Cationic Double-Hydrophilic Model Networks: Synthesis

Double-hydrophilic block copolymers (DHBCs)1 represent a new class of ... Recent studies on DHBC model networks come from our research team and ...
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Biomacromolecules 2003, 4, 1150-1160

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Cationic Double-Hydrophilic Model Networks: Synthesis, Characterization, Modeling and Protein Adsorption Studies Elena Loizou, Aggeliki I. Triftaridou, Theoni K. Georgiou, Maria Vamvakaki, and Costas S. Patrickios* Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus Received September 9, 2002

Group transfer polymerization (GTP) was used for the preparation of eight networks based on two hydrophilic monomers, 2-(dimethylamino)ethyl methacrylate (DMAEMA) and poly(ethylene glycol) methacrylate (PEGMA). Ethylene glycol dimethacrylate (EGDMA) served as the cross-linker, whereas 1,4-bis(methoxytrimethylsiloxymethylene)cyclohexane (MTSMC) was used as a bifunctional initiator. Seven of the networks had linear segments of accurate molecular weight between the cross-links, i.e., they were model networks, whereas the eighth was an equimolar randomly cross-linked network. Five of the seven model networks were based on ABA triblock copolymers with PEGMA midblocks and DMAEMA endblocks, in which the DMAEMA/PEGMA ratio was varied. The remaining two model networks were equimolar isomers, the one based on BAB triblocks (with a DMAEMA midblock) and the other based on the statistical copolymer. The degrees of swelling of all of the networks were measured as a function of pH and were found to increase below pH 7. The degrees of swelling at low pH values increased with the percentage of the DMAEMA monomer, which is ionized under these conditions. These swelling results were confirmed qualitatively by theoretical calculations. Finally, the pH-dependence of the adsorption of the proteins pepsin, bovine serum albumin, and lysozyme onto one of the model networks was studied. Introduction Double-hydrophilic block copolymers (DHBCs)1 represent a new class of copolymers with two different types of hydrophilic blocks, with one of the two blocks being convertible to hydrophobic by a change in the solution conditions (temperature,2,3 pH,4-6 or salt concentration7) or by the introduction of a substrate (macromolecule8 or crystal surface9). The nature of the monomer repeat units in each hydrophilic block can be either nonionic or ionic (or ionizable), leading to three possible binary combinations of units in the two blocks of DHBCs: nonionic-nonionic,2,3 nonionic-ionic,10 and ionic-ionic.5,6,11 Two important properties of these materials are, first, reversible solutioncondition-induced micellization2,3,7,12 and, second, retention of solubility upon complexation,13 the latter leading to several novel applications including drug (and DNA) delivery, colloidal stabilization, and crystal design.14 Although linear DHBCs receive increasing attention,1 DHBC model15 networks,16-18 i.e., cross-linked DHBCs, remain scarcely explored. These DHBC networks are expected to have all of the attributes of linear DHBCs, mentioned above. Moreover, if these networks are model as well, the comparison of their experimental characteristics with theoretical predictions will be more reliable, which will compensate for the considerable effort required for their synthesis. Recent studies on DHBC model networks come from our research team and concern the preparation and aqueous characterization of networks based on positively and * To whom correspondence should be addressed.

negatively charged blocks (polyampholyte networks),19 as well as networks based on negatively charged and neutral blocks.20 The former system comprised end-linked linear ABA triblock polyampholytes,19 whereas the latter was formed by the interconnection of star copolymers.20 The present study involves the preparation by group transfer polymerization (GTP)21-24 of DHBC model networks based on end-linked linear ABA triblock copolymers comprising positively charged and neutral units. These materials can find applications in medicine and biotechnology as drug release systems and as media for protein and DNA purification. One particular advantage of these networks when the applications involve electrostatic binding of negatively charged biomolecules (e.g., DNA) to the positively charged units of the networks is the absence of the total collapse of the network, even at high biomolecule loadings, because of the presence of the nonionic hydrophilic units. Upon their Coulombic binding to solutes of opposite charge, the positively charged units of the network would lose their mobile counterions, with a concomitant reduction in the interchain repulsions and osmotic pressure, leading to shrinkage of that part of the network. However, the nonionic part of the network would remain swollen, as it would not participate in the electrostatic binding, thus securing water retention in the system. This would be analogous to the retention of water-solubility of complexes of linear DHBCs with oppositely charged solutes, and it is the essence of the DHBC systems. In addition to the synthesis, this investigation reports the experimentally measured aqueous degrees of swelling of these networks, which are compared to theoretical

10.1021/bm020102c CCC: $25.00 © 2003 American Chemical Society Published on Web 08/21/2003

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Figure 1. Chemical structures and names of the reagents used for the network synthesis.

values predicted by a thermodynamic model. Finally, the ability of one of these networks to adsorb various proteins is investigated. Experimental Section Materials and Methods. All of the chemicals were purchased from Aldrich, Germany, with the exception of the poly(ethylene glycol) methacrylate (PEGMA) monomer (purity 99% w/w, 0.4% water), having an average of six ethylene oxide units in the side chain,25 that was kindly donated by Laporte Performance Chemicals, U.K. The proteins were purchased from Sigma. Figure 1 shows the chemical structures and names of the materials used for the polymerizations: the monomers, PEGMA, and 2-(dimethylamino)ethyl methacrylate (DMAEMA, 98%), the bifunctional initiator, 1,4-bis(methoxytrimethylsiloxymethylene)cyclohexane (MTSMC), the cross-linker, ethylene glycol dimethacrylate (EGDMA, 98%), and the catalyst, tetrabutylammonium bibenzoate (TBABB). Although the monomers and the cross-linker are commercially available, the MTSMC initiator and the TBABB catalyst are not. Thus, they were both synthesized in our laboratory. MTSMC was prepared by the silylation of dimethyl 1,4-cyclohexanedicarboxylate (97%), accomplished in a two step procedure:26 the reaction of dimethyl 1,4-cyclohexanedicarboxylate with diisopropylamine (99%) and butyllithium (2.5 M in hexanes) in absolute tetrahydrofuran (THF) at -78 °C, followed by the reaction of the mixture with chlorotrimethylsilane (98%) under the same conditions. The TBABB polymerization catalyst was prepared by the reaction of the tetrabutylammonium hydroxide (40% w/w solution in water) with benzoic acid (99%), following the method described by Dicker et al.23 The catalyst powder was stored under vacuum until use. THF (99.8%; Labscan, Ireland) served as the polymerization solvent. It was dried by refluxing it over a sodium/potassium alloy for 3 days, and it was freshly distilled prior to the polymerization. The PEGMA monomer was used as a 50% v/v solution in freshly distilled THF because it is a highly viscous liquid and rather difficult to handle without dilution. The monomers and the cross-linker were passed twice through basic alumina columns to remove inhibitors and acidic impurities. DMAEMA and EGDMA were subsequently stirred overnight over calcium hydride to remove the last traces of moisture and protonic impurities. A free radical inhibitor, 2,2-diphenyl1-picrylhydrazyl hydrate (DPPH, 95%), was also added to DMAEMA and EGDMA (but not to PEGMA) to prevent

thermal polymerization. Finally, DMAEMA and EGDMA were distilled under vacuum prior to the polymerization. DPPH was not added to the PEGMA solution because PEGMA is nonvolatile and it cannot be distilled and separated from the inhibitor. Instead, the PEGMA solution was stirred over calcium hydride for 3 h, and it was syringed through a 0.45 µm filter (to keep any suspended calcium hydride) directly into the polymerization flask. All glassware was dried overnight in an oven at 120 °C and assembled hot under dynamic vacuum. The polymerizations were carried out in 100 mL round-bottom flasks. Catalytic amounts of TBABB (∼10 mg) were transferred to each flask, which was fitted with a rubber septum and purged with dry nitrogen. Freshly distilled THF was transferred from the still to the flask via a glass syringe. Network Synthesis. All of the networks of this study were prepared by GTP at room temperature. The polymerization exotherm was monitored by a digital thermometer and was used to follow the progress of the reaction. The synthesis of the networks was accomplished by the addition of the monomers at the appropriate ratio and in the appropriate sequence. Variation in the relative amounts of the two comonomers resulted in networks of different compositions, whereas variation in the order of addition of the reagents led to networks with different architectures. The synthetic sequences followed for the preparation of the four different network architectures are illustrated in Figure 2. Figure 3 presents the linear precursors to all eight networks, in which the PEGMA units are drawn in white, whereas the DMAEMA units are painted black. Above each linear chain, the corresponding polymer formula appears with the theoretical degrees of polymerization of each block. The central column of Figure 3 shows networks of different compositions. The central row of the same figure depicts the four equimolar isomers, which comprise the BAB and ABA triblocks, the statistical copolymer, and the randomly cross-linked statistical copolymer in which the units of the cross-linker EGDMA are colored gray. The synthetic procedure is illustrated below, where the preparation of the equimolar ABA triblock copolymer-based model network is detailed (Gel51 in Table 1). The procedure comprises a three-step sequential addition, starting with the preparation of the PEGMA midblock, followed by the growth of the DMAEMA endblocks, and completed with the incorporation of the dimethacrylate cross-linker. A 100 mL round-bottom flask, kept under dry nitrogen atmosphere and

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Figure 2. Synthetic sequences followed for the preparation of the four different network architectures (three model networks and one randomly cross-linked network).

Figure 3. Schematic representation of the structures of the linear precursors to the networks. The PEGMA units are shown in white, whereas the DMAEMA units are drawn in black. The EGDMA cross-linker units (appearing only in one structure) are painted gray.

containing a small amount of TBABB (∼10 mg) and freshly distilled THF (36 mL), was charged with MTSMC initiator (0.25 mL, 0.3 g, 0.87 mmol) and PEGMA (11.6 mL of 50 v/v% solution in THF, 17.4 mmol) in this order, under stirring. The polymerization exotherm (25.7 to 31.7 °C) abated within 5 min, a sample was extracted for gel permeation chromatography (GPC) analysis (full monomer conversion, polymer relative Mn ) 8550 g mol-1 and Mw/ Mn ) 1.13), and DMAEMA (2.9 mL, 2.7 g, 17.4 mmol) was added. After the completion of the polymerization of this monomer (exotherm 29.2 to 35.3 °C) and sampling for GPC (full monomer conversion, polymer relative Mn ) 12 100 g mol-1 and Mw/Mn ) 1.14) and proton nuclear magnetic resonance (1H NMR) spectroscopy (50.7 mol % DMAEMA) analyses, EGDMA cross-linker (1.3 mL, 1.4 g, 6.9 mmol) was added (exotherm 33.0-36.3 °C) which led to the gelation of the solution within seconds. The BAB triblock copolymer-based model network (with a DMAEMA midblock) was also obtained using a similar procedure in which the order of addition of the two co-monomers was reversed, whereas the statistical copolymer model network was synthesized by the simultaneous addition of the two comonomers. The randomly cross-linked network of the

statistical copolymer was prepared by the addition of the MTSMC initiator to the THF solution of the monomers, cross-linker, and catalyst, as already shown in Figure 2. Characterization of the Network Precursors by GPC and 1H NMR. Linear homopolymer and copolymer samples were obtained before cross-linking and were characterized in terms of their molecular weight and composition using GPC and 1H NMR. GPC was performed on a Polymer Laboratories system equipped with a PL-LC1120 isocratic pump, an ERC-7515A refractive index detector, and a PL Mixed “D” column (bead size 5 µm; pore sizes ) 100, 500, 103, and 104 Å). The eluent was THF, and it was pumped at 1 mL min-1. The molecular weight (MW) calibration was based on seven narrow MW (630, 4250, 13 000, 28 900, 50 000, 128 000, and 260 000 g mol-1) linear poly(methyl methacrylate) (PMMA) standards. The 1H NMR spectra of polymer solutions in deuterated chloroform were recorded using a 300 MHz AVANCE Bruker spectrometer equipped with an Ultrashield magnet. Determination and Characterization of the Sol Fraction (Extractables). The networks were taken out of the polymerization flasks by breaking the flasks, and they were subsequently washed in 200 mL of THF for 1 week to

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remove the sol fraction (extractables) which was mainly polymeric. The solvent was replaced twice during that time. The THF solution was recovered by filtration each time. The solvent was removed in a rotary evaporator, and the sol fraction was dried in a vacuum oven for 24 h at room temperature. The sol fraction was calculated for each network as the ratio of the mass of the extracted polymer (consisting mainly of un-cross-linked linear chains) divided by the theoretical mass of the gel (estimated as the sum of the masses of the monomers, cross-linker, and initiator fragment). Finally, the sol fractions were characterized in terms of their molecular weight and composition using GPC and 1H NMR, as described in the previous paragraph. Characterization of the Degree of Swelling (DS). The degrees of swelling (DSs) of the networks were measured in THF, neutral water and in aqueous solutions of various pHs covering the range between 2 and 11. The DS was calculated as the ratio of the swollen divided by the dry network mass. All masses were determined gravimetrically. First, the swollen masses of thirteen cubic samples (edge size 0.6 cm; each sample was in a separate glass vial) from each network were measured after equilibration for two weeks in THF. Next, the dry mass of each sample was determined after removing the THF by placing all samples for 48 h in a vacuum oven at room temperature. Then, 5 mL of milli-Q (deionized) water was added to each vial. For each network, seven of its samples were acidified by the addition of a calculated volume (in number of drops) of 0.5 M HCl solution such that degrees of ionization between 14% and 100% were achieved. The calculation was based on the measured dry mass of each sample (from which the number of equivalents of the DMAEMA units can be estimated) and the assumption that all DMAEMA units were not ionized before the addition of HCl. The pHs of these seven samples covered the range between 4 and 6. Three more samples from each network were further acidified to cover the pH range between 2 and 3.5, two samples became alkaline (pH 9-11) by the addition of small volumes of a 0.5 M NaOH solution, and the last sample remained neutral (no acid or base was added to it, just the 5 mL deionized water) and had a pH 7-8. The samples were allowed to equilibrate for 3 weeks and the solution pH and swollen network mass were measured (swollen network mass and pH stabilize in two weeks). The DS of each sample was calculated. All aqueous DSs were determined in triplicate and the averages of the measurements are presented along with the 95% confidence intervals. Calculation of the Degree of Ionization and the pK. The degree of ionization of each sample was calculated as the number of HCl equivalents added divided by the number of DMAEMA unit equivalents (calculated from the network dry mass and composition) present in the sample. The hydrogen ion titration curves were obtained by plotting the calculated degrees of ionization against the measured solution pH. The effective pK of the DMAEMA units of each network was estimated from the hydrogen ion titration as the pH (of the supernatant solution) at 50% ionization. Protein Adsorption Studies. The model network based on the triblock copolymer DMAEMA10-b-PEGMA20-b-

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DMAEMA10 was chosen for the protein adsorption studies. This material is equimolar in DMAEMA and PEGMA, and it, therefore, represents fairly the two constituting monomer repeat units. The ABA (DMAEMA-PEGMA-DMAEMA) rather than the BAB or the random structure was elected because most networks in this study were based on ABA chains. The proteins selected for these studies were pepsin, bovine serum albumin, and lysozyme, which are known to be acidic, slightly acidic, and basic proteins, respectively.27 Fifteen THF-swollen cubic samples of size 0.6 cm from the above network were obtained, and each was placed in a separate vial. All network samples were dried in a vacuum oven at room temperature for 48 h and they were weighed. Subsequently, they were separated into three groups, so that five network samples would be used for each one of the three proteins. Deionized water (5 mL) was added to each sample. For every group of five samples, three were adjusted to degrees of ionization of their DMAEMA units of 0.5, 0.8, and 1.0 by the addition of the appropriate volume of 0.5 M HCl solution as described earlier, one remained neutral, and the fifth became slightly alkaline by the addition of a small volume of a 0.5 M NaOH solution. The samples were allowed to equilibrate for three weeks, and their pHs (of the supernatant solution) and swollen masses were measured in order to determine their DSs. A small volume of a dilute solution of the appropriate protein was transferred to each vial, so that the initial protein concentration in the supernatant was 1 mg mL-1. The vials were allowed to equilibrate for three weeks in a refrigerator. The absorbance of the supernatant solution at 280 nm was measured using a Lambda 10 Perkin-Elmer UV/vis spectrophotometer and the protein concentration was calculated using the relevant calibration curve. The amount of adsorbed protein was calculated from the amount of protein initially added and its final concentration in the supernatant. Finally, the pH of the supernatant and the network swollen mass were measured. Modeling of Network Swelling in Water The aqueous DSs of the model networks were predicted theoretically using a molecular thermodynamic model developed recently by our group.28,29 This model considers the three Gibbs free energy components in the system. These are the elastic and electrostatic energies and the energy of mixing. The elastic energy comprises a Gaussian component and a non-Gaussian component which does not allow the chains to stretch beyond their fully extended length. The electrostatic energy is, for simplicity, described by the osmotic pressure due the translational entropy of the counterions. The mixing energy is composed of the two enthalpic terms describing the interaction of each hydrophilic block with water via the appropriate Flory-Huggins χ parameters and the term due to the translational entropy of water, ignoring the translational entropy of the polymer, following the approach of Flory.30 The total Gibbs free energy is minimized with respect to the polymer volume fraction with the aid of a numerical code written in GWBASIC. The DS at equilibrium is calculated as the inverse of the polymer volume fraction at the free energy minimum.

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Figure 4. Schematic representation of the procedure followed for the synthesis of the model network based on the ABA triblock copolymer DMAEMA20-b-PEGMA20-b-DMAEMA20. White and black colors indicate PEGMA and DMAEMA units, respectively, whereas the “*” symbols represent active polymerization sites. The number of arms at the cross-links is not 3, as shown in the Figure, but probably around 20.

The variable inputs to the code are the number of backbone carbon atoms in each block, taken as twice the corresponding degree of polymerization, and the degree of ionization of the ionizable block. The following constant inputs are also required: first, the polymer volume fraction upon crosslinking, which is equal to 0.2 in all cases (consistent with the network synthesis procedure); second, the number of arms per cross-link, which was taken equal to 20, based on the number of arms in star polymers prepared by GTP and using the same cross-linker31-33 [We have established, however, that, although the value of the number of arms influences the equilibrium free energy, it does not appreciably affect the value of the equilibrium DS.]; and finally, third, the Flory-Huggins interaction parameters χ between water and each of the two blocks. For the water-DMAEMA pair, a χ value of 0.45 was assigned to reflect the marginal watersolubility of the DMAEMA homopolymer known to precipitate in water at ∼35 °C.34 This choice is also consistent with experimentally determined χ values for similar oxygenand-nitrogen-containing acrylic monomers in hydrogels.35 The lower χ value of 0.30 was assigned to the waterPEGMA pair to capture the higher hydrophilicity of PEGMA, whose homopolymers do not precipitate in aqueous solution even after heating to 90 °C.25 This is also in agreement with the χ value of around 0.3 used for PEG in the literature,36,37 which is based on activity measurements for the system PEG-water.38 Results and Discussion Polymerization Methodology. Although the GTP syntheses of statistical39 and diblock40,41 copolymers of PEGMA and DMAEMA have been described recently, there are no reports on the preparation of their ABA triblock copolymers or their model networks, which is the subject of the current investigation. The steps involved in this synthesis are presented schematically in Figure 4, where the synthesis of the network based on the DMAEMA20-b-PEGMA20-bDMAEMA20 triblock copolymer is shown. The use of the MTSMC bifunctional initiator combined with the sequential addition of monomers and cross-linker leads to the successive formation of linear PEGMA homopolymer with both active ends (indicated by asterisks), of the linear DMAEMA20-bPEGMA20-b-DMAEMA20 triblock copolymer with also two active ends, and of the final block copolymer model network. The number of arms at the cross-links of the network is not three, as indicated in the figure, but higher, probably

approximately 20, similar to the number of arms in star polymers also prepared by GTP and using the same crosslinker.31-33 The amount of the EGDMA cross-linker used was eight times the number of moles of the MTSMC initiator, as determined in preliminary investigations in which the synthesis of DMAEMA star homopolymers (with the use of a monofunctional rather than a bifunctional initiator) was optimized.42 Molecular Weights (MWs) and Composition of Precursors. Table 1 shows the relative molecular weights (MWs) and composition of the linear precursors to the networks as measured by GPC and 1H NMR, respectively. Although direct comparison between the GPC number average MWs, Mns, and the theoretically predicted MWs (calculated from the ratio of monomer to initiator) should not be made because of differences in the hydrodynamic properties of the samples and the GPC PMMA calibration standards, the values of the two were found to be in reasonable agreement with each other. Molecular weight distributions (MWDs) were found to be narrow with calculated polydispersity indices (PDIs, Mw/Mn) lower than 1.22. This confirms the size homogeneity of the elastic chains of the networks. The polymer composition was determined from the 1H NMR spectra (not shown) of the copolymers, by ratioing the signal from the three methoxy protons at 3.3 ppm in PEGMA to the six protons in the two azamethyl groups at 2.3 ppm in DMAEMA.25 The percentages of DMAEMA determined by 1H NMR were found to be close to the theoretically calculated percentages of DMAEMA. This confirms that the networks have the desired composition. The DMAEMA20-b-PEGMA20-bDMAEMA20 network was synthesized twice (Gel55 and Gel55R). Table 1 shows that the linear precursors to these two networks have similar experimental MWs and composition, indicating the reproducibility of the synthetic procedure. Percentage, MWs, and Composition of Sol Fraction. Table 2 shows the percentage, relative MWs and composition of the extractables for each network, as measured by gravimetry, GPC and 1H NMR, respectively. With the exception of the randomly cross-linked statistical copolymer network which has a high percentage of extractables (19.6%), all of the other networks exhibit relatively low sol fractions ( 2.9), the slightly acidic bovine serum albumin is negatively charged for pH > 5.1 and positively charged for pH < 5.1, and finally, the basic lysozyme is positively charged for the greatest part of the pH range (pH < 11.0). The pIs of the three proteins are indicated by arrows in the corresponding plots of Figure 13 to facilitate data interpretation. Two other arrows are drawn in the three plots of the figure to indicate the charge properties of the DMAEMA units in the network (obtained from the hydrogen ion titration of this network from Figure 5). These are the pH below which the DMAEMA units start to acquire a positive charge (∼7.2), and the pK of the DMAEMA units in this network which is the pH at which half of the DMAEMA units are ionized (∼4.8). In most cases, Figure 13 indicates that protein adsorption takes place when the DMAEMA units and the protein have an opposite charge. A similar motif of electrostatic/ion-exchange adsorption has also been observed be-

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tween charged methacrylic acid/acrylic acid microgels and several proteins,47 as well as between sodium acrylate gels and multivalent metal cations.48,49 Figure 13a shows the results for pepsin. Pepsin adsorption occurs between pH ∼4 and ∼7, where the protein is negatively charged and the DMAEMA units are positively charged. At pH > 8, where pepsin is again negatively charged but the DMAEMA units are neutral, no adsorption takes place. The absence of adsorption in this latter pH range indicates that other attractive interactions, such as van der Waals or hydrophobic, between the protein and the network are not sufficiently strong. Figure 13b presents the results for the adsorption of bovine serum albumin onto the network. Adsorption is observed within the pH range ∼5-7, where protein and network are oppositely charged. At pH < 5, where the protein and network are both positively charged, and at pH > 8, where albumin is negatively charged and the network is neutral, no protein adsorption was measured. Figure 13c displays the results for lysozyme. Adsorption of lysozyme occurs only at pH ∼ 10, where the protein is positively charged and the DMAEMA units in the network are neutral. Thus, in contrast to Figure 13, parts a and b, the attractive interactions in this case are not electrostatic. However, at pH e 7, where lysozyme and the network are both positively charged, no adsorption takes place, highlighting the importance of electrostatic (repulsive) interactions for the adsorption of this protein too. Conclusions A “living” polymerization technique, GTP, has been employed to prepare double-hydrophilic model networks based on hydrophilic ionizable amine units of DMAEMA and hydrophilic nonionic units of PEGMA, covering a range of compositions and architectures. The DSs of all networks increased by lowering the pH and by increasing the DMAEMA content. This trend was confirmed by theoretical calculations of the DSs. Adsorption studies at different pHs indicated that pepsin and albumin are adsorbed onto the network electrostatically, whereas lysozyme is adsorbed by a nonelectrostatic mechanism. Acknowledgment. This work was supported by the University of Cyprus Research Committee (Grant 20002003). The A. G. Leventis Foundation is gratefully acknowledged for a generous donation which enabled the purchase of the NMR spectrometer of the University of Cyprus. Dr. R. Budd of Laporte Performance Chemicals, U.K., is thanked for kindly donating to us the PEGMA monomer. References and Notes (1) Co¨lfen, H. Macromol. Rapid Commun. 2001, 22, 219-252. (2) Forder, C.; Patrickios, C. S.; Billingham, N. C.; Armes, S. P. J. Chem. Soc., Chem. Commun. 1996, 7, 883-884. (3) Forder, C.; Patrickios, C. S.; Armes, S. P.; Billingham, N. C. Macromolecules 1996, 29, 8160-8169. (4) Bu¨tu¨n, V.; Billingham, N. C.; Armes, S. P. Chem. Commun. 1997, 7, 671-672. (5) Bu¨tu¨n, V.; Bennett, C. E.; Vamvakaki, M.; Lowe, A. B.; Billingham, N. C.; Armes, S. P. J. Mater. Chem. 1997, 7, 1693-1695. (6) Lowe, A. B.; Billingham, N. C.; Armes, S. P. Macromolecules 1998, 31, 5991-5998. (7) Bu¨tu¨n, V.; Billingham, N. C.; Armes, S. P. J. Am. Chem. Soc. 1998, 120, 11818-11819.

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