Langmuir 1991, 7,2412-2414
2412
Hydrophilization of Polystyrene Surfaces with Ethyl(hydroxyethy1)cellulose Martin Malmsten,'*+Bj6rn Lindman? Krister Holmberg3 and Carina Brink% Physical Chemistry 1, Chemical Center, P.O. Box 124, S-221 00 Lund, Sweden, and Berol Nobel AB, S-444 85 Stenungsund, Sweden Received March 6, 1991
Introduction In many practical applications,it is of significant interest to modify the surface properties of materials. This is particularly true in several biomedical applications.' Hence, surface hydrophilization has proven to be very interesting for applications where it is essential to avoid immunoreactions,e.g., in connectionwith transplantations. Hydrophilization is also of a great potential importance for conditioning of contact lenses and in connection with food processing. The basic mechanism in these applications is the reduction of protein adsorption.' This can be achieved in several ways, but one of the most efficient ways, and certainly the easiest one, is by means of adsorption of (water-soluble)polymers. Several requirements must then be met. Firstly, it is essential that the polymer adsorbs strongly, in order to avoid desorption of the polymer in the presence of the protein solution. Secondly,the protein should not be able to adsorb on top of the adsorbed polymer layer. This, in turn, requires elimination of significant electrostatic and hydrophobic attractive interactions between the protein moleculesand the adsorbed polymer layer. Thirdly, the adsorbed polymer layer should be sufficiently thick to eliminate any significant interaction between the protein and the underlying surface. Especially consideringthe two latter requirements, nonionic hydrophilic polymers (e.g., poly(ethylene oxide), PEO) are ideal, and accordingly, these polymers have been extensivelyused for hydrophilization purposes.'J These polymers, however, do not absorb very strongly at hydrophobic surfaces3and desorb easily when exposed to protein solutions. One way to circumvent this is to use block copolymer^^^ (e.g., of the PEO-PPO-PEO type, PPO being poly(propy1ene oxide)). In these polymers, the relatively hydrophobic PPO block acts an an anchor, thus providing extensive and strong adsorption, while the hydrophilic PEO blocks extend into the solution, providing steric stabilization. The major drawback with these block copolymersis their low molecular weight, which facilitates protein-induced polymer desorption and, hence, reduces the efficiency of the "hydrophilization" layer. Furthermore, due to the low molecular weight, these polymers desorb extensively on dilution.3 A high molecular weight polymer of a finite polydispersity, by contrast, does not.6 t t
Experimental Section Materials. The polymer used, EHEC, is a nonionic cellulose ether, which was supplied by Berol Nobel AB,Sweden. The EHEC fraction used has an average molecular weight of 250 000, as determined by light scattering, but a high polydispersity, as evidencedby GPC (results not shown). The degree of substitution of ethyl groups is 1.4, while the molar substitution of ethylene oxide groups is 0.9. The cloud point (CP) of a 0.1 wt % aqueous solution of this particular fraction is 39 "C on heating.' Dry EHEC powder normally contains3-5 % (w/w) ofNaCl (impurity from synthesis) and therefore the EHEC solutionswere dialyzed against membrane filtered water (Millipore,USA) for 5 days. As a dialyzing membrane regenerated cellulose with a molecular cutoff of 6000 was used (Spectrum Medical Industries, USA). After dialysis the polymer was freeze-dried. (6) CohenStuart,M. A.;Coegrove,T.;Vincent,B.Adu. Colloidlnterface Sci. 1986,24, 143. (7) Malmsten, M.; Lindman, B. Langmuir 19W,6, 357. (8)Malmsten, M.; Claesson, P. M.; Pezron, E.; Pezron, I. Langmuir 1990, 6, 1572.
Physical Chemistry 1.
Berol Nobel AB. (1) Kim, S. W.; Fei'en, J. Crit. Rev. Biocompat. 1986, 1 , 229. (2) Merrill, E. w.; ialz man, E. W.; Dennieon, K. A.; Tay, S.-W.; Pekala,R. W.InProfiressinArtificia1Orfiam-l98&ISAOPreee: Cleveland, OH, 1986. (3) Tiberg, F.; Malmsten, M.; Lime, P.; Lindman, B. Langmuir, in nrem. r - --.
One type of polymer, which possesses all the desired properties discussed above, is ethyl(hydroxyethy1)cellulose (EHEC),the adsorption properties of which have been extensively studied previ~usly.~-~l Thus, EHEC adsorbs extensively at hydrophobic surfaces (cf. Figures 1and 2), forming thick adsorbed layers."'O Furthermore, EHEC adsorbs strongly at these surfaces, as evidenced by direct surface force measurements.SJ' On rinsing, only a very small fraction ( < l o % ) of the adsorbed polymer molecules desorbs, due to the high molecular weight and the polydispersity of this p0lymer.~18JlFinally, EHEC is manufactured in large quantities and is inexpensive, and its toxicity is 10w.l~ Accordingly, an increasing interest has been devoted to EHEC as a hydrophilizating agent.13J4 So far, however, no studies, taking the adsorption/ interaction properties of EHEC into account, have been presented. Like other EO-containing polymers and surfactants, EHEC shows a reversed temperature dependent phase behavior and a lower consolutetemperat~re.~J~ The lower temperature phase boundary is often referred to as the cloud point (CP). CP depends on the molecular weight and, more importantly, on the degrees of substitution. It is also affected by cosolutes, like electrolytes, alcohols, and s u r f a ~ t a n t s . ~ JBy ~ Jvariation ~ of these parameters, phase separation temperatures (in water) ranging from less than 0 "C to more than 100 "C can be achieved,which is of particular interest, since the adsorption properties of EHEC are strongly influenced by the proximity to CP.7-11 As CP is approached, the adsorbed amount increases (Figure 2) at the same time as the adsorbed layer ~ o n t r a c t s .The ~ ~ interaction between two EHEC-coated hydrophobic surfaces is strongly repulsive up to temperatures slightly above CP. At temperatures even higher, the interaction is attractive.819 Considering the interesting and fairly well-studied solution and adsorption properties of EHEC, we decided to investigate its "hydrophilization" efficiency. In this introductory study, we further decided to work with polystyrene surfaces, due to their versatile applicability.
(4) Lee, J. H.;Kopeckova, P.; Kopecek, J.; Andrade, J. D. Biomuteriala 1990, 1 1 , 455. (5) Lee, J. H.; Kopecek, J.; Andrade, J. D. J.Biomed.Mater.Res. 1989, 23, 351.
(9) M h t e n , M.; Claesson, P. M. Langmuir 1991, 7,988. (10) Claesson, P. M.; Malmsten, M.; Lindman, B. Langmuir 1991, 7, 1441. (11) Pezron, I.; Pezron, E.; Claesson, P. M.; Malmsten, M. Langmuir 1991, 7, 2248. (12) Communication with the manufacturer. (13) Oleson.J.; Hellsten, M.: Holmberg, - K. Submitted for publication
in Colloid Polym. Sci. (14) Karlseon, Ch.; Carlaeon, A.; Stenberg, M.; Nygren, H. Submitted for Dublication in Colloid Polvm. Sci. 0 5 ) Lindman, B.; Carlaeon, A.; Karlatr6m, C.; Malmten, M. Adu. Colloid Interface Sci. 1990,32, 183.
0743-7463/91/2407-2412$02.50/0 0 1991 American Chemical Society
Langmuir, Vol. 7, No. 10,1991 2413
Notes
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0.00
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0.10
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0.15
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0.20
Conc.lwt %
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030 035 0.20 Conc./%w/w Figure I. Adsorption isotherm (at 30 OC) for EHEC at hydrophobized silica surfaces.' 0
0.05
0.05
Figure 3. Relative adsorption of IgG at polystyrene surfaces at different concentrations of EHEC. Filled triangles represent adsorption at 25 OC, open circles adsorption at 40 OC, and f i e d circles adsorption at 40 OC followed by cross-linking. polymer. Each point represents the mean value of three measurements.
and Discussion Since EHEC possesses most of the properties desired for a hydrophilizing agent, we decided to examine the efficiency with which EHEC reduces protein adsorption. In Figure 3, we show the results of these preliminary experiments. Three different sets of experiments were performed. In the first, hydrophilization was achieved by adsorption at 25 O C , while in the second, the adsorption temperature was 40 OC (CP + 1OC). In the third set of experiments,hydrophilization was achieved by adsorption of EHEC at 40 OC followed by cross-linking of the adsorbed EHEC layer at the same temperature. As can be seen in Figure 3, EHEC is fairly efficient in reducing the IgG adsorption already at 25 OC. At low polymer concentrations, however, there is a reduction of the 'hydrophilization efficiency' (this is true also at 40 OC). This effect is most likely due to the lower adsorbed amount of EHEC at these concentrations (cf. Figure 1). Hence, we infer that plateau polymer adsorption is required in order to obtain a maximum reduction of the IgG adsorption. As the temperature is raised to 40 "C (CP + 1 "C), there is a strong increase in the amount of EHEC adsorbed (Figure2), at the same time as the adsorbed polymer layer becomes more dense.74 As can be seen in Figure 3, this results in afurther reduction of the IgG adsorption. This result may seem somewhat surprising, considering that the EHEC phase separates at this temperature. However, it is in line with previous results, where it was found that the interaction force between hydrophobic surfaces coated with EHEC is repulsiue even at temperatures slightly above CP. This, in turn, was attributed to the possible orientation of the polymer at hydrophobicsurfaces and to the very high polymer concentration in the adsorbed layer at higher temperature^.^^^ Although the analogy with the present result is not complete, similar mechanisms are most likely operative in both cases. In order to further reduce the IgG adsorption, we crosslinked the adsorbed polymer layer after adsorption at 40 "C. As can be seen in Figure 3, this indeed gives an even more pronounced reduction of the IgG adsorption. This result is an interesting one, since it shows that not only the polymer-surface interaction is of importance but also the lateral interactions in the adsorbed polymer layer. Again, this finding ie well in line with previous results on the interaction between EHEC-coatedsurfaces,where the importance of lateral interactions in the adsorbed layer bSUltS
Figure 2. Adsorbed amount of EHEC at hydrophilic and hydrophobized silica surfaces as a function of temperature. The EHEC concentration was 0.1 wt % .7 The polystyrene surfaces (havinga surface charge of 1e-/25-50
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A*) were from NUNC, Denmark. Cross-linking was achieved by the tetraepoxide of ethoxylated (320 EO) di(trimethylo1)propane (M, 14000-15000),16which is a noncommercial product from Berol Nobel AB, Sweden. All chemicals used in the ELISA experiments (immunoglobulin G (IgG), rabbit immunoglobulins, peroxidase-conjugated immunoglobulins (rabbit), and 1,2phenylene diamine dihydrochloride (ODP)) were obtained from Dakopatts, Denmark. Method. Enzyme-linked immunosorbent assay (ELISA) was performed with polystyrene surfaces. The surfaces were first incubated with the EHEC solutions at the experimental conditions desired for 2 h, in order to obtain equilibrium adsorption. Subsequently, the surfaces were rinsed with (preheated) distilled water. The surfaces were then either incubated for 1h with the IgG solution (1.8 X 1O-amg/mL IgG in a 0.05 M phosphate buffer (pH 7.0)) or cross-linked (10% cross-linking agent in 1M NaOH for 5 h) before being contacted with the IgG solution. The surfaces were then rinsed with (preheated) distilled water once more. A mixture of peroxidase-conjugated and *normal" immunoglobulins (1:4) in a buffer containing 0.1 M NaZHPOd, 1.0 M NaCl, and 0.2 wt % Tween 20 (pH 7.0) was added, and the surfaces were incubated for 30 min. Again, the surfaces were rinsed with distilled water. A buffer solution (0.1 M NalHPO, and citric acid (pH 5.0)) containing OPD was added, followed by the addition of Ha02 (30%). The oxidation was terminated after 180 s by addition of 5 M HzSO,. Finally, the absorbance was determined at 490 nm. The relative amount of IgG adsorbed was defined as the ratio of the amount of IgG adsorbed on EHEC treated plates to that on untreated plates and was obtained as the absorbance ratio in the presence and absence of adsorbed (16) Bergatram,K.; Holmberg,K.; Safranj, A.; Hoffman, A. S.; Edgell, M.J.; Hovanes, B. A,; Harris, J. M. Submitted for publication in Biomaterials.
2414 Langmuir, Vol. 7,No. 10, 1991
Notes
on the interaction forcewas clearlydemonstrated by means of surface force measurements.ll
the adsorbed polymer layer, with respect to both dilution and mechanical wear.
Conclusions
Acknowledgment. This work was financed by Berol Nobel AB and the Swedish National Board for Technical Development (STU). Registry No. Polystyrene, 9003-53-6; ethyl(hydroxyethy1)-
EHEC is very efficient in reducing the adsorption of IgG to polystyrene, especially close to CP. Cross-linking of the adsorbed polymer layer enhances this efficiency and is also expected to increase the long-term stability of
cellulose, 9004-58-4.
