Langmuir 1995,11, 2344-2347
2344
Coating Polystyrene Particles by Adsorption of Hydrophobically Modified Dextran C. Fournier, M. Leonard,* I. Le Coq-Leonard, and E. Dellacherie LCPM, URA CNRS 494, ENSIC B.P. 451, 54001 Nancy, France Received March 14, 1995. I n Final Form: May 22, 1995@ In order to decrease their hydrophobicity and thus t o reduce the protein adsorption at their surface, polystyrene-divinylbenzene (PS-DVB) particles have been coated with dextran. To favor its adsorption, the polysaccharide was previously substituted with low concentrations of phenoxy groups. The amount of dextran adsorbed onto the PS-DVB surface was examined as a function of the polymer concentration, and various samples of beads, differing in surface dextran concentration, were prepared. The adsorbed polymeric layer was then chemically cross-linked and its stability was checked in the presence of sodium dodecyl sulfate. The hydrophiliccharacter of the various resulting beads was evaluated toward the adsorption ofbovine serum albumin, a stronglyhydrophobic protein. By this study, the conditionsfor coating polystyrene particles were optimized and the dextran layer was shown t o greatly reduce the nonspecific adsorption of proteins.
I. Introduction Polymer adsorption at solid-liquid interfaces is a widely studied phenomenon as connected with many important processes including colloidal stabilization, flocculation, adhesion, or coating. The reduction of protein adsorption by coating hydrophobic surfaces is of significant importance for many biomedical and biotechnological applications. Particularly, there is a n increasing interest in the use of polystyrene particles for liquid chromatography of biological molecules. These materials exhibit excellent mechanical properties and a good chemical stability over a large range of pH.IJ However, to use this packing material for the chromatography of proteins, it is essential to make the bead surface hydrophilic-except in the reverse-phase mode-in order to avoid irreversible interactions and protein denaturation. The use ofwater-soluble polymers in the adsorbed coating technology turns out to be one of the most efficient methods for the hydrophilization of polystyrene-divinylbenzene (PS-DVB) particles. Thus the adsorption of polyelectrolyte onto charged polystyrene, Le., polystyrene on which ionic groups have been grafted, leads to highly hydrophilic ion-exchange materiaL3 Hydrophilization of PS-DVB can also be achieved by adsorption of nonionic polymers such as ethyl hydroxyethyl c e l l ~ l o s e poly(ethy1ene ,~,~ oxide)-poly(propylene oxide) copolymer^,^,^ poly(viny1 or polyglycerol.1° However, it is well-known that these polymers partially desorb when exposed to other polymers or proteins.11J2 To overcome this problem, low concentra-
* To whom correspondence should be addressed Abstract published in Advance A C S Abstracts, July 1, 1995. (l)Moore, J. J. Polym. Sci. 1964,Part 2, 835. (2) Lee, D. P. J . Chromatogr. Sci. 1982,20,203. (3) Rounds, M. A,; Regnier, F. E. J. Chromatogr. 1988,443, 73. (4) Malmsten, M.; Lindman, B.; Holmberg, K.; Brink, C. Langmuir 1991,7,2412. (5) Malmsten, M.; Tiberg, F. Langmuir 1993,9, 1098. ( 6 ) Bridgett, M. J.; Davies, M. C.; Denyer, S. P. Biomaterials 1992, 13, 411. (7) Lee, J.H.; Kopecekova, P.;Kopecek, J.;Andrade, J.D.Biomaterials 1990,11, 455. (8) Tuncel, A.; Denizli, A.; Purvis, D.; Lowe, C. R.; Piskin, E. J. Chromatogr. 1993,634, 161. (9) Leonard, M.; Fournier, C.; Dellacherie, E. J. Chromatogr. 1996, 664,39. (10) Varady, L.;Mu, N.; Yang, Y. B.; Cook, S. E.; Afeyan, N.; Regnier, F. E.J. Chromatogr. 1993,631, 107. (11)Tiberg, F.;Malmsten, M.; Linse, P.; Lindman, B. Langmuir 1991, 7. , 2723. (12) Stuart C. M. A.; Cosgrove, T.; Vincent, B.Adu. Colloid Interface Scz. 1986,24,143. @
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0743-7463/95/2411-2344$09.00/0
Scheme 1 + H
CI-CH,-CH-CH* '0/
Zn(BF,)* --t pH1
n
HO 8
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1
0-CH,CHOH-CH,-CI Dex-CI
DexP
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tions of hydrophobic groups can be introduced onto the backbone of the hydrophilic polymer. The presence of such groups should favor the adsorption by means ofweak hydrophobic interactions. Furthermore, the polymeric adsorbed layer can be chemically cross-linked and thus stabilized. This paper describes the hydrophilization of macroporous PS-DVB beads by adsorption of a hydrophobically modified dextran (DexP). We report the effect of varying the adsorption and cross-linking conditions on the amount of adsorbed dextran, on the stability of the polymeric layer and on the hydrophilization efficiency with regard to bovine serum albumin (BSA) which is well-known to be strongly adsorbed on PS-DVB surfaces.13
11. Experimental Section PS-DVB particles (Amberchrom CG162s) were a gift from Rohm and Haas (Philadelphia,PA). The specific surface area was 200-300 m2/g,the average diameter was 35pmJ20-60 pm ;angel, and the average pore size was 1000-1400 A (10-3000 A range). The hydrophobic derivative of dextran (DexP) was prepared from dextran T40 (manufacturer data: M , = 40 000, M,, = 25 000) obtained from Pharmacia (Uppsala,Sweden).The introductionof aromatic groups on the polymeric backbone was performed according to Scheme 1. It consists of a two-step reaction. Firstly, dextran was allowed to react with epichlorhydrin (EpCl,25 mmoVg dextran) at pH 1 and 80 "C for 5 h, in the presence of Zn(BF& (50 wt % solution in water, 1 mug de~tran1.l~ The product thus obtained was precipitated once with acetone and 3 times with ethanol. Then the resulting chlorinated derivative (DexC1, 0.65 mmol of CVg) was treated with an aqueous solution of phenol (0.65 mmol/g DexC1) at pH 11and at ambient temperature for 24 h. Then,the crude product was precipitatedtwice with ethanol and treated with 1M NaOH (13)Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers; Plenum Press: New York, 1995; Vol. 2, p 51. (14) Rogovin, Z. A,; Virnik, A. D.; Khomiakov, K. P.; Laletina, 0. P.; Penenzhik, M. A. J . Macromol. Sei. Chem. 1972,A6, 569.
0 1995 American Chemical Society
Langmuir, Vol. 11, No. 7, 1995 2345
Letters for 48 h, in order to remove all the unreacted chlorinated groups. Finally, the solutionwas dialyzed against water and freeze-dried. In the resulting DexP, about 2% of the total saccharide units mol of phenoxy groupdg), as were substituted (1.0 x determined by spectroscopyat 269 nm (E = 1372 L mol-l cm-l). Viscometricmeasurements of DexP solutionswere carried out using an Ostwald-typecapillary viscometer (0.36mm diameter) thermostated at 25 "C. Fluorescence emission spectra of pyrene (1.0 x M in polymer solutions)were recorded in the 350-500 nm range on a SPEX Fluorolog 2 spectrometer (Edison,USA). The excitation wavelength was 335 nm. The weight-averagemolecular weight (M,) of dextran and of DexP were determinedby low angle laser light scattering(LALLS) Milton-Roy,USA) and were experiments in 0.2 M KSCN (-6, found to be respectively 41 000 and 44 000. The hydrophilization of PS-DVB particles was achieved by adsorption of DexP in water (100 mL of solutiodg of dry PSDVB at various polymer concentrations) at 20 "C and for 20 h. Then the suspension was centrifuged and the supernatant collected for the determination of the nonadsorbed DexP by spectroscopy at 269 nm. Cross-linkingof the coated layer was achieved at 20 "C either by epichlorhydrin (EpC1) or by butanediol diglycidyl ether (BDGE). In typical experiments, the coated particles (50 mg of PS-DVB) were allowed to react for 24 h with EpCl or BDGE at various concentrations in 5 mL of 1 M NaOH. Finally, in order to desorb the untigthly bound DexP, the beads were washed with a 2%sodium dodecyl sulfate (SDS)solution (100 mug PSDVB) at 40 "C for 24 h. The final amount of DexP adsorbed on the PS-DVB surface was determined as the differencebetween the initial amount of added polymer and the remaining one in all the supernatants and washing solutions. These results were confirmed by FTIR spectroscopy, using a method previously described? by measuring the absorbance at 795 and 1453 cm-l. In SDS solutions, DexP was determined according to the anthrone method, by spectroscopy at 630 nm.15 After cross-linkingof the coated layer, the modified particles (50 mg of PS-DVB) were incubated for 24 h at 20 "C with 5 mL of bovine serum albumin (BSA) solution (Sigma, St. Quentin Fallavier, France), either in 0.05 M citrate buffer, pH 5, or 0.05 M phosphate buffer, pH 7. The adsorbed amounts of protein were determined from the supernatant absorbance at 280 nm.
7 m
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(15)Scott, T.A.; Melvin, E.H.Anal. Chem. 1953,11, 25. (16)Dong, D. C.; Winnik, M. A. Can. J. Chem. 1984,62,2560.
I
10
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c (m Figure 1. Z1IZs ratio of pyrene fluorescence in the presence of dextran (B) and DexP (0) in water as a function of polymer concentration.
n
M
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111. Results and Discussion
The covalent fxation of phenoxy groups onto dextran leads to a derivative whose amphiphilic properties are clearly evidenced by fluorescence and viscosity experiments. At low concentrations, fluorescence of pyrene is characterized by five peaks (I1to 15)whose relative intensities are very sensitive to the polarity of the medium. In polar solvents such as water, the ratio I l l 3 is close to 1.8 while it is close to 0.62 in nonpolar solvents such as cyclohexane.16 Thus pyrene can be used to indicate the change in environmental polarity and, particularly, to probe the formation of hydrophobic microdomains in amphiphilic polymers chains. In Figure 1, we show the variation of Il/Is vs polymer concentration in pure water for dextran and D e s . For dextran, 11/13 remains in a range of 1.821.84. In opposition, in the presence of DexP, the Ill& value decreases with increasing polymer concentration to a plateau value of 1.1. These results suggest that, a t polymer concentrations higher than 2 g/L, pyrene concentrates in hydrophobic regions of DexP resulting from intra- and intermolecular chain associations. The intrinsic viscosity [VI, corresponding to the extrapolation of the reduced viscosity (vadC)to infinite dilution is slightly smaller for the dextran derivative than for the starting material (Figure 2). Since the average molecular weight of dextran does not change significantly
I
1
0
,
1
1
2
3
4
c (g/W Figure 2. Reduced viscosity of dextran (a) and DexP ( 0 )in water at 25 "C versus polymer concentration. afier the fixation of phenoxy groups, as determined by low-angle laser light scattering (M,= 40 000 and 44 000, respectively), this variation of viscosity may be interpreted in terms of intramolecular hydrophobic interactions between phenoxy groups, leading to shrunken conformations in dilute solutions. After several preliminary tests proving that native dextran does not adsorb on the PS-DVB beads, we observed that DexP, under the same conditions, could be confined on the surface of PS-DVB beads, probably by means of hydrophobic interactions between the phenoxy groups and the PS-DVB surface. The adsorption isotherm of DexP obtained in water (Figure 3) is typically Langmuir-type where the adsorbed amount rapidly increases with polymer concentrations up to a plateau region. The plateau is obtained for a n equilibrium concentration of 20 g//L and a r value of 2 mg/m2 was determined. Although that the fluorescence study shows that DexP chains start to associate in solution a t polymer concentrations higher than 2 g/L, this adsorption isotherm does not indicate aggregates or multilayer adsorptions. Thus one may assume that isolated polymer chains preferentially adsorb to PS-DVB particles until the surface is saturated,
2346 Langmuir, Vol. 11, No. 7, 1995
Letters Table 1. Adsorbtion of BSA onto Various Coated PS-DVB Particles adsorbed BSA Des (mg/m2PS-DVB) (mg/g PS-DVBY 0 203 176 0.3 138 0.9 61 1.2 1.6 40 1.7 39 1.8 36 a
BSA adsorptionexperimentscarriedout in 0.05M citratebuffer
pH 5. Initial BSA concentration = 5 g/L.
n
0
I
I
I
I
10
20
30
40
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I
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DexP equilibrium concentration (g/l) Figure 3. Adsorptionisotherm of DexP on PS-DVB particles in water at 20 "C.
ou 0
I
1
1
I
5
10
15
20
25
BSA equilibrium concentration (g/l) Figure 5. Adsorption isotherm of BSA on coated PS-DVB particles (1.8 mg of DexP/m2 of PS-DVB) in 0.05 M citrate or 0.05 M phosphate buffer pH 7 (0)and on buffer pH 5 (0) uncoated PS (H pH 5 or 0 pH 7).
N E l E2 E3 E4 E5 E6 B1 B2 B3 B4
Figure 4. Percentagesof DexP desorbedfiom coatedparticles (2 mg of DexP/m2 of PS-DVB) during cross-linking (dotted area) and SDS treatment (gray area). For cross-linkingconditions, see Experimental Section. E l to E6 were cross-linked with EpCl (respectively 0.08,O.16,0.4,0.6,0.8, andl.6 mol of EpCVL). B1 to B4 were cross-linked with BDGE (respectively 0.16, 0.4, 0.8, andl.6 mol of BDGEL). N = non-cross-linked dextran layer. within the studied polymer concentration range. The polymer backbone probably adsorbsas loops on the surface and phenoxy groups act as contact points with the particles. In order to avoid the desorption of dextran in the presence of hydrophobic species such as proteins or surfactants, the adsorbed polymeric layer was stabilized by cross-linking, either with BDGE or with EpC1, and the amount of DexP released during the reaction was determined. As can be seen in Figure 4, the higher the crosslinking reagent concentration, the higher the dextran desorption. This effect is more pronounced with BDGE, which is a more hydrophobic molecule than EpC1. Thus cross-linking reagents are expected to compete with phenoxy groups of modified dextran for PS-DVB binding sites. Then the coated particles were washed with a 2% SDS (w/w)solution,in order to desorb the not tightly bound DexP and thus to evaluate the stability of the cross-linked layer. Under these conditions, the amount of dextran released from a non-cross-linked layer approaches 50% (Figure 4). From this study, it is noteworthy that the
chemical nature and the amount of the cross-linking reagent are important parameters. This results in a big difference in the final amount of adsorbed DexP. From our experiments, it can be concluded that reaction with 0.6 M EpCl(O.15 mol/g adsorbed DexP) is a good way to stabilize the dextran layer since only 15% of polymer desorbed after cross-linking and SDS treatment. BSA was used as a test protein to study the hydrophilization efficiency of PS-DVB surfaces. Various samples of beads, differing in surface DexP concentration were prepared, using EpCl as cross-linkirigreagent. Then the adsorption of BSA on these samples was investigated. In these experiments, the initial BSA concentration was 5 g/L and the pH of the medium was 5. At this pH, BSA has no net charge (isoelectric point of the protein) and hydrophobicinteractions are clearly evidenced. As it can be seen in Table 1, the hydrophilization efficiency was found to increase by increasing the adsorbed amounts of DexP since the binding capacity for BSA decreased. Figure 5 shows the BSA adsorption isotherms on coated (coating corresponding to the plateau polymer adsorption) and uncoated PS-DVB beads at pH 5 and 7. Even at relatively high protein concentrations, DexP coating significantly decreases the BSA adsorption since the relative adsorption of the protein, i.e., compared to that onto uncoated surface, is close to 15%a t both pH 5 and 7. These results suggest either that the surface coating is not totally impermeable or that a few hydrophobic groups of DexP are oriented toward the outside of the coated beads and are involved in the protein adsorption. This preliminary work shows that the introduction of phenoxy groups on dextran leads to a derivative which
Letters can be adsorbed on polystyrenic surfaces by means of hydrophobic sites. Then the adsorbed layer can be stabilized by chemical cross-linking. This treatment provides surfaces on which the protein adsorption is strongly reduced. This should find many biomedical and biotechnological applications. Particularly, such a procedure could provide hydrophilic HPLC supports not only for gel filtration but also for ion-exchange, hydrophobic, and affinity chromatography since the dextran layer can be easily derivatized by classical methods. However, before studying such applications, it should be possible to improve the hydrophilization procedure through the
Langmuir, Vol. 11, No. 7, 1995 2347 optimization of both the phenoxy group content and the adsorption conditions of dextran, in order to minimize the amount of residual hydrophobic sites on the coated PSDVB surface and thus to further reduce the protein adsorption.
Acknowledgment. We thank Peter G. Cartier of Rohm and Haas Co. for the gift of the beads used in this research. Thanks also to Dr. M. L. Viriot for his help during the fluorescence measurements. LA9501997
