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Langmuir 1997, 13, 6510-6515
Adsorption of PEO-PPO-PEO Triblock Copolymers at the Solid/Liquid Interface: A Surface Plasmon Resonance Study R. J. Green,† S. Tasker,† J. Davies,‡ M. C. Davies,*,† C. J. Roberts,*,† and S. J. B. Tendler*,† Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, The University of Nottingham, University Park, Nottingham NG7 2RD, U.K., and Johnson & Johnson Clinical Diagnostics, Nightingales Lane, Chalfont St. Giles, Buckinghamshire HP8 4SP, U.K. Received December 11, 1996. In Final Form: August 13, 1997X Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers are used in a wide range of industrial applications from detergents to pharmaceutical materials. We have employed surface plasmon resonance (SPR) to monitor the adsorption of these materials, from aqueous solutions to a model hydrophobic surface. The effect of varying the PPO or PEO block size of the copolymer on adsorption has been investigated, and a linear increase in SPR angle shift with increasing PPO or PEO chain length is observed. The SPR angle shifts show a greater increase in SPR angle with increasing PPO block size compared to an equivalent increase in PEO block size. This has been explained by considering the effect each block has on the dielectric properties of the adsorbed polymer at the interface. The presence of micelles in solution and their influence on Pluronic adsorption has also been considered, and adsorption from micellar solutions has been determined to be dependent on the PPO content of the copolymer.
Introduction Poly(ethylene oxide)n-poly(propylene oxide)m-poly(ethylene oxide)n (PEOn-PPOm-PEOn) triblock copolymers (Pluronics, BASF Co.) form a range of water soluble, nonionic amphiphilic surfactants, varying in their PPO and PEO block size. They have been widely used in the chemical and pharmaceutical industries as detergents,1 colloidal dispersion stabilizers,2 and in cosmetic products. Recently, interest has focused on the properties of adsorbed Pluronic layers and their role in reducing nonspecific protein adsorption and cell adhesion3-6 on biomaterial surfaces. The interfacial adsorption behavior of block surfactants from solution has been extensively studied,7-11 and it is their amphiphilic nature, which arises due to the differing solubilities of the copolymer blocks, that results in their novel properties. The hydrophobicity of the PPO block arises due to the presence of pendant methyl groups; hence the difference in solubility of the block segment is not as marked as many of the more common amphiphilic surfactant molecules. It is accepted that the hydrophobic PPO chains provide the necessary anchor for the polymer * To whom correspondence should be addressed. † The University of Nottingham. ‡ Johnson & Johnson Clinical Diagnostics. X Abstract published in Advance ACS Abstracts, November 1, 1997. (1) BASF Technical Brochure, BASF Co., Parsippany, NJ, 1989. (2) Muller, R. H. Modification of Drug Carriers. Colloidal Carriers for Controlled Drug Delivery and Targeting; CRC Press: Boca Raton, FL, 1991; p 23. (3) Lee, J. H.; Andrade, J. D. Surface Properties of Aqueous PEO/ PPO Block Copolymer Surfactants. In Polymer Surface Dynamics; Andrade, J. D., Ed.; Plenum Press: New York, 1988; p 119. (4) Amiji, M.; Park, K. Biomaterials 1992, 13, 682. (5) Lee, J. H.; Martic, P. A.; Tan, J. S. J. Colloid Interface Sci. 1989, 131, 252. (6) Lee, J. H.; Kopecek, J.; Andrade, J. D. J. Biomed. Mater. Res. 1989, 23, 351. (7) Ligoure, C. Macromolecules 1991, 24, 2968. (8) Shull, K. R. Macromolecules 1993, 26, 2346. (9) Amiel, C.; Sikka, M.; Schneider, J. W., Jr.; Tsao, Y-H.; Tirrell, M.; Mays, J. W. Macromolecules 1995, 28, 3125. (10) Zhan, Y.; Mattice, W. L. Macromolecules 1994, 27, 677. (11) Munch, M. R.; Gast, A. P. Macromolecules 1988, 21, 1360.
S0743-7463(96)02098-7 CCC: $14.00
Figure 1. Schematic of the adsorbed layer of triblock copolymers forming (a) a phase-segregated layer structure with the PPO block poorly solvated and the PEO block well solvated and (b) a “loops”, “tails”, and “trains” conformation to a hydrophobic interface.
molecules to remain adsorbed at the interface and the PEO chains extend into the solvent phase.12 It is the sterically repulsive forces provided by the extended, highly hydrated, brushlike PEO layer at high surface coverages that allows the stabilization of colloidal dispersions2 and gives rise to their protein repellant nature.4,13 At equilibrium the adsorbed layer has been described as a lamellar, phase-separated structure8,9,14 comprising an anchoring film or melt phase (PPO), which is made up of the sparingly soluble polymer segment and an extended well-solvated brush phase (PEO). This model is illustrated in Figure 1, where it is contrasted with the more conventional concept of adsorption of Pluronics as the conformational orientation of molecular “loops”, “tails”, and “trains” depending on the proportions of PEO (hydrophillic) and PPO (hydrophobic) blocks.3 PEO-PPO-PEO triblock copolymers readily form micelles in aqueous solution with a core predominantly (12) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414. (13) Evans, D. F.; Wennerstro¨m, H. The Colloidal Domain; VCH: New York: 1994; p 316. (14) Johner, A.; Joanny, J. F. Macromolecules 1990, 23, 5299.
© 1997 American Chemical Society
Adsorption of PEO-PPO-PEO Triblock Copolymers
PPO and a corona containing mostly PEO. The presence of micelles complicates the adsorption process, by introducing an equilibrium system where there is competition between the adsorption of unimers to the surface and micellar formation.15 The kinetics of layer formation are believed to consist of several limiting kinetic regimes. Initially, adsorption is relatively rapid, as single molecular chains14,16 diffuse to the surface and adsorb at unoccupied sites. This leads to the depletion of free chains in solution, which in turn causes the relaxation14 of the micellar aggregates and the release of single molecular chains. As a consequence, the kinetics cease to be diffusion limited and become governed by the rate of relaxation of the micelles. As the density of adsorbed molecules increases, the adsorbed layer forms a polymer “brush”, which hinders further adsorption because the polymer chains have to permeate through the extended hydrophilic layer to reach the potential adsorption site. Eventually, saturation of the surface sites is achieved, preventing further adsorption. Despite a large amount of work concerning the adsorption of Pluronics, a full understanding of the properties of Pluronics in solution16-19 and adsorbed at interfaces3,4,20-23 has proved elusive. For example micelle formation has been shown to be temperature sensitive and, consequently, the investigation of micellar structure has proven difficult.17 Variations in values of critical micelle concentrations (CMC) reported in the literature have been explained as being predominantly due to the polydispersity of the system,19 but also due to batch variation and the presence of impurities.12,24,25 Studies show that micellization is strongly temperature dependent,12 with the CMC and solubility decreasing as the temperature increases,19 and that the CMC is governed predominantly by the PPO block chain length. It has been reported that increasing the PPO block size decreases the CMC, whereas increasing the PEO chain length will result in a small increase in the CMC.20 Many techniques have been used to characterize block copolymer adsorption, these include ellipsometry,26 photon correlation spectroscopy,27 internal reflection interferometry,15 total internal reflectance fluorescence (TIRF),28 and dynamic light scattering.29 However, few of these techniques have been able to accurately relate the chemical structure of the block copolymer to their adsorption kinetics or the structure of the adsorbed layer. The majority of published work to date has relied on indirect methods such as radiolabeling,4 fluoresence,4 ultraviolet (UV),22 and X-ray photoelectron spectroscopy (XPS)3 to quantify the adsorbed layer in its equilibrated state. In this paper, surface plasmon resonance (SPR) has been used to give an insight into the adsorption of a range (15) Munch, M. R.; Gast, A. P. J. Chem. Soc., Faraday. Trans. 1990, 86, 1341. (16) Zhan, Y.; Mattice, W. L. Macromolecules 1994, 27, 677. (17) Zhang, K.; Khan, A. Macromolecules 1995, 28, 3807. (18) Wu, G.; Chu, B.; Schneider, D. K. J. Phys. Chem. 1995, 99, 5094. (19) Linse, P. Macromolecules 1994, 27, 6404. (20) Alexandridis, P.; Athanassiou, V.; Fukuda, S.; Hatton T. A. Langmuir 1994, 10, 2064. (21) Tiberg, F.; Malmsten, M.; Linse, P.; Lindman B. Langmuir 1991, 7, 2723. (22) Carthew, D.; Buckton, G.; Parsons, G. E.; Poole, S. Pharm. Sci. 1995, 1, 3. (23) Amiji, M. M.; Park, K. J. Appl. Polym. Sci. 1994, 52, 539. (24) Sikora, A.; Tuzar, Z. Makromol. Chem. 1983, 184, 2049. (25) Price, C. Pure Appl. Chem. 1983, 55, 1563. (26) Tiberg, F.; Malmsten, M.; Linse, P.; Lindman, B. Langmuir 1991, 7, 2723. (27) Wu, D. T.; Yokoyama, A. Polym. J. 1991, 23, 709. (28) Lok, B. K.; Cheng, Y.-L.; Robrtson, C. R. J. Colloid Interface Sci. 1983, 91, 104. (29) D’Oliveria, J. M. R.; Xu, R.; Jensma, T.;Winnick, M. A.; Martinhu, J. M. G.; Croucher, M. D. Langmuir 1993, 9, 1092.
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of Pluronics onto a polystyrene substrate. The SPR technique has become over the last few years an effective method for probing molecular interactions at surfaces.30 It is extremely surface sensitive and can give an indication of the degree of surface coverage and thickness of an adsorbed layer even at very low surface densities. Its initial use was for the characterization of molecular organic monolayers,31,32 such as Langmuir-Blodgett films,33 but more recently, given its high surface sensitivity and its ability to examine surfaces under aqueous conditions, SPR has found many applications in the analysis of biospecific interactions.34 For example, antibody-antigen interactions35 and epitope mapping36 have been investigated using this approach. Several articles30,36-38 have described the SPR apparatus in detail and therefore will be outlined only briefly here. A laser beam is directed onto the underside of a silver layer, which has been coated onto a glass prism, where at most angles of incidence it undergoes total internal reflection and is detected by a photodiode detector. However, at a specific angle of incidence, coupling occurs between photons from the laser beam and surface electrons (plasmons) at the metal/solution interface, causing excitation of surface plasmons and a subsequent reduction in the intensity of internally reflected light. The angle at which this minima occurs (the SPR angle) is dependent on the dielectric properties of the substrate, supporting layer, and in particular, the SPR sensor surface, i.e., the refractive index and thickness of the adsorbed layer. Therefore, if adsorption occurs at this interface, an increase in the SPR angle is observed, and by monitoring the SPR angle against time, one can obtain an adsorption profile. SPR analysis is rapidly emerging as an extremely promising tool for probing adsorption processes in real time and can provide valuable information concerning the kinetics of adsorption. Tassin et al.39 were one of the first groups to publish kinetically resolved data for block copolymer adsorption; they examined poly(vinyl-2-pyridine) and polystyrene block copolymers from toluene onto silver. However, to date, there have been no known studies relating the proportion of hydrophobic and hydrophilic blocks on the adsorption behavior of the PEO-PPO-PEO triblock molecule to the kinetics of formation or the physical properties of the adsorbed layer using this technique. In this study, we have investigated the effect varying PPO and PEO block size has on the nature of the resulting physisorbed layer adsorbed onto polystyrene films. Experimental Section Sample Preparation. The silver-coated slides (Johnson & Johnson Clinical Diagnostic, Chalfont St. Giles, U.K.) used in the SPR apparatus were ultrasonically cleaned for 10 min in a dry toluene/acetone (1:1) mixture to remove any physisorbed contamination. A solution (0.5 w/v) of polystyrene (2000 MW, Aldrich Chemical Co. Ltd., Dorset, U.K.) in toluene was prepared, (30) Davies, J. Nanobiology 1994, 3, 5. (31) Pockrand, I.; Swalen, J. D.; Gordon, J. G.; Philpott, M. R. Surf. Sci. 1978, 74, 237. (32) Swalen, J. D.; Gordon, J. G.; Philpott, M. R.; Brillante, A; Pockrand, I.; Santo, R. Am. J. Phys. 1980, 48, 669. (33) Suzuki, J. D. J. Mol. Electron. 1986, 2, 155. (34) Malmqvist, M. Nature 1993, 361, 186. (35) Mayo, C. S.; Hallock, R. B. J. Immunol. 1989, 120, 105. (36) Lundstro¨m, I. Biosens. Bioelectron. 1994, 9, 725. (37) Raether, H. Physics of Thin Films; Academic Press: New York, 1977; Vol. 9, p 145. (38) Kooyman, R. P. H.; de Bruijn, H. E.; Eenik, R. G.; Greve, J. J. Mol. Struct. 1990, 218, 345. (39) Tassin, J. F.; Siemens, R. L.; Tang, W. T.; Hadziiannou, G.; Swales, J. D.; Smith, B. A. J. Phys. Chem. 1993, 93, 2106. (40) Chattoraj, D. K.; Birdi, D. S. Adsorption and the Gibbs Surface Excess; Plenum Press: New York, 1984; p 285.
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Table 1. Properties of the Pluronic Surfactants Used in This Study and Their SPR Angle Shifts for Adsorption to Polystyrenea amt molecular blocks (mol)
pluronic surfactant
av mol wt
EO
PO
EO
L61(2) L63(2) P65(2),(3) F68(2) P84(3) L101(1) P103(1),(3) P105(1) F108(1) P123(3)
2000 2650 3400 8350 4200 3800 4950 6500 14000 5750
3 10 19 75 22 7 20 38 128 21
30 30 30 30 39 56 56 56 56 67
3 10 19 75 22 7 20 38 128 21
CMC (% w/v) at 35 °C (ref 12)
CMT (°C) at 0.1% w/v (ref 12)
1
46
0.15
37
0.002 0.005 0.15 0.001
24.5 27 36 21
max. SPR angle shift prior to buffer wash (mDA)
av final SPR angle shift (mDA)
1800 325 150 200 285 235 240 250 255 160
20 ( 11 (n ) 18) 65 ( 4 (n ) 12) 86 ( 19 (n ) 33) 83 ( 23 (n ) 15) 104 ( 10 (n ) 15) 109 ( 17 (n ) 18) 126 ( 23 (n ) 21) 178 ( 23 (n ) 21) 128 ( 16 (n ) 12)
a Series of Pluronics used in this study: two series with increasing PEO chain length and constant PPO block size of (1) 56 PPO molecular blocks and (2) 30 PPO molecular blocks; (3) one series with increasing PPO and approximately constant PEO of 20 molecular blocks.
and approximately 50 nm thick polymer films were spun cast on the silver slides at 1000 rpm. The prepared slides were then stored in a desiccator prior to use. PEO-PPO-PEO triblock copolymers (BASF Co., Parsippany, NJ) were obtained and used without further purification, the proportions of each molecular block for the materials are shown in Table 1. The Pluronic solutions (0.1% w/v) were prepared using a stock solution of aqueous phosphate buffer (10 mM, pH 7.4). Instrumentation. The SPR system (Johnson & Johnson Clinical Diagnostics, Chalfont St. Giles, U.K.),30 used a monochromatic laser light source (780 nm) focused onto a glass slide coated with a 50 nm silver film. Index matching between the prism and the silver-coated glass slide was achieved by using index matching oil (Stephens Scientific). The totally internally reflected laser light was analyzed by a two-dimensional array of charge-coupled detectors (CCD), and the data were recorded using an IBM PC compatible computer. Three thermoregulated flow channels were formed on the substrate surface by means of a liquid flow cell pressed onto the slide, and the whole arrangement was kept watertight using a rubber sealing ring. The solution under investigation (0.9 mL) was passed over the substrate at a constant flow rate (4 µL/s) using a motorized pump.
Results and Discussion Initially, the effect of PPO and PEO block size on the formation of the adsorbed Pluronic layer was investigated. To monitor the effect the PEO chain length had on adsorption to polystyrene, two Pluronic series were chosen with increasing PEO block size and a constant PPO block, with 56 or 30 PPO molecular units, respectively. The effect of increasing the PPO block size was studied by investigating the adsorption of a series of Pluronics with increasing PPO block size and a constant PEO chain length of approximately 20 molecular units. The Pluronics selected for this study are detailed in Table 1. The SPR adsorption profiles for each series are shown in Figures 2-4. Parts a and b of Figure 2 show the adsorption of Pluronics with increasing PEO content and 56/30 molecular blocks of PPO. The group of Pluronics with 20 PEO units and an increasing proportion of PPO are shown in Figure 3. Let us initially consider the adsorption of the 56 PPO molecular block series with increasing PEO content (Pluronics L101, P103, P105, and F108) shown in Figure 2a. We observe a rapid increase in the SPR angle after approximately 20 s of solution flow as the adsorbate enters the sample cell and comes into contact with the surface. This corresponds to the adsorption of copolymer molecules from solution where the abundance of potential adsorption sites gives rise to the rapid rate of adsorption. It is particularly noticeable in this series of materials that the adsorption rate remains relatively constant for each of the Pluronics studied. As the proportion of occupied
Figure 2. SPR adsorption profile for (a) the 56 PPO block Pluronic series, (b) the 30 PPO molecular block series with increasing PEO chain lengths, and (c) Pluronic L61 to a polystyrene surface.
surface sites increases, the rate of increase of the SPR shift slows. The onset of equilibrium is surprisingly rapid,
Adsorption of PEO-PPO-PEO Triblock Copolymers
Figure 3. SPR adsorption profile for the 20 PEO block series of (a) P65, (b) P84, (c) P103, and (d) P123 to a polystyrene surface.
Figure 4. SPR adsorption profiles of Pluronic P105 from (a) a range of solution concentrations (0.001-1% w/v) and (b) a 0.1% w/v solution at 24 °C (dotted line) and 34 °C (solid line) to a polystyrene surface.
and this represents the inhibition of further adsorption due to the saturation of the surface. This has been termed the “brush-limited regime” where the well-solvated PEO chains prevent the further adsorption of the Pluronic molecules and which is observed as a plateau in the SPR adsorption profile.14,15 The rapidity with which an equilibrium level of adsorption was attained is due to the intrinsic kinetics of block copolymer adsorption, which tend to be rapid, and the flow arrangement of the SPR cell, which accelerates the adsorption process.30 Once equilibrium has been established, the surface is washed through twice with fresh buffer, removing material loosely associated with the surface or close enough to affect the SPR signal. A direct comparison of the SPR profiles in Figure 2a shows that the increasing proportion of PEO relative to PPO for each material has no discernible effect on the initial rate of adsorption or the maximum SPR angle shift observed prior to the buffer wash. However,
Langmuir, Vol. 13, No. 24, 1997 6513
the final SPR angle shifts (mDA, millidegree angle), taken as the difference between the initial SPR angle prior to adsorption and the final SPR angle after the buffer wash, were found to increase with increasing PEO content of the copolymer, suggesting an increase in adsorbed layer thickness. A similar trend is seen in the other series (Figure 2b) showing the SPR adsorption profiles of the second series of Pluronics with constant PPO (30 molecular blocks) and increasing PEO content. The L63, P65, and F68 Pluronics all produced adsorption profiles closely related to the 56 PPO block series. However, in contrast to this series, the initial rates of adsorption and the maximum shift values immediately prior to the buffer washing stage were found to differ for each of the Pluronics. However, the final postwash SPR shifts also showed an increase in value with an increase in PEO content. The SPR profile of L61, shown in Figure 2c, deviates from the remainder of the series. It is characterized by a large initial adsorption step that continually increases often beyond the range of the photodetector array, thus preventing the SPR angle from being measured. Subsequently, the buffer wash stage removes all nonadsorbed and loosely bound Pluronic material and leads to a return of an SPR angle measurement and a final SPR angle shift of less than 100 mDA. Unfortunately, the loss of the SPR angle during the experiment results in the inability to produce a reproducible final SPR angle shift for this Pluronic. This behavior will be discussed in detail later in this text. The SPR plots for the Pluronics series (P65, P84, P103, and P123) of constant PEO and hence increasing PPO are shown in Figure 3. Again, the initial rates of adsorption and maximum shift values prior to the washing stage differed within this range of Pluronics, yet the final postwash shifts increased in line with increasing PPO content. Differences in apparent trends between each series may be accounted for by considering the Pluronic’s solution behavior. The kinetics of adsorption of Pluronics at the solid-liquid interface is thought to be dependent on the dynamic equilibrium between polymer molecules free in solution (unimers), those already adsorbed at the surface, and those contained in micelles.15,39 On the basis of this information, the molecular association of Pluronic molecules in solution would be expected to have a significant affect on the adsorption kinetics10 and many studies12,15,39 have suggested that the rate of adsorption is governed by the relaxation kinetics of the micelles as well as the adsorption kinetics of the free molecules. Micelle formation has been shown to be dependent on the proportion of PPO and PEO in the copolymer structure as well as the overall molecular weight of the surfactant. Increasing the PPO block of the Pluronic decreases the CMC and critical micelle temperature (CMT), whereas increasing the PEO chain length enables the polymer molecules to remain stable in solution without the need for micellization and results in an increasing CMC and CMT41 value. Micellization has been shown to be strongly temperature dependent, and the CMC is governed predominantly by the PPO block chain length. Hence higher molecular weight Pluronics with high proportions of PPO would be expected to self-aggregate more readily. CMC and CMT values12 for some of the Pluronics investigated here have been included in Table 1. These values cannot be assumed (41) Cosgrove, T.; Crowley, T. L.; Mallagh, L. M.; Ryan, K.; Webster, J. R. P. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1989, 30, 370. (42) Davies, J.; Allen, A.; Bruce, I., Burrows, Y., Heaner, P. J.; Hemming, F.; Nunnerley, C. S.; Skelton, L. Proceedings of the International Symposium on Surface Properties of Biomaterials, Butterworth-Heinmann: Oxford, U.K., 1994; p 117.
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absolute, due to batch variation and the polymers’ polydispersities, and are therefore used as guidelines only. The quoted CMC values for the Pluronics suggest a link between the formation of micelles and the rates of adsorption in the data presented in Figures 2-4. The series of Pluronics presented in Figure 2a (PPO ) 56 molecular blocks), all have similar adsorption profiles and exhibit the most rapid initial change in the SPR shift, which seems independent of molecular weight or PEO content. At the concentration (0.1% w/v) and temperature (34 °C) at which this study was performed, we can assume that each of these Pluronic solutions exhibited some micellar behavior, thus explaining their similarity in adsorption. This is in agreement with the work of Tassin et al.,39 who observed greater rates of adsorption from micellar solutions. The materials with constant PEO content (approximately 20 molecular blocks) in Figure 3 do not follow the same trend, and this may be attributed to the fact that P103 and P123 are micellar in nature whereas P65 and P84 do not form micelles under the experimental conditions of this study. To confirm the influence of micellization on the formation of polymer brushes, the adsorption of some Pluronics at concentrations and temperatures above and below their CMC and CMT have been investigated. The series of Pluronics (PPO ) 56) resulted in adsorption profiles that appeared to be independent of the nature of the adsorbate solution. The adsorption of Pluronic P105 at a range of concentrations and temperatures is displayed in Figure 4. The concentration dependence shows an increase in the rate of adsorption with increasing concentration irrespective to whether the adsorbing solution was at a concentration above or below its CMC value. The final SPR angle shifts at each concentration are consistent with adsorption to a saturated surface. When considering adsorption of the Pluronic at two temperatures selected above (34 °C) and below (24 °C) the CMT value,12 one observes that the presence of micelles has no effect on either the rate or the extent of adsorption. This observation was not seen for all the other Pluronics studied. Indeed, this work suggests that Pluronics with smaller PPO molecular blocks are affected by the presence of micelles in solution. For example, both Pluronics L61 and L63 show considerable dependence on the micellar nature of the adsorbate solution. At higher concentrations, Pluronic L63 exhibits adsorption profiles similar to that observed in Figure 2c for L61, and at lower concentrations the adsorption profiles of both Pluronics resemble a more conventional appearance. This observation may be explained when considering the stability of the micelles in solution. Pluronics with smaller PPO block sizes form micelles less readily in an aqueous environment than Pluronics with a larger hydrophobic PPO block. Micelles from Pluronics with a small central PPO block are less stable, thus pushing the equilibrium toward the breakup of micelles and the adsorption of unimers, resulting in enhanced adsorption. The adsorption of Pluronic L61 (at 0.1% w/v and 34 °C) is a prime example of this. The adsorption of Pluronic L61 may be explained by considering the cloud point temperature of this Pluronic solution. The cloud point temperature of L61 for a 1% and 10% w/v solution of the material is 24 and 17 °C, respectively.1 A 0.1% w/v solution of L61 prepared at room temperature may be considered as turbid once it is warmed to 34 °C in the SPR analysis cell. This would cause an increase in the refractive index of the overall Pluronic solution. An increase in the refractive index of the solution within the detection limits of the SPR sensor surface will result in an increase in shift of the SPR angle, since SPR monitors changes in dielectric properties of the medium
Green et al.
Figure 5. Effect of temperature on the adsorption of Pluronic L61 to a polystyrene surface, showing adsorption at 34 °C (dotted line) and 24 °C (solid line).
Figure 6. Graph showing the SPR angle shifts against PPO/ PEO molecular block size.
above the silver surface to a sampling depth of at least 100 nm. The SPR adsorption of a 0.1% w/v solution of Pluronic L61 at 34 and 24 °C, shown in Figure 5, indicates a large difference in the adsorption profiles at the two temperatures. At 24 °C, the Pluronic solution is below its cloud point and adsorbs to the surface in a normal manner, where a plateau in the adsorption profile is observed and a final SPR angle shift can be determined. The adsorption behavior at the elevated temperature of 34 °C reveals what may be misinterpreted as the continual adsorption of the Pluronic but in fact reflects the increase in the refractive index due to the clouding of the polymer solution. The final SPR angle shift for each Pluronic (adsorbed from 0.1% w/v solution at 34 °C) is determined in the presence of fresh buffer solution, after the buffer washes, and is independent of the refractive index of the corresponding Pluronic solution; therefore, they can be used to provide an indication of the relative thicknesses of adsorbed Pluronic layers. The average final SPR shifts for each of the Pluronics are shown in Table 1 with the exception of L61. To compare the effects the PEO and PPO blocks have on the thickness of the adsorbed layer, the SPR angle shifts (mDA) of the PPO ) 56 and PEO ) 20 series have been plotted against the molecular number of the increasing PEO or PPO blocks, respectively, in Figure 6. A linear relationship is clearly demonstrated between the SPR angle shift observed and the increasing size of the PEO/PPO blocks for both series, respectively. A difference is noticeable between each of the series when the rate of increase in SPR angle with respect to increasing PEO/PPO chain length is considered. Increasing the PPO block size results in an increase in the SPR angle shift 3 times that observed when increasing the PEO block size. This observation can be explained if we consider the effect an adsorbed Pluronic layer has on the dielectric properties
Adsorption of PEO-PPO-PEO Triblock Copolymers
of the near-interfacial region of the substrate, since SPR monitors changes in the dielectric properties rather than increases in layer thickness. The SPR response is dependent on effects of each copolymer block. The size of the PPO block has a larger effect on the SPR response, since it is largely dehydrated and orientated nearer to the interface than the highly hydrated PEO block. It is, therefore, possible that the Pluronic layer consists of a structure similar to that illustrated in Figure 1a, with the hydrophobic PPO layer forming a densely packed dehydrated layer at the interface, and highly solvated PEO chains extending well out into the aqueous phase. The refractive index of the dehydrated PPO layer will be higher and have a greater effect on the SPR angle than the hydrated PEO layer that is further away from the surface. Therefore, an increase in the thickness of the PPO layer will result in a larger SPR angle shift than an equivalent increase in the PEO layer. The SPR angle shift corresponding to the increase in one PEO or one PPO unit can be calculated from the graph of Figure 6. One of the plots in Figure 6 shows the increase in SPR angle with increasing PEO chain length and constant PPO content of the polymer (56 molecular blocks). The gradient of this will correspond to the SPR angle shift due to the increase of two PEO units, one for each of the PEO chains in the Pluronic. The plot’s intercept with the y-axis will lead to the SPR angle shift required for the adsorption of a PPO-only polymer containing 56 PPO molecular blocks. This leads to the values of 1.78 mDA per PPO block and 0.31 mDA per PEO unit. Repeating this procedure for the SPR angle shift plot of increasing PPO content leads to the values of 1.67 mDA per PPO block and 0.39 mDA per PEO block. The SPR angle shifts calculated from both plots are consistent and support our hypothesis that the PPO block has a greater effect on the shift of the SPR angle than the extending hydrophilic PEO chains. Conclusions This study has investigated the adsorption of a range of Pluronic surfactants to a model hydrophobic surface by SPR. Using this technique, we have probed the copolymers adsorption in situ, providing valuable information to the rate and the extent of adsorption. From close observation of the resulting SPR adsorption profiles, we have been able to investigate the effect the PEO and PPO chain lengths and the micellar nature of the solution have on adsorption. A detailed study into the effect of micellar solutions on Pluronic adsorption was performed by monitoring the Pluronics’ adsorption at concentrations and temperatures above and below their CMC and CMT values. This resulted in the conclusion that the central hydrophobic PPO block, as well as being the predominant factor determining the formation of micelles,19 is also the predominant factor for determining the effect the micellar nature of the solution will have on adsorption. We have shown that increasing the PPO content of the polymer leads to a decrease in the effect micelles in solution have
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on adsorption. The adsorption of Pluronics containing a 56 PPO block is predominantly independent of the solution’s micellar nature, whereas Pluronics with a lower PPO content, such as P65 and L63, show signs of enhanced adsorption from a solution containing micelles. This has been explained by considering the stability of micelles in aqueous solutions, suggesting that Pluronics containing smaller PPO blocks form less stable micelles and, therefore, their micellar relaxation kinetics increase, pushing the solution equilibrium toward unimer adsorption. When looking at the SPR angle shifts produced for each Pluronic series, a linear increase in SPR angle shift was observed with increasing either the PEO or PPO block of the Pluronic. Increasing the PPO block size, however, caused a greater SPR angle shift than increasing the PEO block size. It would appear that the PPO layer is a densely packed nonhydrated layer adsorbed at the hydrophobic interface and increasing the PPO content of the polymer affects the dielectric properties immediately above the layer to a greater extent than an equivalent increase in the highly hydrated PEO layer, thus leading to a larger shift in the SPR angle. This work highlights the ability of SPR to monitor adsorption processes in real time, providing valuable information on the adsorption process. It also highlights the need of care when interpreting SPR adsorption data, so as not to confuse an increase in SPR angle shift as a quantitative increase in adsorbed layer thickness alone, but to consider the SPR measurement as an increase in the dielectric properties of the near-interfacial area immediately above the SPR sensor surface. Therefore, although the SPR technique is unable to provide direct quantitative data upon the exact nature of the adsorbed layer, it has successfully highlighted valuable trends of adsorption with respect to the variation of solution concentration and the size/proportion of individual copolymer blocks. The successful characterization of this Pluronic layer has led to further work probing protein interactions with this potential biomaterial coating, determining the effect the PEO and PPO chain lengths have on protein inhibition.43 It can, therefore, be envisaged that the employment of SPR could be used to rapidly screen a wide range of polymeric surfaces and adsorbate interactions,44,45 including monitoring specific protein interactions, polymer film hydration, and the rates of degradation of biodegrading polymers. Acknowledgment. The authors would like to acknowledge the financial support of the Brite Euram program and the EPSRC/DTI Nanotechnology LINK Initiative with Kodak Limited, Oxford Molecular Group plc., and Fisons plc. LA962098U (43) Green, R. J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. submitted to J. Biomed. Mater. Res. (44) Chen, X.; Davies, M. C.; Roberts, C. J.; Shakesheff, K. M.; Tendler, S. J. B.; Williams, P. M. Anal. Chem. 1996, 68, 1451. (45) Shakesheff, K. M.; Chen, X.; Davies, M. C.; Domb, A.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.. Langmuir 1995, 11, 3921.
