Amphiphilic Block Copolymers as Bile Acid Sorbents - American

Biomacromolecules 2002, 3, 124-132

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Amphiphilic Block Copolymers as Bile Acid Sorbents: 2. Polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride): Self-Assembly and Application to Serum Cholesterol Reduction† Neil S. Cameron,‡ Adi Eisenberg,*,‡ and G. Ronald Brown§,| Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montre´ al, Que´ bec, Canada H3A 2K6, and Department of Chemistry, Brock University, 500 Glenridge Avenue, St. Catharines, Ontario, Canada L2S 3A1 Received August 3, 2001; Revised Manuscript Received November 1, 2001

This paper presents morphological studies and preliminary bile salt binding properties of the new amphiphilic diblock copolymer polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride) (PS-bPTMEACl)1 (see Figure 1), a derivative of PS-b-poly(tert-butylacrylate) (PS-b-PtBuA). In an aqueous environment, PS-b-PTMEACl forms simple spheres (∼20 nm diameter), large compound micelles (>100 nm diameter), and larger, more complex architectures as presented and discussed below. The colloidal stability with respect to sodium chloride and as a function of particle concentration is also considered. Finally, PSb-PTMEACl aggregates were prepared and tested as an alternative to the commercially available bile salt sequestrant resins that target coronary heart disease due to elevated cholesterol levels. Electron micrographs were employed to visualize the colloid-based polyelectrolyte-biosurfactant interaction and chromatographic separation analytical methods were used to quantify the sequestration. The results indicate that although at this preliminary stage they require laborious preparation, self-assembled aggregates may present an interesting alternative to the clinically used bile salt sequestrants. Introduction Coronary heart disease (CHD) is the leading killer in North America.2 In Canada alone, it is responsible for slightly more than one-third of all deaths and is the third leading cause of death under the age of 75.3 The effects of heart disease are not, however, limited to mortality: chronic pain or discomfort, restricted activity, disability, and unemployment as well as a profound impact on family life, sexuality, psychological health, and reaction to treatment (negative side effects) are among the nonfatal yet important possible health implications. As described in the preceding paper,1 bile salt sequestrants (BSS) represent one of the three main strategies for serum cholesterol, and thereby CHD, reduction. The present paper addresses the self-assembly and preliminary bile salt binding properties of a new amphiphilic diblock copolymer where the water-insoluble block is polystyrene and the water-soluble block is a polyacrylamide that is N-functionalized with ethylenetrimethylammonium chloride, polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride) (PS-b-PTMEACl)1 (see Figure 1). Although other diblock copolymer systems were potential candidates for this study, we chose a polystyryl core-forming * To whom correspondence may be addressed: e-mail, adi.eisenberg@ mcgill.ca; phone, (514) 398-6934; fax, (514) 398-3797. † This work was presented in part at the 73rd ACS Colloids and Surfaces Symposium (M.I.T., 1999). ‡ McGill University. § Brock University. | Deceased.

Figure 1. Polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride).

block because of the stable aggregates it yields and an ammonium-bearing coronal block, since earlier work in this laboratory has established that trimethylammonium groups, when attached to a polymeric backbone through a hydrophobic spacer and an amide link, are effective bile salt sequestrants both in vitro and in vivo.4 BSSs function in the gastrointestinal (GI) tract as quasi-selective sequestrants for bile salts, thereby amplifying their excretion. In turn, the liver replaces the excreted bile salts by modifying endogenous cholesterol molecules from which they are biosynthesized. Scope of the Paper. Self-assembly of amphiphilic block copolymers has been a topic of active research for several decades5 and the systematic study of asymmetric amphiphilic copolymers was initiated and has been followed by Eisenberg et al.6-9 The groups of Alexandridis,10 Antonietti,11 Bates, Discher, and Hammer,12 Liu,13 Maskos,14 Manners and Winnik,15 Mo¨ller,16 and Wooley,17 among others, are also active in this field. Fundamentally, the self-assembly of amphiphilic diblock copolymers resembles that of smallmolecule surfactants which have been shown to form a variety of morphologies including spheres, vesicles, rods,

10.1021/bm015596c CCC: $22.00 © 2002 American Chemical Society Published on Web 12/13/2001

Biomacromolecules, Vol. 3, No. 1, 2002 125

Amphiphilic Block Copolymers as Bile Acid Sorbents Table 1. Summary of Polymers Synthesized, Their Dimensions, and Aggregate Diametersa PSn-b-PTMEAClm (n - mcoupling agent)

NR4+ bearing units, φ

SSS

LCM

PS

total

80-330DCC 95-65DCC 175-65DMED 175-35DMED 385-80C2O2Cl2

0.81 0.40 0.28 0.16 0.17

26 33 60 c 45

93 75 118 250, 328 73

20 24 43 43 86

103 39 59 51 116

diameters, d (nm)

chain lengths (nm)b

a n and m ) degree of polymerization (average); φ ) ammoniumbearing unit fraction; SSS ) small simple sphere; LCM ) large compound micelle; d ) diameter measured from TEM micrographs. b Theoretical planar zigzag (fully extended) conformation. c None.

bilayers, and bicontinuous structures.18 Self-assembly can be considered to be under the control of three free energy contributions: a term for the hydrophobic phase that is excluded from the solvent, a term for the hydrophilic phase which is solvated and is often charged, and a term for the interface which will be minimized within the limits of the system. In the case of copolymers, these components are better described as core-block stretching, interfacial energy, and charged coronal-block repulsion. Among the asymmetric amphiphilic diblock copolymers, more than 30 morphologies have been identified but their classification is nontrivial since some are the result of kinetic trapping due to the constraints of macromolecules.9 We now describe the self-assembly of a new amphiphilic block copolymer, PS-b-PTMEACl, in block-selective solvents that provided small spheres, vesicles, large compound micelles (LCM), and other architectures. A survey of these aggregates is presented and discussed as a function of relative block length. The trimethylammonium-bearing copolymer differs significantly over the polystyrene-b-poly(acrylic acid) (PS-b-PAA)-type systems, reported previously, not only in that the charge on the corona-forming block is cationic but also in that the full ionization of the hydrophilic block is expected. While ionizable systems such as PS-b-PAA are tunable by addition of acid or base, by comparison, PS-bPTMEACl is expected to be comparatively immune to the effects of pH. The hydrophobic spacer between the charge and the amide link to the backbone of the corona-forming block (in this case, ethylene) can be exchanged for more, or indeed less, hydrophobic moieties. This type of solvophobic polyelectrolyte has been explored recently by Waigh et al.19 Preliminary assays on suspensions of these aggregates suggest limited binding of bile salts, as discussed below. Experimental Section Copolymer Synthesis. The parent materials were synthesized via anionic polymerization and were functionalized with N,N-dimethylethylenediamine (DMED) as described in the preceding paper1 and summarized in Table 1. Self-Assembled Aggregate Preparation. Following purification via dialysis, the polymer was lyophilized and the resulting powder was dissolved in DMF (e1% w/w). Deionized water was then added at a rate of about 1 drop every 10 s until the water content reached 6-10%. The

resulting turbid or blue-tinged suspension was then dialyzed against distilled water for several days with frequent water changes to remove DMF. In previous studies,20 salt was added before micellization. In this study, however, we added sodium chloride to a suspension of micelles after dialysis in order to confirm the solution stability of the colloidal system in the presence of simple electrolytes. As a control, a stock suspension of micelles was diluted with deionized water and also with an aqueous solution of sodium chloride to a final NaCl concentration of 5 mM. The resulting samples were then analyzed via transmission electron microscopy (TEM). Transmission Electron Microscopy. The TEM instrument used was a Phillips EM410 operating at an acceleration voltage of 80 kV. A drop of aggregate-containing suspension was deposited on a copper grid coated first with a thin film of Formvar and then with carbon. The specimen was left to permit evaporation of water at atmospheric pressure for several minutes. The sample concentration was chosen to maximize grid coverage without excess impingement, overlapping, and possible flocculation. Residual water was “wicked” away with a piece of absorbent paper (Kimwipe) and the grids were dried and shadowed with palladium/ platinum at an angle of about 30° (an Edwards or Baltzers evaporator). Image Analysis. TEM negatives were scanned directly at 150 dpi, 200% enlargement, as gray scale images. Calibrated automated area measurements were carried out (SigmaScan Pro 5.0, SPSS Inc.) to measure 100 or more aggregates per sample. An estimation of the diameters was made using simple algebra based on the reasonable assumption that the area of each aggregate was circular. The resulting data were manipulated into histograms (SigmaPlot 4.0/5.2, SPSS Inc.) and were peak-fitted (PeakFit 4.0, SPSS Inc.) with Gaussian bell curves to determine average diameters and modality. Bile Salt Sorption Studies. Since Brown et al. have an extensive history of bile salt sorption studies,21,26 efforts were made to replicate previous experimental conditions. Equilibrium bile acid concentrations were determined with reverse-phase high performance liquid chromatography (HPLC). The flow rate of the pump (LKB Bromma 2248(10)) was typically set to 1.0 mL/min, and the eluant was a Millipore-filtered binary mixture of methanol and deionized water (MilliQ) at a volume ratio of 80:20. Glacial acetic acid was added to a final concentration of 0.1 M in order to protonate the bile salt. The analyte was detected with a Waters 410 differential refractometer. Standard solutions of bile salts were prepared in 2.5 or 5.0 mM tris(hydroxymethyl)amino ethane (Tris hereafter) buffer adjusted to pH 7.2 with 0.1 M HCl. For calibration, aliquots were simply filtered and loaded onto the injection loop (50.0 µL) and a linear relationship between peak area and bile salt concentration was observed. Attempts at microfiltration of the copolymer-containing suspensions resulted in either immediate filter blockage or no polymer retention. Therefore, measuring the equilibrium bile salt concentration (Ceq) following incubation with copolymer aggregates required centrifugation. Aliquots of bile salt and

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Figure 2. Schematic representation of a simple sphere where a few symbolic polystyryl core-forming blocks (dashed lines) are shown for clarity and a TEM micrograph of simple spheres formed from PS385b-PTMEACl80.

copolymer-containing suspension (1 mL) were placed in Epindorf tubes and were subjected to 14k rpm for 2 min (Fischer Scientific Micro14), which cleared the polymer from the suspension. Unbound bile salts participating in true micellization were not appreciably affected by this procedure. Results and Discussion Primary characteristics. A defining characteristic of a micelle is the critical micelle concentration (cmc) above which aggregation occurs yet an equilibrium exists between aggregated and free molecules. It should be noted that styrene has a relatively high glass transition temperature (Tg ∼ 100 °C) and, furthermore, is not wetted in water. Therefore, as water is added and the styrene is segregated from the increasingly aqueous environment, the architecture becomes essentially “frozen” at some point. During the aggregation process, the system is, however, at (or near) equilibrium. Since the cmc for such a polymer in pure water is so low as to be essentially meaningless, it is a simplification to refer to these aggregates as micelles. Nevertheless, given that these aggregates resemble morphologies seen in true micelles where the aggregates and unimers are in dynamic equilibrium, block copolymer aggregates are conventionally referred to as micelles. Primary spheres of PS-b-PTMEACl formed in aqueous media are expected to have a styrene core and ammoniumcontaining corona. The electron density contrast between core and corona-forming blocks was insufficient to distinguish the two via TEM (see Figure 2). The predominance of primary micelles can be indirectly observed on sample preparation. As one might expect from light scattering, due to the colloidal size and low concentration of the particles, they create a blue-tinged turbid suspension. Typically, as samples with progressively shorter coronal blocks are induced to self-assemble, the aggregation number and particle diameter increase until there are insufficient core-forming blocks that are long enough to space-fill the core without insurmountable stretching energy penalties. The length of the core-forming block, the aggregation number, the solubility characteristics of the corona-forming block, and the sample polydispersity, therefore, regulate the size of this smallest aggregated morphology. Note that for these anionically synthesized block copolymers, polydispersity indices (PDIs) on the order of 1.1 were measured. Even at these low PDIs, a substantial fraction of the total polymer chains have a degree of polymerization that equals or exceeds twice the degree of polymerization at the number average molecular weight (Mn). Therefore, the chain dimensions of an

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Figure 3. Transmission electron micrographs (TEM) demonstrating the morphologies within a family of PS-b-PTMEACl: left, LCMs from PS175-b-PTMEACl35; right, mixed morphologies from PS175-bPTMEACl65.

average coil do not necessarily limit the simple sphere morphology since only a few chains must reach the center of any given aggregate.27 When the chain dimensions of the longest chains no longer permit simple spheres, other morphologies predominate. With the samples employed in this study, LCMs, which resemble bulk reverse micelles, were evidently the preferred morphology. Visually, a suspension of these larger particles is more opaque. For the largest particles, settling was observed, though resuspension involved only gently shaking the sample vial. Considering that for PS-b-PAA, when the mole fraction of acid units exceeds 10%, only primary spheres are observed under standard conditions and that in all cases for PS-bPTMEACl, the mole fraction of charge-containing groups equals or exceeds 16%, we might expect to see only primary spheres. Indeed, primary spheres were observed for all samples except PS175-b-PTMEACl35 (see Figure 3, left). For the PS175-b-PTMEACl65 sample (see Figure 3, right), the modal diameter of smallest aggregates was near 60 nm, well above the diameter one would expect for aggregates from PS-b-PAA of similar block lengths; however, as discussed above, sample polydispersity, halide counterion effects, the presence of trace homopolymer, and solubility issues with the coronal block19 can have a profound effect on aggregation. Nevertheless, given that these nominally primary spheres are quite large, it is perhaps less surprising that the PS175-b-PTMEACl35 sample gives a population of LCMs where primary micelles are not possible due to the short coronal chains that favor higher aggregation numbers. The large “transitional” primary spheres of PS175-bPTMEACl65 are composed of chains that, when fully extended, are ∼60 nm long (average). Indeed, these spheres may be nascent LCMs where the solubility of the coronaforming block, which has hydrophilic and hydrophobic components as well as H-bonding sites and charged moieties, led to LCMs in the binary solvent mixture. Furthermore, since the sample preparation requires slow addition of a nonsolvent for the core-forming block to a macromolecularly disperse solution in a common solvent for both blocks, the first chains to phase separate will be any homo-PS and the longest of the copolymer macromolecules. These “large” small spheres were observed in the presence of true LCMs (≈120 nm mean diameter, see Figure 3, right), which may better reflect the preferred morphology of the average coils following the selective self-assembly. Thus, we infer that 65 coronal units per chain is near the solubilizing limit for

Amphiphilic Block Copolymers as Bile Acid Sorbents

primary spheres in this polymer family under present preparative conditions. By comparison, the related PS170-bPAA33 material (for which the coronal block is very hydrophilic) has been independently studied7 and forms only small spheres in pure water with a diameter of 25 ( 1.4 nm. Asymmetric amphiphilic PS-b-PAA is a convenient paradigm system since it is in the strong segregation limit, it has been studied in detail, and these materials have been shown to provide a wealth of self-assembled morphologies. During the process of self-assembly, the core-forming PS blocks will tend to a self-avoiding tethered random walk, the coronal blocks will repel each other, both as a function of steric constraints near the core-corona interface and of partial ionization of the carboxylic acid moieties, and the hydrophilic-hydrophobic interface will tend to a minimum value within the space-filling constraints of the core-forming block. The morphology dimensions are a direct result of these factors at equilibrium and kinetic trapping. Similarly, the observed morphologies and their dimensions for PS-bPTMEACl are a function of the same energetic and kinetic factors; however, the coronal block offers several additional parameters as compared with its PAA cousin,19 which evidently can have a profound effect on copolymer selfassembly. Where PAA is partially ionized in water, quasicomplete ionization of the coronal block for PTMEACl is expected due to the tethered N,N,N-trimethylammonium groups. Furthermore, the coronal pendants offer not only intrachain interactions and halide counterion effects but also hydrophobic associative tendancies due to the ethylene spacer in each pendant, as well as possible hydrogen bonding. Decreased coronal-chain repulsion leads to increased aggregation numbers and observed aggregate dimensions. LCM Formation Mechanism. Significantly, large compound micelles are accessible from PS-b-PTMEACl samples when the corona-forming block is much longer than that for similar PS-b-PAA samples. LCMs, such as those produced from PS175-b-PTMEACl35, can form by at least two pathways: (i) On addition of nonsolvent for one of the blocks, the nonsoluble blocks aggregate to create a continuous phase with islands of corona-forming blocks nested throughout and surrounded by a coronal shell at the core-solvent interface. (ii) Alternatively, simple spheres can form, but corona-chain flocculation or interpenetration occurs and thus the continuous phase is coronal blocks with pockets of insoluble blocks (Figure 4). These two alternatives correspond to the extremes of morphology observed in the bulk for diblock copolymers.28 Bulk behavior dictates that the largest block will form the continuous phase. The especially rough texture of the LCMs arising from PS385-b-PTMEACl80 (see Figure 5) suggests that in this case, coronal-block interaction is the primary mode of formation in effect. This mode of aggregation is somewhat counterintuitive given the charge-bearing coronas; however, the hydrophobic pendant spacer and possible hydrogen bonding between amide groups may compete with electrostatic repulsion. The PS175-b-PTMEACl35 sample formed the least visually complicated LCMs by TEM, but this observation needs some explanation as PS385-b-PTMEACl80 has the same mole


Figure 4. Schematic representation of chain entanglement and direct LCM formation pathways.

Figure 5. TEM micrographs of various large compound micelles (LCMs): A, PS175-b-PTMEACl35; B, PS175-b-PTMEACl65; C, PS385b-PTMEACl80; D, PS80-b-PTMEACl330.

fraction of acrylamide units but forms primary spheres. In this latter case, the styrene block is more than twice as large as the styrene in the PS175 family and therefore the free energy of stretching for the core blocks is perhaps less important. If significant “killing” between the PS block polymerization and the acrylate block addition occurred, residual homo-PS may contaminate this sample. Hence, for PS175-b-PTMEACl35, the force balance between stretching of the core blocks, extension of the corona blocks and the interface energy, and the possibility of residual homo-coreforming polymer dictate that primary micelles are energetically too expensive to permit their formation. Bilayer Structures. The formation of nonequilibrium structures including vesicles and lamellae was also observed (see Figure 6). While primary spheres and LCMs dominated, other structures were occasionally observed suggesting that kinetic trapping also occurs. It is probable that in the process of formation, some aggregates do not have sufficient time to achieve a near-equilibrium structure. Since the cores eventually become glassy following self-assembly and continued water addition, macromolecular dynamics and chain transfer processes are dramatically slowed and are ultimately “frozen”. For PS-b-PAA in DMF/water mixtures, the critical water concentration (cwc) is typically on the order of 4% (w/w) and at 10% water (w/w), morphological “averaging” appears to be virtually nonexistent over 24 h.29

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Figure 6. TEM micrographs of baroque structures: A, PS80-bPTMEACl331 shell structure of diameter (300 nm); B, PS385-bPTMEACl80 dimpled architecture (55 nm); C, PS175-b-PTMEACl65 interconnected vesicles with simple spheres; D, PS175-b-PTMEACl65 micrograph enlargement from C (rotated 90° counterclockwise) showing especially interconnected spheres or “pearl necklaces” (upper right) and corpuscular vesicles (center).

From these dramatically slowed kinetics we infer that single chain exchange is not possible under these conditions. Indeed we expect the extrapolated cmc for these polymers in pure water to be less than 10-30.30 This is encouraging for the potential application of copolymer aggregates in the GI tract where single chains present potential toxicity. At water concentrations below and slightly above the cwc, the annealing time between water drop additions would have to be substantially increased (standard conditions were ∼2%/ min) in order to entirely avoid the kinetic oddities of the PS-b-PTMEACl and related systems. Figure 6A shows a hollow sphere with internal surface features. The emptiness of this structure can be inferred from the electron transparency of the subject and the opacity of its rim. Were it not for this polymer void, the surface texture and occlusions would be much more difficult to observe. Figure 6B focuses on a dimpled aggregate that may be a collapsed, thick-walled vesicle. Figure 6C demonstrates a mesoscopic bilayered architecture that hides a significant population of much smaller primary spheres and vesicles. The center of Figure 6C is enlarged and presented in Figure 6D, where the corpuscular features (6D image center) and “pearl necklace” nascent rods (6D image upper right) are more obvious. Though these aggregates were rare, they do indicate that PS-b-PTMEACl can offer the same sort of morphological variation exhibited by the PS-b-PAA and PSb-PEO crew cut systems that have been studied so intensively.6 Population Histograms. When the populations of aggregates (see Figure 7) are put in the context of their parent chain maximum dimensions, understanding the observed morphologies becomes simpler. As long as adequate, sufficiently long core-forming blocks are present to volumefill the sphere, then simple “micelles” are a likely possibility, as discussed above. In practice, it is easy to distinguish simple spheres from higher order morphologies, especially when both are present since the simple spheres are so much smaller than the structurally more complex LCMs. The population histograms presented in Figure 7 offer two interpretations: a profile with a single peak and tailing or a bimodal distribution. Given the visual impact of the micrographs, it seems most reasonable to assign multiple peaks to the histogram profiles, with the maxima at small diameters referring to the simple spheres, and the larger populations to the LCM aggregates.

Figure 7. Population histograms for PS-b-PTMEACl. Peak maxima are indicated.

The histograms in Figure 7 are presented in order of decreasing corona-forming repeat unit mole fraction φ. The uppermost histogram in Figure 7 corresponds to PS80-bPTMEACl330, where φ is 0.81. For this sample, the long corona-forming blocks limit the aggregation number by electrostatic repulsion and crowding. The simple sphere diameter for this polymer is therefore relatively small (mode ) 30 nm). However a substantial fraction of the aggregates is present as LCMs with an average diameter of 93 nm. The following sample, PS95-b-PTMEACl65, has a similar size of core-forming block, but φ is 0.40. With fewer corona-forming units, the decreased crowding in the corona favors an increase in the aggregation number, as reflected in the slightly larger simple spheres (33 nm), and very few LCMs are observed. When φ is 0.28 (PS175b-PTMEACl65), the modal small sphere diameter is 60 nm, i.e., substantially increased (see Figure 3, right). These large simple spheres can be considered transitional, since the related PS175-b-PTMEACl35 sample, where φ is 0.17, yields no small spheres and forms only LCMs (see Figure 3A). Finally, when the hydrophobic styrene block is sufficiently long to space fill the core of a simple sphere as dictated by the aggregation number-determining corona-forming block, small spheres are once again formed as demonstrated for PS385-b-PTMEACl80, where φ is also 0.17 (see Figure 5C). Comparison of the size distributions for PS175-bPTMEACl35 and PS175-b-PTMEACl65 shows that by changing only the hydrophilic block length, drastic morphogenesis can be induced. For diblock systems within the same morphological regime (for example, simple spheres), the aggregate diameter is primarily dependent on the core-forming block: PS80-b-PTMEACl330 and PS385-bPTMEACl80 follow this trend, where short styrene blocks form small primary aggregates (average diameter ) 30 nm)


and long styrene blocks form large primary aggregates (45 nm). In order for simple spheres to form, there must be sufficient ammonium-bearing units to solubilize the styrene, to limit the aggregation number; also the PS blocks must not be obliged to stretch beyond the limits imposed by the free-energy penalty to space-fill the core. For PS175-bPTMEACl65, where there are evidently insufficient ammonium-bearing groups, transitional and LCM spheres selfassemble (60 and 120 nm) leading to the higher-order aggregates formed by PS175-b-PTMEACl35 (250 and 328 nm). Trends in the LCM diameters for all five samples are less easily explained since, unlike sphere and rod diameter, or bilayer wall thickness, which are limited by block length, their formation is governed by secondary considerations such as polymer concentration. From the outset, it seems counterintuitive that a polymer sample of low polydispersity, once induced to self-assemble, would form a mixture of aggregates. However there are examples of mixed morphologies that have been shown to arise from wide coexistence regions in the phase diagrams between pure morphologies.9 Selfassembly for PS-b-PTMEACl is more complex than for PSb-PAA due to the range of possible interactions within and among the corona-forming blocks. The compositional heterogeneity of the coronae suggests that the most probable explanation for the various LCMs presented here is that at some stage in the self-assembly process, they were thermodynamically stable, and on continued addition of nonsolvent for PS, they became kinetically trapped. It is also possible that in the anionic synthesis of the parent diblock copolymers in the PS175 family, some killing on the addition of the acrylate may have slightly contaminated the sample with traces of homopolystyrene. Work in our laboratory has confirmed that the addition of homo-core-forming polymer leads to core swelling for PS-b-PAA.31 The baroque structures may also be best understood by drawing parallels to the paradigm PS-b-PAA system. We have demonstrated that PS-b-PAA is extremely sensitive to counterion valence and concentration.20 Adding CaCl2, for example, during the formation of aggregates from PS410-bPAA13 at extremely low concentration (as few as 0.03 Ca2+ ions per repeat unit) induces vesicle formation from a polymer that forms simple spheres in the absence of added ions. Due to the added complexity of the amidation for PSb-PTMEACl, it is possible that despite repeated dialysis and the IR evidence for essentially complete functionalization, these samples were very slightly contaminated by reaction byproducts or ionic species beyond our detection limit. Due to the hydrophobic spacer between the backbone and the ammonium group, we expect the polymer to be even more sensitive than PS-b-PAA to minute changes in environment; thus, because of the nontrivial effect of the interactions, some samples may initially appear eccentric. Effect of Simple Salt on Coronas. Preliminary investigation of micelle stability demonstrates that the aggregates are robust. Addition of sodium chloride before and during micellization to a block polyelectrolyte such as PS-b-PAA induces morphogenesis. Previous studies show that a polymer that self-assembles to form simple spheres with deionized water as the precipitant (e.g., PS410-b-PAA25) can be induced


Figure 8. TEM micrographs for PS385-b-PTMEACl80: A, trace salt; B, 5 mM salt.

Figure 9. TEM micrograph of PS385-b-PTMEACl80 with high polymer and salt content-induced impingement.

to form short rods, long rods, vesicles, and large compound vesicles (LCVs) by adding salt to solvent mixture.8 Similar morphogenic effects are to be expected for PS-b-PTMEACl. However in the present work, where an indication of colloidal stability was required, sodium chloride was added after micellization and dialysis. In water, we expect the polystyrene core to be glassy and solvent-free, and as a consequence we expect the micelles to be stable. It should be noted that, in general, block copolymer micelles appear to be intrinsically stable. Even when the glass transition temperature (Tg) of the core-forming block is very low, as can be the case for polybutadiene-bpoly(acrylic acid) (PBD-b-PAA), LCMs, rods, and primary structures are formed.32 In this context it is not surprising that the addition of small amounts of salt to “frozen” aggregates (primary and transitional spheres) of PS-bPTMEACl perturbs at most only the coronal dimension in solution without disrupting the integrity of the structure (see Figure 8). The samples are virtually identical. An early TEM experiment, where the small-sphere concentration was too high to give isolated spheres on the grid, led to the micrograph in Figure 9. The intimate contact of these micelles is an artifact of the TEM grid preparation. Nevertheless, it remains clear that the polymer concentration was higher than for the other samples, yet each sphere retains coherence. Indicative TEM Study. The first indication that block copolymer assemblies could function as bile salt sequestrants was strikingly visual. We think we have some understanding as to the how and why of bile salt and sequestrant interactions, but it was profoundly satisfying to see the effect of bile salt addition to a suspension of nanospheres prepared

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Figure 10. TEM micrographs of PS385-b-PTMEACl80 samples prepared in the presence of (A) 5 mM NaCl, (B) 0.6 mM NaGC, and (C) 1.2 mM NaGC. The simple electrolyte slightly reduces the diameter of the aggregates as compared with the salt-free sample (see Figure 8). However, the addition of bile salt surfactant molecules, even in very low concentrations, causes agglomeration.

Cameron et al.

Figure 12. TEM micrographs of PS-b-PTMEACl self-assembled architectures (EDC mediated functionalization): A, PS80-bPTMEACl330 showing primarily simple small spheres though nascent rods and lamellae (one perforated) are observable; B, C, and D, PS80-b-PTMEACl130 larger aggregates and agglomerates clearly dominate, but small spheres and bilayer structures are also present. Table 2. A Summary of PS-b-PTMEACl Binding Experiments

Figure 11. Schematic representation of the bridging interaction between copolymer aggregates (Agg) by stylized bile salts (many substituents and molecular details omitted for clarity).

from the PS385-b-PTMEACl80 sample (see Figure 10). To a stock suspension of aggregates, NaCl and solutions of sodium glycocholate (NaGC) in Tris buffer were added. The suspensions to which NaCl was added were unchanged to the unaided eye, in keeping with the observations reported above. However, the addition of NaGC ultimately led to macrophase separation over the course of several hours. One would expect a polyelectrolyte chain to collapse somewhat on the addition of salt due to electrostatic shielding of the charges on the polymer.33 However, the macrophase separation of the samples treated with NaGC is clearly much greater than those observed on addition of NaCl and appears to be the result of surfactant-mediated agglomeration. It seems reasonable to suggest that ion exchange, electrostatic shielding, and the mechanisms of bile salt self-assembly (see Figure 11) cooperatively lead to the macrophase separation. Bile salt micelles may bridge copolymer spheres, or bile salts may render the polymer surfaces hydrophobic ultimately leading to precipitation. The entropy accompanying the release of microions will favor aggregation. High Polymer Concentration Sorption Study: HPLC. The data discussed above give a qualitative indication of the bile salt binding potential of these copolymer aggregates; however, to measure the bile salt sequestration, higher initial bile salt concentrations with more polymer to achieve substantial differences between initial and equilibrium GC concentrations were required due to instrumental detection limits. A water-soluble carbodiimide (EDC, see above) was employed to convert two related samples of PS-b-PAA to PS-b-PTMEACl (see TEM micrographs, Figure 12). Once these aggregates were isolated in pure water, the samples were lyophilized and aliquots of NaGC solutions were tested with sufficient polymer to ensure a significant decrease in Ceq (NaGC). The dry copolymer aggregates (∼10 mg) were added directly to the NaGC/Tris solutions (2.0 mL) to avoid potential errors resulting from dilution, and HPLC injections were carried out in duplicate. Under these condi-

polymer

initial -NMe3Cl [NaGC]

GC bound

GC

per g of

bound

PSn-PTMEAClm

mass

(n-m)coupling agent

(mg)

(µmol)

(mM)

(mM) (mmol/g)

chain

80-130EDC 80-330EDC 80-330EDC 80-330EDC 80-330DDC 80-330DDC

9.9 3.0 10.6 10.6 0.5 0.5

13.1 14.0 49.5 49.5 2.3 2.3

0.62 0.62 0.62 2.53 1.5 3.0

0.43 0.48 0.42 2.12 1.39 2.85

1.3 6.7a 2.3 5.6 31a 43a

Ceq

polymer 0.039 0.096a 0.038 0.077 0.43a 0.59a

per

a Values to be interpreted with caution due to the mass of polymer in the assay.

tions, PS80-b-PTMEACl330(EDC) reduces the NaGC concentration by up to one-third of the initial concentration. The quantitative binding results for all polymers tested using HPLC analysis of the Ceq (NaGC) are given in Table 2. The first four lines of Table 2 give data for the polymers functionalized using EDC and the last two concern the DCCmediated polymers. The data show that when nearly equal masses of polymers are used, the sample with a higher mole fraction of TMEACl units is twice as effective per chain at removing NaGC from solution showing that the sorption per chain is related to the coronal block length. The difference in aggregate morphology between PS80-b-PTMEACl130(EDC) and PS80-b-PTMEACl330(EDC) does not seem to have a strong influence on bile salt anion sorption. Very low polymer concentrations seem to result in the best ratio of bound GC (mmol) to polymer mass (g), but direct comparison of the efficacy of the DCC and EDC derived polymers is complicated by the limited amount of polymer available for the study. PS80-b-PTMEACl330(DDC) assays nominally employed ∼0.5 mg of polymer, whereas the EDC-mediated sample assays tested 3-10 mg copolymer. A secondary differencecausing effect may involve the assay buffer concentrations. For compatibility with ongoing investigations into the nature of bile salt sorption,34 5.0 mM Tris was used for the EDCderived polymers whereas dilution gave 2.5 mM Tris for the DCC-derived samples. Bile salts are sequestered most effectively from pure water, less well from Tris buffer, and phosphate buffer surrenders bile salts under protest. As the buffer concentration increases, the ionic strength of the


solution increases and the sorption decreases although, for these experiments, this effect is expected to play only a minor role.24 Conclusions Morphology Summary. We have presented a novel amphiphilic diblock copolymer that self-assembles into nanoand mesostructures composed of a polystyrene hydrophobic phase and a tethered cationic trimethylammonium chloride containing hydrophilic phase. The most prevalent observed morphologies are simple small spheres and LCMs. However, more complex, apparently kinetically trapped structures are occasionally observed and indicate a rich field of morphological investigation. Among the interesting facets of this polymer is the hydrophilic corona-forming block with hydrophobic characteristics, which makes PS-b-PTMEACl more sensitive than PS-b-PAA to precipitation and renders the coronal block less “able” to solubilize the styrene blocks into the binary solvent mixture. The ammonium-containing block is also interesting because of the secondary amide link between the ammonium group and the backbone and the implied potential for hydrogen bonding. Hydrophobic interactions and hydrogen bonding make flocculation a reasonable mechanism for LCM formation. Once the plasticizing common solvent is completely removed from the system via dialysis, the micelle architecture is stable, though the coronas may be induced to collapse slightly in the presence of sodium chloride. Even at high polymer concentration, the aggregates seem to maintain coherence. Binding Summary. N,N,N-Trimethylammoniumethylenefunctionalized PS-b-poly(acrylamide) samples are shown to be bile salt sequestrants. The ion exchange sorption of biosurfactants into the highly charged hydrophilic coronas of the self-assembled nanoaggregates derived from this family of polymers has been established. Furthermore, indications of a significantly high capacity are suggested by the data. Synthetic and practical challenges notwithstanding,1 these materials do participate in ion-exchange reduction of free glycocholate when placed in a glycocholate solution. Selfassembled aggregates of the parent copolymer can be isolated by lyophilization and, on addition of water, resuspend immediately to give a turbid “solution”. When the fraction of available ammonium groups is very low compared with the absolute number of glycocholate molecules, up to 40 binding sites per chain consisting of 330 potential sites may be occupied (assuming that each bound molecule completely occupies one site). Given the region of restricted chain mobility due to crowding near the core-corona interface, the true number of available sites will be somewhat less than the degree of polymerization of the sorption block. When a greater quantity of PS80-b-PTMEACl330 copolymer was tested, 5.6 glycocholate anions were bound per polymer chain. Although these diblock copolymer assemblies cannot yet compete in vitro with the cross-linked resin, QPDA-12,1,25 which sequesters virtually all GC- under experimental conditions similar to those described above, in vivo their efficiency may be improved due to the phase separation


phenomenon seen in Figure 10. There is reason to believe that in the GI tract, these assemblies might aggregate and therefore compete more effectively against the extremely efficient enterohepatic recirculation of bile salts in the lower small intestine. With further tailoring, one would expect to improve on the raw binding properties of these materials. In summary, despite the absence of a long hydrocarbon spacer used in previous sequestrants and the expected concomitant dramatic increase in binding constant,25 these materials show some promise as bile salt sequestrants. The primarily electrostatic attraction between glycocholate and polyelectrolyte as well as the partition of glycocholate between the highly polar aqueous medium and the hydrophilic, but less polar, matrix favors surfactant sorption and ion exchange. Electron microscopy visually confirms the effect of the addition of biosurfactants to nanoscale aggregates formed from amphiphilic diblock copolymers. It is in the study of these interactions that these aggregates may prove most fruitful. Acknowledgment. The authors are grateful for financial support in the form of operating grants from the Natural Science and Engineering Research Council, Canada and Fonds FCAR, Quebec, and N.S.C. is particularly pleased to acknowledge the J. W. McConnell foundation and Sigma Xi (Canada). The authors also thank Carl Bartels, Muriel Corbierre, and Professor Donald Paterson for fruitful discussions. The conceptual basis for this work, i.e., the possibility of using self-assembled block copolymer simple spheres as bile acid sorbents, was first suggested in the M.Sc. thesis of F. Asgari. In Memoriam. G. Ronald Brown died unexpectedly just as this manuscript was being completed. His input to the work was invaluable, and he will be sorely missed as a supervisor, friend, and collaborator. A generation of students and colleagues at all levels benefited from his insightful, thoughtful, and caring interactions. The personal and scientific loss is immeasurable. Supporting Information Available. Binding isotherm of NaGC sorption to PS(80)-b-PTMEACl(330)DCC. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Cameron, N. S.; Eisenberg, A.; Brown, G. R. Biomacromolecules 2002, 3, 116. (2) Grundy, S. M. JAMA, J. Am. Med. Assoc. 1986, 256, 2849-2858. (3) Heart and Stroke Foundation of Canada, The Changing Face of Heart Disease and Stroke in Canada 2000. Ottawa, Canada, 1999. (4) St-Pierre, L. E.; Brown, G. R.; Wu, G. U.S. Patent 5,374,422, 1994 and patents cited therein. (5) See for example: (a) Tuzar, Z.; Kratochvil, P. Surface and Colloid Science; Matijevic, E., Ed.; Plenum Press: New York, 1993; Vol. 15, pp 1-83. (b) Price, C. DeVelopments in Block Copolymers; Goodman, I., Ed.; Elsevier Applied Science: London, 1982; Vol 1, pp 39-80. (c) Selb, J.; Gallot, Y. DeVelopments in Block Copolymers; Goodman, I., Ed.; Elsevier Applied Science: London, 1985; Vol. 2, pp 27-96. (6) Zhang, L.; Eisenberg, A. Science 1995, 268, 1728. (7) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168. (8) Zhang, L.; Yu, K.; Eisenberg, A. Science 1996, 272, 1777. (9) Cameron, N. S.; Corbierre, M. K.; Eisenberg, A. Can. J. Chem. 1999, 77, 1311. (10) Svenson, B.; Olsson, U.; Alexandridis, P. Langmuir 2000, 16, 1689.

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(11) Chernyshov, D. M.; Bronstein, L. M.; Bo¨rner, H.; Berton, B.; Antonietti, M. Chem. Mater. 2000, 12, 114. (12) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143-1146. (13) Ding, J.; Liu, G. Macromolecules 1999, 32, 8413. (14) Maskos, M.; Harris, J. R. Macromol. Rapid Commun. 2001, 22, 271. (b) Rheingans, O.; Hugenberg, N.; Harris, J. R.; Fischer, K.; Maskos, M. Macromolecules 2000, 33, 4780. (15) Raez, J.; Barjovanu, R.; Massey, J. A.; Winnik, M. A.; Manners, I. Angew. Chem., Int. Ed. 2000, 39, 3862. (16) Spatz, J. P.; Mo¨ssmer, S.; Mo¨ller, M. Angew. Chem., Int. Ed. 1996, 35, 1510. (17) Huang, H.; Kowalewski, T.; Remsen, E. E.; Gertzmann, R.; Wooley, K. L. J. Am. Chem. Soc. 1997, 119, 11653. (18) Evans, D. F.; Wennerstro¨m, H. The colloidal domain: where physics, chemistry, biology and technology meet; VCH Publishers: New York, 1994. (19) Waigh, T. A.; Ober, R.; Williams, C.; Galin, J.-C. Macromolecules 2001, 34, 1973-1980. (20) Zhang, L.; Eisenberg, A. Macromolecules 1996, 29, 8805. (21) Zhu, X. X. Ph.D. Thesis, McGill University, Department of Chemistry, Montre´al, 1988. (22) Wu, G. Ph.D. Thesis, McGill University, Department of Chemistry, Montre´al, 1990.

Cameron et al. (23) Zhu, X. X.; Brown, G. R.; St-Pierre, L. E. J. Pharm. Sci. 1992, 65. (24) Leonard, D.; Clas, S.-D.; Brown, G. R. React. Polym. 1993. 131140. (25) Wu, G.; Brown, G. R.; St-Pierre. L. E. Langmuir 1996, 466-471. (26) Asgari, F.; Brown. G. R. Int. J. Bio-Chromatogr. 1997, 2, 249267. (27) Nguyen, D.; Zhong, X. F.; Williams, C. E.; Eisenberg, A. Macromolecules 1994, 27, 5173-5181. (28) (a) Matsen, M. W.; Bates, F. S. Macromolecules 1996, 29, 7641. (b) Khandpur, A. K.; Fo¨rster, S.; Bates, F. S.; Hamley, I. W.; Ryan, A. J.; Bras, W.; Almdhal K.; Mortensen, K. Macromolecules 1995, 28, 8796. (29) Zhang, L.; Shen, H.; Eisenberg, A. Macromolecules 1997, 30, 10011011. (30) Shen, H.; Eisenberg, A. J. Phys. Chem. B 1999, 103, 9473-9487. (31) Zhang, L.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 1999, 37, 1469. (32) Yu, Y.; Zhang, L.; Eisenberg, A. Langmuir 1997, 13, 2578. (33) Grosberg, A. Y.; Khokhlov, A. R. Statistical physics of macromolecules; AIP Press: New York, 1994. (34) Williams, C. K.; Galley, W. C.; Brown, G. R. Personal communication, McGill University, 2000.

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Amphiphilic Block Copolymers as Bile Acid Sorbents - American

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