structure of surface-adsorbed layers in relation to the stability of the sterically stabilized latex dispersions. Although PEO can adsorb on hydrophobic materials, we showed that the adsorption of copolymers was dominated by hydrophobic interactions between the phenoxy groups and the polystyrene surface. We found that the two-phase structure of the copolymer layers was largely determined by the surface density of the anchoring groups, the repulsive intralayer PEO interactions, and the repulsive interactions between PEO and dextran chains. Through side-on configurations, the adsorbed DexP derivatives lead to high packing densities, whereas the DexP-PEO adsorbed copolymers lead to a less densely packed two-phase layer, with an outer PEO shell and an inner dextran layer. The different structures of adsorbed layers are schematically represented in Figure 1. At similar polymer coverages, DexP-PEO provides thicker coatings because of the great extension of the PEO chains. In particular, when the average distance between PEO chains is smaller than the radius of gyration, Rg, of the PEO molecule, the PEO chains are highly stretched, because of lateral repulsions and repulsive interactions with the noncompatible dextran inner layer. However, the influence of the spacing between PEO chains on layer thickness is significantly different from that theoretically predicted by the Alexander-de Gennes expression.33,34 The objective of the present study was to determine the effect of the adsorbed polymer architecture, the surface chain density, the number of anchoring points, and the interfacial layer thickness on the extent of protein adsorption. As previously, the substrate used in this work was a polystyrene latex with an average particle diameter of 180 nm. Because the surface of polystyrene is hydrophobic, BSA can bind to it by nonspecific hydrophobic interactions, and thus, it was used as a test protein for studying the hydrophilization efficiency of coated PS surfaces. Adsorption experiments were also carried out using Con A as another test protein. As the Con A molecule exhibits a specific affinity for glucose-containing carbohydrates (e.g., dextran), its use (28) Fournier, C.; Le´onard, M.; Dellacherie, E.; Chikhi, M.; Hommel, H.; Legrand, A. P. J. Colloid Interface Sci. 1998, 198, 27. (29) Passirani, C.; Ferrarini, L.; Barratt, G.; Devissaguet, J. P.; Labarre, D. J. Biomater. Sci., Polym. Ed. 1999, 10, 47. (30) Morra, M.; Cassineli, C. J. Biomater. Sci., Polym. Ed. 1999, 10, 1107. (31) Rouzes, C.; Gref, R.; Le´onard, M.; De Sousa Delgado, A.; Dellacherie, E. J. Biomed. Mater. Res. 2000, 50, 557. (32) De Sousa Delgado A.; Le´onard M.; Dellacherie E. J. Biomater. Sci., Polym. Ed. 2000, 11, 1395. (33) Alexander, S. J. J. Phys. (Paris) 1977, 38, 983. (34) De Gennes, P. G. J. Phys. (Paris) 1976, 37, 1443.

Scheme 1. Chemical Structure of DexPz-PEOx

allowed us to probe the presence of dextran on the external surface of the modified nanoparticles. Experimental Section Materials. The hydrophobic derivatives of dextran (DexP) were prepared from dextran T40 (manufacturer’s data: Mn ) 28 500 g/mol, Mw ) 42 800 g/mol) obtained from Pharmacia (Uppsala, Sweden). The fixation of phenoxy groups on the dextran backbone was performed as previously described.31 The phenoxy content of the polymers was determined by UV spectroscopy at 269 nm and by 1H NMR spectroscopy in D2O. Monomethoxy poly(ethylene oxide) (PEO) 5000 (Mn ) 5500 g/mol, Mw ) 5600 g/mol) was obtained from Aldrich (St. Quentin Fallavier, France). A method for preparing well-defined PEOsubstituted dextrans (Dex-PEO) has been already described by Hoste et al.35 A slightly modified method was used to prepare PEO-substituted DexP (DexP-PEO).32 The chemical structure of DexP and DexP-PEO is presented in Scheme 1. Monodisperse emulsifier-free polystyrene nanospheres (180 nm) were prepared by a modification of the procedure of Goodwin et al.36 The latex particles were purified by repeated dialysis against EtOH/water (1/1 v/v) and then water. Adsorption of Dextran Derivatives onto PS Nanoparticles. The hydrophilization of PS particles was achieved by adsorption of DexP or DexP-PEO in water, at the polymer plateau coverage (1.25 g of DexP or DexP-PEO in 125 of mL water per gram of dry PS) at 20 °C for 20 h. Then, the suspension was centrifuged at 34 000g for 30 min, and the supernatant was collected for a determination of the nonadsorbed dextran by spectroscopy at 269 nm. The adsorbed DexP layers were stabilized by cross-linking with 0.4 M epichlorohydrin (EpCl) in NaOH (1 M NaOH, 180 mL/g of PS) for 24 h. The adsorbed DexP-PEO layers were (35) Hoste, K.; Bruneel, D.; De Marre, A.; De Schrijver, F.; Schacht, E. Macromol. Rapid Commun. 1994, 15, 697. (36) Goodwin, J. W.; Hearn, J.; Ho, C. C.; Ottewill, R. H. Colloid Polym. Sci. 1974, 252, 464.

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stabilized by cross-linking with 0.05 M divinyl sulfone (DVS) for 4 h under milder alkaline conditions (0.01 M NaOH, 180 mL/g PS) to avoid hydrolysis of the urethane linkage between PEO and the dextran chains. All reactions were carried out at ambient temperature. Then, the suspensions were dialyzed against water for 48 h to remove the NaOH, DVS, or EpCl byproducts. Finally, the particles were washed twice with water by centrifugation. The final amount of DexP or DexP-PEO adsorbed on the polystyrene surface (Γ, in mg/m2) was determined as the difference between the initial amount of polymer added and the polymer remaining in all of the supernatants and washing solutions. Particle Characterization. The nanosphere size distribution was studied in 10-3 M NaCl at 30 °C by photon correlation spectroscopy (PCS) experiments, and the electrophoretic mobility (µe) was determined in NaCl as a function of ionic strength. The experiments were carried out on a Zetasizer 4 (Malvern Instruments, Malvern, U.K.). The zeta potential (ζ) was calculated from the electrophoretic mobility using the modified Booth equation.37 This equation allows for the calculation of the zeta potential for any k and a values, where k-1 is the Debye length and a is the radius of the particles, whereas the classical Smoluchowsky and Huckel equations are applicable only under two limiting cases, i.e., ka > 100 and ka < 0.1, respectively. The electrokinetic layer thicknesses (δel) were calculated from the evolution of the zeta potential vs k. The interfacial PEO concentration, σPEO, and the average distance between PEO chains, sPEO, were calculated from the amount of adsorbed DexP-PEO and the PEO weight fraction in the copolymer. The PEO volume fraction in the adsorbed layer was estimated from the interfacial PEO concentration and the PEO layer thickness δel/PEO. The stability of the adsorbed dextran layers was assessed after extensive washing of the particles in water and incubation in 1% sodium dodecyl sulfate (SDS) or 0.7 g/L BSA solutions (75 mL/g PS). After 24 h at room temperature, the amount of released dextran was determined according to the anthrone method by spectroscopy at 630 nm.38 The colloidal stability of latex suspensions in the presence of added electrolyte or at increasing temperature was assessed by turbidimetry. Typically, 20 µL of dispersion was added to 3 mL of NaCl solution (from 1 × 10-4 to 4 M) or 0.6 M Na2SO4. Each sample was allowed to stand for 40 min, and its absorbance was measured over the range 450-700 nm at 50-nm intervals. The slope n of the straight-line log(optical density) versus log(wavelength) plot was taken as an indication of particle size.39 Flocculation was evidenced by the sharp decrease in n values. BSA Adsorption Experiments. BSA adsorption experiments were carried out in 3.5 × 10-2 M phosphate buffer at pH 7. The nanoparticles (40 mg of PS) were tumbled for 20 h at 20 °C with 5 mL of BSA solution (final BSA concentration ) 0.7 g/L). The adsorbed amounts of protein were determined according to the Bradford method.40 Con A Adsorption Experiments and Affinity Precipitation. The stock protein solution was prepared by dissolving 0.93 g of powder containing 15% active Con A in 100 mL of pH 5 phosphate solution (0.02 M phosphate, 2 × 10-4 M CaCl2 and 2 × 10-4 M MnCl2), followed by centrifugation at 34 000g for 60 min to remove the insoluble species. The protein solution (5 mL) was mixed with 5 mL of coated latex (8 mg/mL) (final Con A concentration ) 0.7 g/L). The reaction mixture was stirred for 2 h at 20 °C. The adsorbed amount of Con A was determined according to the Bradford method, and the colloidal stability of the suspension was assessed by turbidimetry, as described above.

Results and Discussion Properties of DexP- and DexP-PEO-Coated Nanoparticles. Table 1 presents the characteristic data for (37) Deshiikan, S. R.; Papadopoulos, K. D. Colloid Polym. Sci. 1998, 276, 117. (38) Scott, T. A.; Melvin, E. H. Anal. Chem. 1953, 25, 1656. (39) Meadows, J.; Williams, P. A.; Garvey, M. J.; Harrop, R. J. Colloid Interface Sci. 1992, 148, 160. (40) Bradford, M. M. Anal. Biochem. 1976, 72, 248.

De Sousa Delgado et al. Table 1. Characteristic Data for DexP-PS and DexP-PEO-PS Particles

sample

phenoxy content (DS)a

PEO content (DS)b,c

DexP2-PS DexP11-PS DexP16-PS DexP2-PEO6-PS

2 11 2 2

DexP11-PEO2-PS

11

DexP11-PEO6-PS

11

6b 4.6c 2.5b 2.7c 4.9b 5.8c

Γd (mg/m2)

De (nm)

δel f (nm)

2.4 6.8 6.2 2.3

192 209 200 223

10 15 8 17

4.3

223

18

3.2

236

21

a DS ) degree of substitution, i.e., number of phenoxy groups per 100 glucose units, determined by UV spectroscopy. b,c DS ) degree of substitution, i.e., number of MPEO 5000 chains per 100 glucose units, determined by (b) UV spectroscopy and (c) 1H NMR spectroscopy. d At surface plateau coverage. e Particle diameter determined by photon correlation spectroscopy after polymer adsorption. Initial diameter of PS nanoparticles ) 180 nm. f Electrokinetic layer thickness.

DexP- and DexP-PEO-coated nanoparticles (DexP-PS and DexP-PEO-PS, respectively), in terms of the adsorbed amounts (Γ) and compositions, particle diameters, and layer thicknesses. In the previous study,32 two different methods were used to determine the adsorbed layer thicknesses. The hydrodynamic layer thickness, δH, was determined from PCS measurements by measuring the diameters of uncovered and covered particles, whereas the electrokinetic layer thickness, δel, was obtained from zeta potential measurements, using the Eversole and Boardman equation.41 Uncoated polystyrene particles bear negative charges, which can be related to the presence of sulfate groups that originate from the use of the persulfate initiator during emulsion polymerization. The electrostatic consequences of neutral polymer (DexP or DexP-PEO) adsorption were clearly evidenced by a comparison of the ζ values at various ionic strengths. In all cases, adsorption of dextran derivatives results in an effective charge screening. The evolution of the ζ values at increasing NaCl concentrations depends on the interfacial layer thickness, which, in turn, depends on both the amount and the conformation of the adsorbed dextran derivative. In this paper, only electrokinetic layers thicknesses, δel, are compared because PCS measurements led to erroneous results after the nanospheres had been washed and centrifuged, as a result of the removal of smallest particles. Stabilization of Interfacial Dextran Layer. The stability of the coated layers was then assessed under various conditions. After DexP or DexP-PEO adsorption, the particles were extensively washed with water to desorb the loosely bound polymer. Then, the suspensions were incubated in the presence of SDS or BSA solutions. The amounts of polymer released under these different conditions were determined, and a significant polymer loss (3080%) was evidenced, depending on the chemical composition of the adsorbed dextran derivative and the incubation conditions. Therefore, to avoid the desorption of polymer in the presence of hydrophobic species such as proteins, the adsorbed layers were stabilized by chemical cross-linking of the dextran chains. To determine the optimum stabilization conditions, we varied the amount and nature of the cross-linking reagent (epichlorohydrin or divinyl sulfone), and the amount of dextran derivative released during the reaction was measured. As previously (41) Eversole, W. G.; Boardman, W. W. J. Chem. Phys. 1941, 9, 798.

Protein Adsorption on Dex and Dex-PEO-PS Nanoparticles

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Table 2. Characteristic Data for DexP and DexP-PEO Adsorbed Layers after Stabilization Cross-linking sample

Γa (mg/m2)

δelb (nm)

δel/PEOc (nm)

ΓPEOd (mg/m2)

CCCe (M)

DexP2cl-PS DexP11cl-PS DexP16cl-PS DexP2cl-PEO6-PS DexP11cl-PEO2-PS DexP11cl-PEO6-PS

1.6 4.5 5.0 1.1 4.0 2.4

8 12 7 9 14 16

6 12 15

0.7 1.7 1.4

>4 >4 3.5 1.5 >4 >4

a Surface coverage. b Electrokinetic layer thickness. c Electrokinetic PEO layer thickness, calculated as described in (32). d PEO surface coverage, calculated from the PEO weight fraction in the copolymer. e NaCl critical coagulation concentration.

described, cross-linking with epichlorohydrin under welldefined conditions proved to be an efficient way to stabilize adsorbed DexP layers.42 However, the alkaline conditions required for the reaction to occur (1 M NaOH) led to partial hydrolysis of the urethane functions in the DexP-PEO copolymers. From our experiments, we concluded that reaction with divinyl sulfone (DVS) under milder basic conditions (10-2 M NaOH) was a better method of stabilizing the adsorbed DexP-PEO layers. Polymer desorption was shown to occur during course of the the reaction and to increase as the polymer content in the phenoxy groups decreased, presumably because of competition between DVS or EpCl and the phenoxy groups for the polystyrene surface binding sites. Regardless of the cross-linking conditions, no particle aggregation was observed, as evidenced by particle size and SEM measurements, except for DexP2-PEO6-PS. In this case, a few aggregates were formed but they were discarded by filtration before more thorough sample characterization. The suspensions were then incubated with SDS or BSA solutions to evaluate the stability of the cross-linked layer. In most cases, the amounts of released polymer in the presence of SDS were significantly reduced when compared with those released under similar conditions from nonstabilized adsorbed layers. In the presence of BSA, no polymer desorption was observed. Characteristic data for DexP-PS and DexP-PEO-PS after DVS cross-linking (DexPcl-PS and DexPcl-PEOPS, respectively) are given in Table 2. The colloidal stability of the suspensions was examined at various NaCl concentrations. Above 0.1 M NaCl, suspensions of bare PS particles are no longer stable because of the screening of the surface charge by salt ions. Therefore, flocculation due to van der Waals attraction occurs when the salt concentration increases. In contrast, no flocculation was observed at NaCl concentration up to 4 M for un-crosslinked DexP-PS and DexP-PEO-PS suspensions, as a result of steric stabilization by osmotic and elastic repulsion potentials. Similar results were obtained after cross-linking, except for the DexP2cl-PEO6-PS suspension, which exhibited a lower critical coagulation concentration (CCC) than the corresponding un-cross-linked particles did. This difference can be easily explained by the relatively high polymer desorption (up to 50%) during the DVS reaction and subsequent decrease in polymer density on particle surface, leading to a decrease in steric repulsion. The colloidal stability of coated polystyrene particles was also examined in 0.6 M Na2SO4 as a function of temperature. Under these conditions, DexP-PS suspensions were found to be stable at any investigated tem(42) Fournier, C.; Le´onard, M.; Le Coq-Le´onard, I.; Dellacherie, E. Langmuir 1995, 11, 2344.

Figure 2. Dependence of the stability coefficient n on temperature for samples coated with DexP2-PEO6 (O), DexP11PEO2 (0), and DexP11-PEO6 (]) and for corresponding crosslinked samples (b, 9, and [, respectively).

perature, i.e., up to 90 °C. DexP-PEO-PS suspensions, however, behaved differently. As PEO exhibits a lower critical solubility temperature, an increase in temperature reduces its solvency, so that flocculation of un-cross-linked DexP-PEO-PS was observed at increasing temperature.32 The suspensions started to become destabilized at temperatures lower than those observed for the phase separation of corresponding DexP-PEO samples. Although this behavior evidences the phase separation of dextran and PEO in the adsorbed layers, we did not find any correlation between the PEO calculated density in the adsorbed layers and the suspension flocculation temperature, presumably because of the presence of the underlying dextran layer with some loops protruding into the PEO layer. The dependence of the suspension stability on temperature is shown in Figure 2. As can be seen, DexP11cl-PEO2-PS and DexP11cl-PEO6-PS suspensions start to become destabilized at temperatures lower than those observed for the corresponding un-cross-linked samples, despite their lower PEO contents resulting from polymer desorption during the cross-linking reaction. This effect is probably a direct consequence of an increase in the underlying dextran layer stiffness. One might expect that DVS cross-linking reduces the mobility and solvency of the adsorbed dextran chains and, consequently, reduces their contribution to the elastic and osmotic potential repulsion according to the model proposed by Ottewill and Walker.43 Thus, the flocculation temperature of the suspensions becomes less dependent on the presence of the underlying dextran layer. In particular, one can observed that the starting flocculation temperature of DexP11cl-PEO6-PS (∼13 ° C) correlates with the measured cloud point of a PEO solution (10 °C) with a similar PEO volume fraction (∼0.09 g of PEO /cm3). Note that, after cross-linking, DexP2cl-PEO6-PS suspensions are no longer stable in 0.6 M Na2SO4, regardless of the temperature. This result is consistent with the low CCC of DexP2cl-PEO6-PS suspension in NaCl. BSA Adsorption. BSA was used as a test protein to examine the effects of the composition and architecture of the DexP or DexP-PEO layers on the extent of nonspecific protein adsorption. Table 3 shows the amounts of adsorbed BSA on DexPcl-PS and DexPcl-PEO-PS. What is interesting to note is that the distances between PEO chains, sPEO, in both DexP11cl-PEO2-PS and DexP11cl-PEO6-PS samples are quite similar and smaller (43) Ottewill, R. H.; Walker, T. Kolloid Z. Z. Polym. 1968, 227, 108.

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Table 3. Effect of the Characteristics of Adsorbed Polymeric Layers on Protein Adsorption

sample PS DexP2cl-PS DexP11cl-PS DexP16cl-PS DexP2cl-PEO6-PS DexP11cl-PEO2-PS DexP11cl-PEO6-PS

σPEOa sPEOb -ζc (chain/nm2) (nm) (mV) 0.08 0.20 0.17

3.5 2.2 2.4

64 10 8 6 7 2 2

adsorbed adsorbed BSA Con A (mg/m2) (mg/m2) 2.0 1.6 0.3 0.0 1.9 0.3 0.8

4.6 2.5d 1.4d ndd 1.0 0.4 0.4

aσ b PEO ) interfacial PEO concentration. sPEO ) spacing between PEO chains. c ζ ) zeta potential in 10-2 M NaCl. d Particle flocculation.

than the radius of gyration, Rg, of the PEO molecule, resulting in extensive chain overlap. Rg was estimated as

Rg ) 0.181N0.58 (nm) where N is the number of repeat units in the poly(ethylene oxide) chain.44 Such a relation provides a radius of gyration of 2.8 nm for PEO 5000. We showed previously that sPEO ≈ Rg is the point at which PEO chains start to extend away from the polystyrene surface resulting in chain stretching. Thus, according to Sofia et al.,12 one might expect that, under these conditions, BSA will not be able to adsorb on the substrate surface. In fact, considering these samples, significant BSA adsorptions were observed, particularly on DexP11cl-PEO6-PS. In contrast to surfaces mostly covered with linear PEO, those coated with DexP11 and DexP16 present markedly higher protein repulsion, despite their smaller layer thicknesses and slightly higher electrophoretic mobilities. In particular, no BSA adsorption was observed on DexP16cl-PS. This result represents an example of the protein-rejecting ability of surface-side-bound dextran, in agreement with the findings of O ¨ sterberg et al.8 It also indicates that the adsorbed layer thickness and reduced electrokinetic effects are not the main parameters governing protein adsorption, at least for the studied samples. In a first approach, the important decrease in BSA adsorption on DexP layers, compared to that on DexPPEO layers, could be related to the larger amount of adsorbed polymer as protein adsorption is very sensitive to the availability of exposed surface. However, at similar polymer coverages, the DexP16 layer proves to be more efficient in reducing BSA adsorption than the DexP11 and DexP11-PEO2 layers. Moreover, this cannot account for the low-repelling properties of DexP11cl-PEO2-PS and DexP11cl-PEO6-PS as complete surface coverage is expected with half-overlapping grafted PEO chains (sPEO < Rg).12 In fact, the amount of BSA adsorbed on the different samples is directly related to the number of anchoring phenoxy groups in the DexPcl or DexPcl-PEO layers, whereas no direct effect of the grafted PEO chains related to the steric repulsion phenomenon was evidenced. As shown in Figure 3, the higher the interfacial concentration of phenoxy groups, regardless of the coating polymer, the lower the nonspecific BSA adsorption. These results led us to conclude that BSA can penetrate the loosely packed dextran layers, i.e., those containing low amounts of adsorbed phenoxy, as well as the PEO brushes, thus confirming the recent conclusions of Vert and Domurado45 that serum albumin and PEO samples with (44) Kawagushi, S.; Imai, G.; Susuki, J.; Miyahara, A.; Kitano, T. Polymer 1997, 38, 2885. (45) Vert, M.; Domurado, D. J. Biomater. Sci., Polym. Ed. 2000, 11, 1307.

Figure 3. Relative BSA adsorption on DexPcl-PS and DexPclPEO-PS particles as a function of interfacial phenoxy group concentration, as calculated from the amount of adsorbed polymer and its total content in phenoxy groups.

molar mass