Surface Modifications with Adsorbed Poly(ethylene oxide) - American

Chapter 25

Surface Modifications with Adsorbed Poly(ethylene oxide)-Based Block Coploymers

Downloaded by UNIV OF ROCHESTER on November 6, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch025

Physical Characteristics and Biological Use Karin D. Caldwell Center for Biopolymers at Interfaces, Department of Bioengineering, University of Utah, Salt Lake City, UT 84112 Pluronic surfactants with varying PEO block lengths are found to adsorb to polystyrene nanoparticles. Stable adsorption complexes can be formed with similar packing densities, allowing an evaluation of chain length on the physical and biological properties of the coating. The ad-layer thickness at maximum surface coverage increases monotonically with increasing chain length, and the chain dynamics parallels this increase. Various complexes have been examined for their ability to repel fibrinogen, a capability that likewise increases with increasing chain length up to 129 EO units (F108), in analogy with previous observations on covalently modified surfaces. At maximum repulsion efficiency, the fibrinogen uptake is two orders of magnitude below that of the bare surface. By end-group activating the PEO chains, proteins and other ligands can be tethered to the surface, where they remain structurally intact. Surfaces modified in this manner have shown to support the growth of anchor dependent cells. Ever since the middle of the 1960's, biochemists have known to precipitate selected proteins from complex mixtures by controlled additions of polyethylene glycol (PEG, PEO). This convenient procedure led to careful and systematic studies (7-5) which established that the PEG molecule was preferentially solvated in aqueous media, effectively excluding other solutes from its solvation sphere in proportion to their size. Specifically, Ingham was able to show (3) that for a given protein, in his case human serum albumin (HSA ), precipitation from solutions of a fixed protein concentration was accomplished at ever smaller weight concentrations of polymer the higher the PEG molecular weight. This exclusion effect was shown to reach its maximum for molecular weights around 6000 Da. Subsequently, he expanded the study to include proteins of a variety of sizes and concluded that there was a general increase in the exclusion efficiency with an increase in protein size. Although later investigations (6,7) have confirmed that PEG interacts only weakly with most proteins, it was clear already from the early studies (1,2) that PEG was unique among water soluble polymers in its ability to exclude, concentrate, and under special conditions precipitate proteins, even those of limited stability, without inflicting losses in their biological activity. Due to its ability to sterically exclude other macromolecules and particles, PEG 400

© 1997 American Chemical Society

In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


25.

Surface Modification with Adsorbed Block Copolymers

CALDWELL

401

has in recent years come to be used extensively as a stabilizer of colloidal systems and as a protective coating of surfaces intended for contact with protein containing fluids. Since its general inertness precludes direct adsorption as a means of forming a stable complex with the surface, the most common strategy for achieving a PEG-based protection has involved a covalent attachment of the linear PEG (or polyethylene oxide, PEO) chains to the surface. Surfaces modified in this manner were carefully examined by several groups (8-10) to determine what constructs might be most effective in suppressing the adsorption of proteins and cells. Of prime importance was the establishment of a relationship between repulsion efficiency and PEO chain length. Here, the pioneering work by Nagaoka (8) indicated that the positive effects of increasing the PEO molecular weight reached a plateau around 5,000 Da, a result that has since been confirmed by many others (9,10). It is interesting to note that these findings suggest the surface-attached PEO to behave in a manner similar to PEO in solution, as discussed above, even though the presence of the substrate must place significant constraints on both structure and dynamics of the polymer chain. The covalent surface modification with PEO is attractive from the point of view of generating a stable product whose properties are readily evaluated. However, for steric reasons, it is less clear whether the substitution of chains of different lengths proceeds to one and the same degree and, therefore, whether the observed plateau is a true phenomenon or an artifact due to incomplete close-packing of the longer chains and a resulting exposure of certain bare areas to contacting proteins. Such areas would likely display residual reactive groups, previously introduced to the surface in large excess to allow a desirable degree of substitution to occur even for long chains with their low concentration of active terminal groups. These concerns have suggested a different approach to surface modification with PEO, namely one based on adsorption. While PEO itself is slightly hydrophobic, as suggested above, and therefore showing weak tendencies to adsorb to hydrophobic surfaces in aqueous environments, these adsorption complexes are relatively unstable, and the PEO is easily displaced by proteins and other, more strongly adsorbing, compounds. However, the adsorption of PEO containing block copolymers is by now a well known route to surface protection. By linking PEO chains of different molecular weights to hydrophobic anchor blocks of different lengths and composition, one can obtain surface coatings that vary extensively, both in stability and repulsion efficiency (11-16). Of particular interest were the early observations by Ilium and others (17-20) regarding the reduced macrophage uptake and the in vivo tolerance of polystyrene latex particles coated with polymeric triblock surfactants of the poloxamer type, i.e. compounds of the general composition (EO) -(PO) -(EO) whose polypropylene oxide (PPO) center block is highly hydrophobic and serves to anchor the PEO chains to the hydrophobic substrate. From this work it has become clear that adsorptive coatings may be generated that have some highly interesting and useful protein repellent properties. Since the adsorption is likely to be strongly regulated by the length of the PPO block, the possibility exists of generating surfaces with one and the same molar concentration of PPO, and hence of PEO, regardless of the lengths of the latter chains. Poloxamer-coated surfaces might therefore provide some valuable insights into the physical characteristics of an optimally repulsive PEO surface layer. The work by Nagaoka (8) had not only shown an increased repulsion efficiency of PEO chains of increasing length, but had also demonstrated that the longer chains were considerably more mobile than the shorter ones. This observation led to the formulation of a mechanism for the repulsion which has its base in the large configurational entropy conferred on the interface by the chain dynamics. In order to adsorb to the surface, an approaching macromolecule or particle would have to constrain this motion by reducing the space available for the polymer chains, and thereby reduce the entropy of the system. In the absence of a strong attraction such a reduction would be prohibitive, and the surface would therefore be protected in proportion both to the dynamics of the polymer chains and the size of the approaching m

n

m



402

POLY(ETHYLENE GLYCOL)

particle. This notion was explored in some theoretical detail by Andrade and coworkers (21,22), and more recently by Torchilin et al. (23) who showed that the flexible PEO molecule is more effectively protecting a liposome surface from protein fouling than a, comparatively more rigid, dextran molecule of similar molar mass. In contrast to the notion that molecular dynamics play a significant role in protecting surfaces from protein adsorption stands the notion that the key to surface protection is the reduction of interfacial energy. This can effectively be accomplished by covering the surface with a perfectly close-packed layer of hydroxy-terminated alkanes, exemplified by the self-assembled monolayers (SAM) formed on a gold substrate if the alkane contains a thiol as its second terminus. Work by Whitesides and others (24-26) clearly demonstrates that hydroxylated or EO-substituted surfaces are less prone to protein uptake than surfaces covered by alkanes terminated in hydrophobic groups. However, comparing protein uptakes between laboratories can easily lead to erroneous conclusions, and it is therefore difficult to promote one mechanism over another. From a practical standpoint, however, the versatility of the adsorptive coatings outweighs that of the SAM:s which require highly specific substrates for their formation. Despite much work in the area of surface protection, many questions still remain unanswered. For some time to come, surface chemists will be attempting to sort out questions concerning the chemical and physical nature of the most effective surface coatings. Specifically, the importance of interfacial chain mobility, close-packing, and layer thickness will continue to intrigue the workers in this field. Analytical Strategies Even though many blood contacting surfaces in need of protection are of a flat geometry, the flat surface is undesirable from an analytical perspective, in that it generally presents an inconvenient surface-to volume ratio and leads to the adsorption of only minute amounts of surfactant or protein. Although situations present themselves in which the quantification of flat surface deposits is unavoidable, demanding labeling with radioactive isotopes for the required sensitivity, our own work has by and large relied on adsorption to uniformly sized polystyrene (PS) nanoparticles. By means of an example, a sample of 200 nm PS latex particles presents just above 71 cm of surface area per m&of solids. Despite the fact that these particles carry a substantial amount of surface charge, displaying zeta potentials of the order of 50 mV, their uptake of the non-ionic poloxamers is in relatively good agreement with that seen on neat polystyrene discs (27). The composition of ad-layers that form on particulate substrates can be found either through direct analysis, carried out after centrifugation and wash procedures have eliminated all loosely adsorbed material. The residual adsorption complex is therefore similar to that observed in an actual use situation. The alternative approach involves an indirect analysis based on the composition of the supernatant after removal of the particulates. This mode of analysis tends to over-estimate the surface uptake, as it will include even loosely associated components. One group of analytical techniques that has been relied upon to perform a direct determination of the surface concentration of adsorbed surfactant involves labeling, either with I or with a pyridyl disulfide group that can be quantified spectroscopically following reductive cleavage of the disulfide bond with release of the thiopyridone (28). Another has its base in the field-flow fractionation (FFF) strategy, whereby bare 2

125

or coated particles are forced to migrate through a thin separation channel (100-250 μπι in thickness) under the constant influence of an externally applied field (29). If the field is a sedimentation field, a particle's movement in the field is reflective of its mass. Even subtle differences in particle mass, such as those deriving from the adsorption of a surface layer, result in different positions within the sharply pointed parabolic flow



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403

profile formed by the mobile phase, and hence of different transport velocities through the analytical cell While in transit through the channel the particle is continuously washed by the mobile phase, and as it elutes its residence time is recorded as an exact measure of its mass. By comparing elution positions of the bare and coated particles, one can directly calculate the surface concentration per particle, without introducing either labeling artifacts or errors due to particle losses during wash. The uptake of protein, either on surfactant-coated or bare particles, is regularly determined by amino acid analysis (30) of the coated particles. At times, the uptake is also evaluated following radioiodination of the proteins to be adsorbed. Indirect methods for assessing surfactant uptake include the colorimetric Baleux method (31), in addition to the thiopyridone quantification mentioned above, while indirect methods for protein quantification involve either radiolabelling, amino acid analysis, or the colorimetric micro-BCA technique (32). The ad-layer thickness is determined from the difference in size between bare and coated particles. This determination is done either by flow FFF (33), a technique which yields the particle's diffusion coefficient from its retention in a hydraulic field, or by photon correlation spectroscopy (PCS)(34), preferably on particles having undergone prior fractionation by FFF with removal of aggregates that invariably form as the large surfactant molecules bridge pairs of particles during adsorption. The mobility of the PEO chains, attached to the particle surface via the adsorbed PPO-block, can be followed by electron spin resonance spectroscopy, following the introduction of a spin label, such as the proxyl radical, into the terminal hydroxyls of the PEO (35). The slower the movements of these labeled ends, the longer are the correlation times, τ, observed in the measurement. Model Systems Surfactants. The poloxamer surfactants used in this study were of the Pluronic type produced by the BASF Corporation and kindly donated to us. Although these products exist in a variety of block lengths, we selected four with reasonably comparable PPO blocks, but with PEO blocks of widely varying lengths, as seen in Table 1. Of the four, Pluronic Ρ105 is highly prone to micellization in aqueous solution, while the other three are not. Table I. Physical properties of selected Pluronics studied here. M W represents the molecular weight of Pluronic, and m and η represent the number of monomer units of polyethylene oxide and polypropylene oxide respectively. Pluronics

MW

m

η

F108

14,600

129

56

F88

11,400

104

39

F68

8,400

76

30

P105

6,500

37

56

Adsorption Conditions. In the original phase of this study, the adsorption was always carried out from surfactant solutions whose concentrations were 4% by weight, using adsorption times of 24 hours or longer. Both in terms of time and concentration



404


these conditions were excessive, as can be seen from Figures 1 and 2. Figure 1 displays two adsorption isotherms recorded for Pluronic F108 (36). The one labeled a) is obtained from the product, as delivered by the manufacturer. The non-ideal features of this isotherm suggested the presence of some impurity. Indeed, from size exclusion chromatography it became clear that the product consisted of two components, one about half the size of the other. Chemically, the two appeared identical, to judge from their IR spectra. The larger of these components was collected after fractionation and used to yield the isotherm labeled b). Both isotherms coincide at the plateau, which is reached at solution concentrations of between 0.01 and 0.02% using adsorption times of 24 hours. It is therefore reasonable to assume that adsorptions at the significantly higher concentration of 4% (initially) produced surfaces that were exclusively populated by the larger component. The rate of adsorption of Pluronic F108 was analyzed by two direct methods (37). The first of these was the above mentioned spectroscopic technique and involved the adsorption of a pyridyl disulfide labeled surfactant. In this case, samples were harvested at specified times, beginning with a five minutes exposure, and were then extensively washed on a Centricon filter. The wash procedure took a minimum of 15 minutes, so the early time points are extremely uncertain. The second method was based on quantification by sedimentation FFF. Also here, the first time points are weak, since a sample needs to relax into its equilibrium position in the separator in the absence of flow, before the analysis can begin. This relaxation process requires 20 minutes under the chosen conditions. Nevertheless, as seen from Figure 2 both methods indicate that the plateau value is reached after about five hours. The adsorption process is clearly bi-phasic, with a rapid first phase in which the surface is populated to about 75%, and a slow second phase in which molecular rearrangements occur that permit a better close-packing on the particle surface. In on-going studies, we are using the FFF analysis method in its electrical mode to investigate the rapid phase of the kinetics. During electrical FFF (elFFF) the relaxation is sufficiently fast to allow a continuous separation process without the type of flow interruptions required for the corresponding sedimentation analysis. The elFFF method retains particles in accordance with their charge, and due to the high zeta potential of the bare particles, they are significantly retained even at weak fields, as seen in Figure 3 (Y-S Gao, personal communication). Upon adsorption of the uncharged surfactant the particle's charge is screened which results in a weaker retention, as seen in the figure. It is interesting to note that even at maximum surface coverage there is still a residual charge on the particle, presumably reflective of areas in between PPO blocks that, for steric reasons, remain uncovered. Since, upon injection in the elFFF channel, the charged particle is immediately separated from the uncharged surfactant, retention in this system is a direct reflection of the degree of surface coverage obtained at a given reaction time. Sampling of the particle-surfactant mixture can at present take place in 10 seconds (37), which allows a closer look at the first rapid phase of the adsorption kinetics (see Figure 4 ) . It is remarkable that complexes are formed in such short times which are capable of withstanding the extensive washing that takes place during the subsequent separation process. Stability of the Adsorption Complex. Due to the absence of any covalent linkages between surfactant and substrate, the adsorption complex is inherently reversible in nature, although the activation energy for release appears to be large. Since one of the practical uses of surfactant coated surfaces is to render them nonadsorptive to proteins, bacteria, and other cells, it is important to establish their stability not only in a pure aqueous environment, but also in the presence of proteins that might interact more strongly with the particle and thereby displace the surfactant. Figure 5 shows the retention of radiolabeled Pluronic F108 by a particulate substrate following washing and suspension in three different media, namely phosphate buffered saline (PBS), PBS with a 0.8% content of human serum albumin, and human whole plasma


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2.5

405

τ

0.00

0.01

0.02

0.03

0.04

UnadsorbedF108 [%] Figure 1. Adsorption isotherms of Pluronic F108 surfactant on PS 252 nm latex. A. Surfactant as delivered; B. High molecular weight fraction collected after GPC on a Superose 12 column from Pharmacia

0 * 0

1

500

1

1000

•

1 1500

Coating Time (min.) Figure 2. Adsorption kinetics of Pluronic F108 on PS 261 nm latex. The surfactant concentration is 4% by weight. The two analytical techniques are both "direct methods" The upper trace represents results from a spectrophotometric quantitation after filtration, and the lower represents data from sedimentation FFF.



406


350

0 H 0

•

1

20

»

«

«

40

«

»

60

Retention Volume (ml) Figure 3. Retention in elFFF of bare and Pluronic F108 coated PS 165 nm latex. The applied voltage was 1.89 V across a 127μπι thick separation channel. The carrier fluid was DI water.



25. CALDWELL


Day 1

Day 2

Day 3

Figure 5. Stability of Pluronic F108 coated PS latex particles, suspended in various media. The surfactant was end-group labeled with a I-Bolton Hunter reagent. (Adapted from ref. 33). 125


407


408


(33). In the first two media there is no evidence of any surfactant loss during three days of observation. The third environment produces a rapid and significant loss of surfactant The exact nature of the displacing entity is not clear. However, SDS gel electrophoresis has indicated a strong presence of some low molecular weight compounds which we have suspected to be apo-lipoproteins. It is interesting to note that the careful analytical work by Blunk (38) identifies several apo-lipoproteins as being preferentially adsorbed on latex particles coated with a comparable poloxamer. Even after equilibration with human plasma, there is no change in the hydrodynamic size of Pluronic F108 coated PS latex particles (33), as illustrated by the flowFFF (flFFF) fractograms of Figure 6. As mentioned earlier, the flFFF technique separates samples based on their diffusivity, or hydrodynamic size. The figure shows elution positions for bare and F108-coated latex particles, as well as for the same coated particles equilibrated in plasma diluted to different degrees. While there is no change in size of the coated particles, the corresponding bare particles have experienced extensive aggregation, as seen in the figure. A highly significant proof of the retention of a protective layer of Pluronic F108 under in vivo conditions was reported by Tan et al. (39), who found the half life of clearance of coated PS latex particles, 73 nm in diameter, to increase to 13 hours, compared to less than 30 minutes for the bare particles. Although surfaces adsorption-coated in this manner are less stable than those covalently modified, the stability appears adequate for many short-term uses. In case a more extended exposure to plasma is needed, the Pluronic surfactants can be covalently attached to their substrate by means of the gamma-radiation procedure used for sterilization, as shown by Amiji and Park (40). Ad-layer Characteristics Surface Density. The four Pluronic compounds of Table 1 were adsorbed to an array of particles, ranging in size from 69 nm to 394 nm, using the 4% concentration/24 hour adsorption time protocol discussed above. In this process a certain curvature effect was noted (29), in that any one surfactant appeared to adsorb with a lesser concentration on the smaller particles. Curvature effects in ad-layer thickness had previously been observed by Baker and Berg (34). In comparing surface concentrations of the different surfactants, it is therefore necessary to use carrier particles of one and the same size. As seen in Figure 7, the four surfactants adsorb with comparable surface concentration on a 69 nm PS latex. Similarities in surface concentrations were also seen for the other particle sizes under investigation (29). Although the PEO blocks vary in length by more than a factor three, this is not reflected in the close-packing on the surface, which instead appears to be governed primarily by the length of the anchor block. The fortuitous similarity in surface concentration allows a direct evaluation of the effect of PEO chain length on protein repulsion, to be discussed below. The mentioned curvature effect in surface density was found through direct analysis of the amount of surfactant present on the particles after adsorption under the above conditions. Attempts at demonstrating this effect using an indirect analysis protocol were unsuccessful (36). Ad-layer Thickness. In accord with observations by Baker and Berg (34) we found the ad-layer thickness to vary with substrate size in such a way that the PEOchains on the smaller particles form a more collapsed layer than those attached to the larger spheres. Given the observed differences in surface concentration, this could be explained by a transition from a "mushroorrT-like conformation at the low surface concentration to a more "brush"-like conformation at the higher concentrations, to use the de Gennes nomenclature (41). The layer thicknesses measured for the different surfactants on the 69 nm substrate are shown in Figure 8, which also includes a trace outlining the diameters which PEO-blocks of various lengths would assume iffreein solution. The latter data derive from observations by Bhat and Timasheff (42), and the


25.

CALDWELL


PS272 PS272-Plasma(l:30) PS272F


PS272-Plasma(l:l) PS272-Plasma(l:6) PS272-Plasma(l:30) 20

25

30 Vr(ml)

o loo 200 300 400 500 600 700 Size (nm) Figure 6. FlFFF fractograms of PS 272 nm after various treatments: Counted from the top, the first represents untreated particles, the second represents particles exposed to human plasma with a particle:protein weight ratio of 1:30, the third represents F108 coated particles, the fourth, fifth, and sixth represent coated particles after 4 h of contact with plasma at the specified particle:protein weight ratios. (Reproduced with permission from ref. 33. Copyright 1996, Elsevier Science B.V..)

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Surface Modifications with Adsorbed Poly(ethylene oxide) - American

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