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Langmuir 1999, 15, 5136-5140
Molecular Interactions of Biomolecules with Surface-Engineered Interfaces Using Atomic Force Microscopy and Surface Plasmon Resonance Simon L. McGurk, Rebecca J. Green,† Giles H. W. Sanders, Martyn C. Davies, Clive J. Roberts,* Saul J. B. Tendler, and Philip M. Williams Laboratory of Biophysics and Surface Analysis, School of Pharmaceutical Sciences, The University of Nottingham, Nottingham NG7 2RD, U.K. Received December 31, 1998. In Final Form: April 13, 1999 We have used albumin-modified atomic force microscope (AFM) tips to probe interactions with a range of hydrophilic polymer brush surfaces and protein. Copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) (Pluronics) adsorbed onto polymer interfaces have been shown in previous studies to modify adsorption properties of blood components [using surface plasmon resonance (SPR) and AFM]. Here we have employed protein-coated AFM probes to study a series of PEOPPO-PEO-coated interfaces prepared with a range of PEO and PPO molecular weights. Subsequent force-distance experiments have shown a good correlation between the forces of adhesion of an albuminfunctionalized AFM probe with the various PEO-PPO-PEO surfaces and the adsorption trends of albumin onto these polymeric surfaces observed with SPR. The data suggest that the size of the hydrophobic PPO segment of the Pluronic is a major determinant of the polymer protein resistance properties. In addition, as the PEO layer density increased, a reduction of interaction force was measured because of the formation of a steric barrier from the PEO polymer brush. Such studies suggest that AFM may be employed as a novel method to assess “biocompatibility” and to rapidly screen surface-engineered surfaces with micrometer spatial resolution.
Introduction The fate of in vivo polymeric devices depends critically on the surface chemistry employed. Recognition of foreign materials by a living body is a consequence of the adsorption of plasma proteins and subsequent platelet and leucocyte activation, hence inducing blood coagulation, fibrinolysis, and thrombus formation. In the case of drugdelivery particles, these actions result in the foreign device being quickly and efficiently removed from the body.1 While many drug-delivery systems implement selective interactions, poor biocompatibility remains a major problem for other drug-delivery techniques. In recent years surface biomedical engineering has taken two approaches to overcome these problems: increasing the biocompatibility of such engineered surfaces and promoting selective interactions for the use in immunosensors and drugtargeting systems.2-4 A major step forward in the search for increased biocompatibility was the development of block surfactants. One of the most common and widely used of these is a triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) PEOn-PPOm-PEOn (manufac* Corresponding author. Tel 44 (0)115 9515063, Fax 44 (0)115 9515110, Email
[email protected] † Current address: Polymer & Colloids Group, Department of Physics, Cavendish Laboratory, University of Cambridge, Madingley Road, Cambridge CE3 0HE, U.K. (1) Poot, A. A.; Beuleling, T. In Modern Aspects of Protein Adsorption on Biomaterials; Missilis, Y. F., Lemm, W., Eds.; Kluwer: The Netherlands, 1991, pp 29-38. (2) Ikada, Y. In Polymers of Biological and Biomedical Significance; Shaleby, S. W., Ikada, Y., Langer, R., Williams., J., Eds.; ACS Symposium Series 540; American Chemical Society: Washington, DC, 1994; pp 35-48. (3) Williams, D. F., Vol. Ed. In Materials Science and Technology 14, Medical and Dental Materials; Cahn, R. W., Haasen, P., Kramer, E. J., Eds.; VCH: Weinheim, Germany, 1992; pp 1-27. (4) Davis, S. S.; Hunneyball, I. M.; Illum, L.; Radcliffe, J. H.; Smith, A.; Wilson, C. G. Drugs Exp. Clin. Res. 1985, 11, 633.
tured by BASF under the name Pluronics) (Figure 1). Initially developed for their use in the chemical, pharmaceutical, and cosmetics industries,5 they have since been recognized for their possible role as surface blockers to protein encrustation.6 It is the differing solubilities of the two components which give rise to their novel properties, with PPO being hydrophobic and PEO hydrophilic. The accepted theory explaining its proteinrepellant nature describes how the hydrophobic PPO chain provides the necessary anchor for the polymer molecule to remain adsorbed at an interface. The PEO chain is then allowed to develop into the solvent phase7 creating steric repulsive forces provided by the extended, highly hydrated, brushlike PEO layer at the surface, hence giving rise to its protein-resistant nature. Previous studies have shown that physisorbed PEO may be dislodged by more surface-active biomolecules such as fibrinogen, suggesting that the PPO portion of the copolymer has great stabilizing importance.8 There have been many studies implementing Pluronic copolymers for stabilization of colloidal dispersions,9 site-specific targeting of colloids,10,11 and proteinrepellant coating for biomedical implants.12 In response to this growth in activity, many techniques have also been developed and adapted to understand both the adsorption of the surfactant copolymers and their protein-resistant (5) BASF Technical Brochure; BASF Co.: Parsippany, NJ, 1989. (6) Lee, J. H.; Martic, P. A.; Tan, J. S. J. Biomed. Mater. Res. 1989, 23, 351. (7) Alexandridis, P.; Holwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414. (8) Amiji, M.; Park, K. Biomaterials 1992, 13, 682. (9) Brindley, A.; Davis, S. S.; Davies, M. C.; Watts, J. F. J. Colloid Interface Sci. 1995, 171, 150. (10) Norman, M. E.; Williams, P.; Illum, L. Biomaterials 1993, 14, 193. (11) McGurk, S. L.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M., in preparation. (12) Green, R. J.; Tasker, S.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Langmuir 1997, 13, 6510.
10.1021/la981788q CCC: $18.00 © 1999 American Chemical Society Published on Web 06/19/1999
Molecular Interactions of Biomolecules
Figure 1. Chemical structure of the Pluronic polymers.
behavior. Ellipsometry,13 X-ray photoelectron spectroscopy,14 and fluorescence techniques15 have been employed to investigate the adsorption characteristics, and reflectometry,16 SDS-PAGE gels,10 and radiolabeling8 have been implemented for protein-resistance studies. In this paper we have used two complimentary techniques, atomic force microscopy (AFM) and surface plasmon resonance (SPR), to explore the extent of protein resistance displayed by a range of Pluronic copolymers. Significant advances have been made in SPR over the past few years as a biophysical biosensor, enabling adsorption processes at surfaces of many soluble and insoluble systems to be monitored in real time with extreme accuracy. Davies has described many potential applications of SPR to probe interaction characteristics of material surfaces.17 SPR has also been used to measure the optical properties of metal films,18 to provide information about adsorbed films and molecules onto surfaces such as Langmuir-Blodgett films,19 the adsorption of gas molecules,20 and protein adsorption to Al, Cu, and modified Ag film surfaces,21 and to study dynamic interactions of the molecule-ligand-receptor type,22 the adsorption of micellar polymeric systems and subsequent protein adsorption,23 and other areas of biocompatibility.24 In SPR the angle at which incident light induces surface plasmons is directly related to the dielectric properties of the material in contact with the sensor surface. Therefore, a material being deposited on the surface of the SPR sensor will result in a shift in the resonant angle of the laser and is measured in millidegree angles (mDA). Previous studies have shown that the magnitude of the shift of the SPR angle is dependent on the thickness of the deposited film.25 In this study we have adsorbed layers of different molecular weight Pluronics onto a polystyrene-modified silver SPR sensor and investigated their protein-resistant character. In addition to SPR, AFM was utilized in a novel fashion to probe the interaction of a protein-coated probe with the same Pluronic-coated surfaces. AFM offers an ideal tool for protein adsorption studies, in aspects of both high spatial resolution and convenient operation environment,26 and has been extensively utilized to quantify and (13) Tiberg, F.; Malmsten, M.; Linse, P.; Lindman, B. Langmuir 1991, 7, 2723. (14) Lee, J. H.; Andrade, J. D. Polymer Surface Dynamics Andrade, J. D., Ed.; Plenum Press: New York, 1988; pp 119-136. (15) Amiji, M.; Park, K. J. Appl. Polym. Sci. 1994, 54, 539-544. (16) Schroen, C. G. P. H.; Stuart, C.; van der Voort Maarschalk, K.; van der Padt, A.; van’t Reit, K. Langmuir 1995, 11, 3068. (17) Davies, J. Nanobiology 1994, 3, 5. (18) De Bruijn, H. E.; Kooyman, R. P. H.; Greve, J. Appl. Opt. 1992, 31, 440. (19) Lawrence, C. R.; Martin, A. S.; Sambles, J. R. Thin Solid Films 1992, 208, 269. (20) Leidburg, B.; Nylander, C.; Lundstrom, I. Sens. Actuators, B 1983, 4, 299. (21) Silin, V. I.; Balchytis, G. A.; Yakovlev, V. A. Opt. Commun. 1993, 97, 19. (22) Bondeson, K.; Frostell-Karlsson, A.; Fagerstam, L.; Magnusson, G. Anal. Biochem. 1993, 214, 245. (23) Davies, J.; Allen, A.; Burrus, Y.; Bruce, I.; Heaney, P. J.; Hemming, F. A.; Nunnerley, C. S.; Skelton, L. Proceedings of the International Conference of Surface Properties of Biomaterials; Butterworth-Heinemann Ltd.: Cambridge, U.K., 1994; pp 117-132. (24) McGurk, S. L.; Green, R. J.; Hartshorne, M. S.; Stolnik, S.; Davies, M. C.; Davis, S. S.; Illum, L.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. in preparation. (25) De Bruijin, H. E.; Kooyman, R. P. H.; Grewe, J. Appl. Opt. 1993, 32, 2426.
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locate specific molecular interactions with a sensitivity to single molecular bonding forces.27-33 The interaction between the probe and the sample when carrying out such force-distance experiments is dependent on both the probe and sample surface chemistry, geometry, and solution environment. Nevertheless, these probe-sample interactions have been studied at length using a variety of approaches.34-38 In this study we control the surface chemistry and probe geometry by attaching human serum albumin (HSA)-functionalized glass spheres to the AFM cantilevers. To determine the level of HSA interaction with a particular Pluronic, the magnitude of the nonspecific adhesion forces between such a protein-coated probe and the Pluronic-coated surface was then determined. The advantages of using a functionalized bead as opposed to a coated AFM probe include an increase in the probesample interaction area, and hence force magnitude, and a reduction in problems arising because of variable probe geometry and sample coating inconsistencies. However, in using such a probe, spatial resolution of the AFM, while still high, is reduced to approximately a few micrometers. This method of force measurement with a probe of large radius has been termed continuum force microscopy (CFM), where the forces being measured are not atomic but are across a continuum.39 In this paper we show that force-distance experiments performed with AFM display excellent correlation with the results obtained from SPR. The adsorption data obtained from SPR has revealed how varying the length of PPO changed the adsorption profiles of proteins to various Pluronic polymer coatings. The magnitude of this interaction between protein and Pluronic samples is in agreement with force-distance experiments and is part of an ongoing project to develop rapid screening methods to explore the in vitro capabilities of biomaterials. Materials and Methods Sample Preparation. A solution (0.5% w/v) of polystyrene (PS; MW ) 2000 g mol-1, Aldrich Chemical Co. Ltd., Dorset, U.K.) was used to coat the SPR slides (Ortho Clinical Diagnostics, Chalfont St. Giles, U.K.). PS films approximately 50 nm thick were prepared by spin casting the polymer solution onto the SPR sensor at 1000 rpm. Copolymers of the Pluronic range (BASF Co., Parsnippany, NJ) were selected because their varied protein adsorption properties.40 These polymers are shown in Table 1. The Pluronic solutions (0.1% w/v) were prepared using aqueous phosphate buffer (10 mM, pH 7.4). SPR Analysis. SPR analysis was performed on an instrument constructed by Ortho Clinical Diagnostics (Ortho Clinical (26) Hansma, H. G.; Hoh, J. H. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 115. (27) Hoh, J. H.; Cleveland, J. P.; Prater, C. B.; Revel, J. P.; Hansma, P. K. J. Am. Chem. Soc. 1992, 114, 4917. (28) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415. (29) Moy, V. T.; Florin, E.-L.; Gaub, H. E. Science 1994, 266, 257. (30) Stuart, J. K.; Hlady, V. Langmuir 1995, 11, 1368. (31) Lee, U. G.; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 771. (32) Allen, S.; Chen, X.; Davies, J.; Davies, M. C.; Dawkes, A. C.; Edwards, J. C.; Roberts, C. J.; Sefton, J.; Tendler, S. J. B.; Williams, P. M. Biochemistry 1997, 36, 7457. (33) Chen, X.; Davies, M. C.; Roberts, C. J.; Tendler; S. J. B.; Williams, P. M. Langmuir 1997, 13, 4106. (34) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1991; Chapters 9-15. (35) Tabor, D.; Winterton, F. R. S.; Winterton, R. H. S. Proc. R. Soc. London, A 1969, 312, 435. (36) Derjaguin, B. V.; Rabinovish, Y. I.; Churaev, N. V. Nature 1978, 272, 313. (37) LeNeveu, D. M.; Rand, R. P.; Parsegian, V. A. Nature 1976, 259, 601. (38) Prieve, D. C.; Frej, N. A. Langmuir 1990, 6, 396. (39) Gracias, D. H.; Somorjai, G. A. Macromol. 1998, 31, 1269. (40) Green, R. J.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 1997, 18, 405.
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Figure 2. Optical photograph of the bead attached to the AFM cantilever.
Figure 3. SPR adsorption profiles of albumin on a model PScoated SPR slide. The SPR trace levels out at approximately 120 mDA, suggesting a monolayer albumin coverage.
Table 1. monomer units Pluronic
MW
PEO
PPO
PEO
L35 L61 F127
1900 2000 11500
11 3 98
16 30 67
11 3 98
Diagnostics, Chalfont St. Giles, U.K.). It utilizes a monochromatic laser light source (780 nm) focused onto a glass slide coated with approximately 50 nm of silver. Optical coupling between the prism and the SPR slide was achieved with index matching oil (BDH, Poole, U.K.). The instrumentation has been described elsewhere in detail,40 but in short the totally internally reflected laser light was analyzed by a two-dimensional array of charge-couple detectors (CCD) and the data were recorded with an IBM PC computer. Three flow channels were employed on the surface of the SPR sensor by means of clamping a water-tight, thermoregulated (33 ( 0.1 °C) liquid flow unit onto the sensor surface. AFM Cantilever Adaption and Calibration of Interactional Forces. Individual AFM cantilevers (Topometrix Corp., Saffron Walden, Essex, U.K.) were modified by adhering a single spherical bead to the probe end of the cantilever (see Figure 2). Amine functional glass beads purchased from Polysciences Inc. (Warrington, PA) were utilized to enhance the interaction area of the probe for force-distance experiments. The diameter of these beads was 47 ( 2 µm, and they had a density of 2.48 g cm-3. They were adhered to the cantilever with Aryldite-Rapid Set Epoxy (Ciba-Giegy Plastics, Duxford, U.K.) using micromanipulation techniques. The bead-adapted cantilevers were left for 24 h to allow the adhesive to cure. Protein was immobilized to the bead by activating the amine termini on the bead with gluteraldehyde (1% solution for 2 h). The tip was then washed and incubated in a solution of albumin (0.1 wt %, Sigma, Dorset, U.K.) for 12 h. Before force-distance experimentation, the tip was rinsed in phosphate buffer (pH 7.4, 10 mM) to remove any loosely bound albumin. AFM Analysis. Force measurements were recorded using a Topometrix Explorer (Topometrix Corp., Saffron Walden, Essex, U.K.). Experiments were performed in freshly prepared filtered phosphate buffer (10 mM, pH 7.4). The force measurements were obtained between albumin-functionalized modified AFM probes and various polymer-coated SPR slides. The spring constant (k) of the individual cantilevers was determined using the resonant frequency method outlined by Cleveland et al.41
Figure 4. SPR data illustrating the effect of adsorbed Pluronic on the subsequent adsorption of albumin compared to PS. The pluronic L35 suppressed the adsorption of albumin to approximately 35%. The Pluronic L61, having the greater PPO segment and less PEO segments than its L35 counterpart, managed to reduce the adsorption of the albumin to approximately half this amount. The higher-molecular-weight Pluronic, F127, completely inhibited adsorption.
SPR Investigation of Pluronic Coatings. The SPR experimental protocol involved a two-stage analysis. First, the soluble Pluronic system was adsorbed onto the surface of the SPR sensor and, second, the surface was treated with albumin (0.05 wt %) to grade the affinity of the surface for this protein. PS was chosen as the model hydrophobic substrate, to produce a strong interaction between the hydrophobic segment in the Pluronic system and sub-
strate. Figure 3 shows the extent of interaction between albumin and PS. The SPR trace leveled out at approximately 120 mDA. This result suggests a monolayer albumin coverage was achieved with a strong interaction between albumin and PS.12 The three Pluronic systems chosen were selected because of their varied protein-resistant properties. The molecular weights and block sizes of the chosen Pluronics are shown in Table 1. In Figure 4, it is possible to see the effect of adsorbed Pluronic on the subsequent adsorption of albumin. It is clear that all of the Pluronic systems chosen considerably reduce the extent of albumin adsorption. Pluronic L35 suppressed the adsorption of albumin to an extent of 35% of monolayer coverage, achieving an SPR shift of approximately 30 mDA. However, the much higher molecular weight Pluronic, F127, was able to completely inhibit adsorption. The Pluronic L61, having the greater PPO segment and less PEO segments than its L35 counterpart, managed to reduce the adsorption of the albumin to 17% of monolayer coverage. Previous studies have outlined two competing factors in the protein-resistant nature of Pluronic polymer systems.8,10 The first theory argues that a large PPO portion of the polymer is the underlying key.8 This enables the Pluronic to achieve a strong hold to a surface which is resistant to desorption and also allows a relatively small segment of PEO to extend into the aqueous media, creating the steric barrier necessary for protein resistance. Recently in support of this theory, Green et al. have shown that for low-molecular-weight Pluronics a significant, yet not complete, reduction in albumin adsorption is observed whereas higher-molecular-weight Pluronics completely inhibit adsorption and that it is the increase in the PPO block size of the copolymer which increases protein resistance.42 The second theory maintains that it is not
(41) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 403.
(42) Green, R. J.; Davies, M. C.; Roberts, C. J.; Tendler S. J. B. J. Biomed. Mater. Res. 1998, 42, 165.
Results and Discussion
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Figure 5. AFM force-distance curves showing the adhesion of a functionalized tip to (a) PS and (b) PS/albumin surfaces. The hysteresis between the approach and retract traces in part a indicates strong adhesion.
the size of the PPO segment which has utmost importance but the size of the PEO segment.10 Here it is explained that a larger PEO segment gives rise to better protein resistance because of the increased steric effect. The L35 and L61 data presented here support the critical role that the PPO segment has in imparting protein resistance and also suggest that an additional issue of packing density is required to fully explain the increased protein resistance. As the molecular weight and hence size of the Pluronic polymer molecule increases, the adsorbed packing density decreases because of the steric effect of the polymer. For such circumstances, complete protein resistance is achieved when the PEO chains are increased in molecular weight (F127). Where smaller chains exist (L61) there is an insufficient surface density of the hydrophilic PEO polymer to create a steric barrier. AFM Investigation of Pluronic Coatings. To ensure appropriate correlation with the SPR data, PS-coated silver SPR sensors were utilized as substrates for the AFM force-distance experiments. The silver SPR substrate has been shown to be quite rough and, therefore, not ideally suited to force-distance experiments; however, when coated with a thin PS layer of polymer, all underlying features are completely masked.12 The Pluronic systems were then deposited onto this PS layer and subsequent force-distance experiments performed. AFM imaging of these polymers and protein adsorption studies has been previously recorded12,43 where it was shown that albumin exhibited a monolayer covering of the PS coating after SPR investigation. AFM investigation of those Pluronics employed here show that a complete coverage of the silver slides is achieved.43 Force-Distance Experiments on the PS Surface. The level of the adhesion between the albumin-functionalized bead-modified tips was validated by performing force-distance experiments on the PS surface alone and PS surfaces treated with albumin. Figure 5 illustrates the magnitude of interaction of an albumin-functionalized tip with the PS surface before and following albumin treatment. The profiles of such force data have been explained previously,33 but in short the tip is brought (43) Green, R. J. Protein/Polymer Interactions investigated by Surface Plasmon Resonance. Thesis, The University of Nottingham, Nottingham, U.K., 1996.
Figure 6. AFM force-distance data showing the adhesion of a functionalized tip to PS-, L35-, L61-, and F127-coated surfaces.
toward the surface indicated by the horizontal line. The contact regime is illustrated by the sloped line of constant gradient and is caused by the deflection in the cantilever as more force is applied up to a preset setpoint. Upon retraction, an adhesion force may be seen if the sloped line carries on past the point of original contact and a hysteresis is developed (as displayed in Figure 5a). This shows that the adhesion force between the tip and the substrate is large enough to overcome the spring force of the cantilever. It is not until the spring force exceeds the adhesion force that the tip eventually “snaps” back to the original noncontact position (termed the “snap-off” point). The size of this hysteresis is directly related to the size of the adhesion force (large hysteresis ) large force). When the retract trace mimics the approach trace (as displayed in Figure 5b) and no hysteresis is obtained, this indicates no adhesion between the tip and the surface. The SPR experimental values indicated a large adsorption affinity of the albumin protein molecules for the PS surface. The AFM in correlation displays a large adhesion force between the functionalized tip and the PS surface to a value of 7.0 ( 0.7 nN (n > 50). No adhesion forces were detected between the albumin-treated surface and the functionalized tip. This adhesion force is significantly larger than previously recorded adhesion between albumin-functionalized tips and PS.33 The explanation for this comes from the increased surface contact area of the bead attached to the cantilever. Separate AFM adhesion experiments with standard tips on these polymer brush systems would be difficult to compare because of varying tip geometry. When using a large bead attached to the cantilever, there is a much-reduced possibility of this probe developing through the PEO layer to the underlying PPO. In addition, it is possible that sharp asperities on functionalized tips may probe between the PEO brushes to the underlying hydrophobic PPO segments, producing false adhesion data.
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Figure 7. Graph showing a summary of the adhesion forces between the AFM probe and the modified surfaces.
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SPR data, we believe this is due to the increased size of hydrophobic portion in the polymer molecule, enabling it to bind more securely to the hydrophobic PS surface. This theory is further supported with the introduction of the Pluronic polymer F127. Here there is a large hydrophobic portion, enabling secure hydrophobic binding of the molecule from solution. There is also a large hydrophilic section, compared to L61, ensuring adequate steric resistance for reduced nonspecific protein adsorption. All of the forces measured between the modified tip and various surfaces have been summarized in Figure 7. Interestingly, an extremely good correlation was produced for the SPR and AFM data as shown in Figure 8. The data in each set were normalized by taking the maximum adhesion force of 7 nN and equating this to the maximum adsorption value of 115 mDA for the albumin on the PS-coated sensor. All of the other data values were graded linearly accordingly. It is clear from this chart that the forces of adhesion obtained from the AFM mimic the adsorption values recorded from the SPR. Hence, not only do these data provide new insights into the effects of molecular weight, surface coverage, etc., but they also introduce a novel fashion of screening biocompatible surfaces. Such AFM probes facilitate rapid screening possibilities and provide new information by virtue of the increased spatial resolution, raising the potential of mapping ‘biocompatibility” on heterogeneous surfaces. Conclusions
Figure 8. Graph representing the normalized SPR adsorption data and AFM adhesion data from Pluronic-coated surfaces.
Force-Distance Experiments on the Pluronic Systems. Figure 6 illustrates the effect of the Pluronic coatings on the adhesion characteristics of the proteinfunctionalized tip. The higher-molecular-weight Pluronic, F127, completely masked all adhesion nature between the modified tip and the surface. This is shown by the retrace following the in-trace throughout the movement of the cantilever and no hysteresis at the “pull-off” point. However, for the other two polymers L35 and L61, adhesion was demonstrated, albeit at a reduced level. The L35 and L61 polymers reduced adhesion to 2.8 and 1 nN, respectively. This equates to approximately 40% and 15% of the complete monolayer coverage for L35 and L61, assuming a direct correlation between the adhesion force and the protein coverage. L35 has a ratio of 11-16-11 (PEO-PPO-PEO) yet displayed a reduced effect of protein-resistance behavior compared to L61, which has a ratio of 3-30-3 (PEOPPO-PEO), even though the PEO chain is 3 times as long. As in the case of the conclusions drawn from the
In this study, we have shown that experiments from the AFM and the SPR have independently demonstrated the nature of the interaction between albumin and Pluronic polymer brush systems. We have proved that, using a probe with large surface contact area, it is possible to obtain accurate measurements of adhesion on softsegment polymer systems which could otherwise prove difficult to investigate with standard AFM techniques. These data support the supposition that the size of the hydrophobic segment of the Pluronic is the major determinant of the polymer protein-resistance properties and also that polymer packing density as affected by the polymer molecular weight is a factor. Such a combined AFM and SPR approach is being applied to develop an increased understanding of interactions of advanced drugdelivery systems such as bioadhesive polymers and polymer therapeutic systems designed for targeting sitespecific delivery. Acknowledgment. M.C.D., C.J.R., S.J.B.T., and P.M.W. thank the BBSRC for a studentship for S.L.M. and for a postdoctoral fellowship for G.H.W.S. and the EU Commission for studentship for R.J.G. LA981788Q
