Association Behavior of Star-Shaped pH-Responsive Block

pubs.acs.org/Langmuir © 2009 American Chemical Society

Association Behavior of Star-Shaped pH-Responsive Block Copolymer: Four-Arm Poly(ethylene oxide)-b-Poly(methacrylic acid) in Aqueous Medium E. He,† C. Y. Yue,†,‡ and K. C. Tam*,§ † School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, ‡Singapore-Massachusetts Institute of Technology Alliance (SMA) and §Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Canada N2L 3G1

Received December 9, 2008. Revised Manuscript Received February 12, 2009 A four arm pH-responsive poly(ethylene oxide)-b-poly(methacrylic acid) block copolymer was synthesized by atom transfer radical polymerization technique. The conformation transition over the course of neutralization was investigated using a combination of potentiometric and conductometric titrations, dynamic and static light scattering, and transmission electron microscopy. The multiarm block copolymer existed as an extended unimer at high pH due to the negatively charged carboxylate groups and hydrophilic poly(ethylene oxide) segments. The block copolymers self-assembled into core-shell micelles and large spherical aggregates that flocculated at low degree of neutralization (R). Such behavior is controlled by the fine balance of electrostatic, hydrophobic, and hydrogen bond interactions. The hydrodynamic radius (Rh) of the aggregates was approximately 84 nm at R of 0.3, and it decreased to 63 and 46 nm at R ∼ 0.2 and 0.1, respectively, as a result of the reduced electrostatic interaction between ionized carboxylate groups. The thermodynamic parameters obtained from isothermal titration calorimetric technique in different salt concentrations indicated that the energy to extract a proton from a charged polyion was reduced by the addition of salt, which favors the neutralization process.

Introduction Stimuli-responsive polymers have attracted increasing attention in biomedical and drug delivery systems because of their attractive and tunable properties.1-4 Various chemical or physical stimuli, such as pH, ionic strength, temp erature, light, electric and magnetic fields, will alter the interactions or energetics of self-assembly and polymer chain conformation.5,6 In the presence of external stimuli, the conformation, solubility, degradation, or association of polymeric chains will change accordingly.7-9 The different pH environments in different tissues and cellular compartments suggest that pH is an important parameter that should be considered in the design of a polymeric system for biomedical applications.10,11 Poly(methacrylic acid) (PMAA) possesses pH-responsive properties, where the conformation and solubility of chain segments in aqueous *To whom correspondence should be addressed. E-mail: mkctam@ uwaterloo.ca. Fax: 519-746-4979. (1) Kumar, A.; Srivastava, A.; Galaev, I. Y.; Mattiasson, B. Prog. Polym. Sci. 2007, 32, 1205–1237. (2) Hoffman, A. S.; Stayton, P. S. Prog. Polym. Sci. 2007, 32, 922–932. (3) Schmaljohann, D. Adv. Drug Delivery Rev. 2006, 58, 1655–1670. (4) Alarcon, C. D. H.; Pennadam, S.; Alexander, C. Chem. Soc. Rev. 2005, 34, 276–285. (5) Checot, F.; Rodriguez-Hernandez, J.; Gnanou, Y.; Lecommandoux, S. Biomol. Eng. 2007, 24, 81–85. (6) Lai, J. J.; Hoffman, J. M.; Ebara, M.; Hoffman, A. S.; Estournes, C.; Wattiaux, A.; Stayton, P. S. Langmuir 2007, 23, 7385–7391. (7) Bellomo, E. G.; Wyrsta, M. D.; Pakstis, L.; Pochan, D. J.; Deming, T. J. Nat. Mater. 2004, 3, 244–248. (8) Boyer, C.; Bulmus, V.; Liu, J. Q.; Davis, T. P.; Stenzel, M. H.; BarnerKowollik, C. J. Am. Chem. Soc. 2007, 129, 7145–7154. (9) Kostina, Y. V.; Bondarenko, G. N.; Alent’ev, A. Y.; Yampol’skii, Y. P. Polym. Sci. Ser. A 2007, 49, 77–88. (10) Wagner, E. Pharm. Res. 2004, 21, 8–14. (11) Rapoport, N. Prog. Polym. Sci. 2007, 32, 962–990.

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media can be manipulated by pH.12,13 The chains adopt an extended random coil conformation at high pH with a larger hydrodynamic volume induced by Coulombic repulsive forces between ionized carboxylate groups. In contrast, the hydrophobic interactions between methyl groups resulted in a lower hydrodynamic volume due to the suppressed hypercoiled morphology at low pH. Biocompatible and hydrophilic poly(ethylene oxide) (PEO) was grafted to PMAA chain segments to stabilize the polymer complexes for possible applications in drug delivery.14-17 In addition, hydrogen bond interactions provide an additional control of the conformation within the polymer complexes, which permit the manipulation of various interactions that control the behavior of PMAA chains in solution.18,19 Various types of linear pH-responsive PEO-based copolymers have been reported, such as PEO-poly(propylene oxide) (PPO),20,21 PEO-poly(2-(diethylamino)ethyl (12) Satturwar, P.; Eddine, M. N.; Ravenelle, F.; Leroux, J. C. Eur. J. Pharm. Biopharm. 2007, 65, 379–387. (13) Bromberg, L. E.; Ron, E. S. Adv. Drug Delivery Rev. 1998, 31, 197– 221. (14) Neugebauer, D. Polym. Int. 2007, 56, 1469–1498. (15) Schweizer, S.; Taubert, A. Macromol. Biosci. 2007, 7, 1085–1099. (16) Nakashima, K.; Bahadur, P. Adv. Colloid Interface Sci. 2006, 123, 75– 96. (17) Soo, P. L.; Eisenberg, A. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 923–938. (18) Lee, S. C.; Lee, H. J. Langmuir 2007, 23, 488–495. (19) Kharlampieva, E.; Sukhishvili, S. A. Polym. Rev. 2006, 46 377–395. (20) Chiappetta, D. A.; Sosnik, A. Eur. J. Pharm. Biopharm. 2007, 66, 303– 317. (21) Bromberg, L.; Alakhov, V. Y.; Hatton, T. A. Curr. Opin. Colloid Interface Sci. 2006, 11, 217–223.

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methacrylate) (PDMAEMA),22 PEO-poly(ethylenimine) (PEI),23,24 PEO-poly(lactide) (PLA),25,26 and PEO-poly (ε-caprolactone) (PCL).27 However, the polymer architecture that defines the shape of a single polymer chain could play an important role since it defines the physicochemical properties of the polymer chains in solution.28 The star-shaped multiarm PEOs possess three-dimensional branched structures that may offer interesting properties in the self-assembly and association of polymeric chains in the presence of other small molecules.29,30 The higher densities of terminal functional groups on the block copolymer chains induce stronger interactions compared to the linear structure of identical molecular weights. The star-shaped four-arm PEO-b-PDEAEMA formed well-defined core/shell micelles in aqueous solution.31 This polymeric system can be efficiently used to condense negative charged plasmid DNAs because of the dominant electrostatic interactions between plasmid DNAs and positively charged PDEAEMAs at physiological pH condition. By attaching MAA segments to biocompatible star-shaped PEO chains, we obtained high density pH-responsive functional groups capable of binding cationic drug via electrostatic interactions. The multiarm polyacids undergo conformational transition with degree of ionization and ionic strength of the media, both of which have not been extensively studied. In this study, atom transfer radical polymerization (ATRP) was used to synthesize a star-shaped block copolymer, fourarm PEO-b-PMAA. The dissolution and association mechanism of the star polymer in different salt environments were examined. The dissociation constant and Gibbs free energy (ΔG) were derived from potentiometric and conductometric titration data, and the impact of ionic environment on the chain conformation and self-assembly are discussed. The critical micelle concentration (CMC) of the block copolymer was investigated to determine a suitable polymer concentration range for potential biological applications. The particle size of the polymer clusters during the course of neutralization, hydrodynamic radius (Rh), and radius of gyration (Rg) were determined by light scattering. The interactions between the polymer and alkali were elucidated through thermodynamic quantification using the isothermal titration calorimetric (ITC) technique. At high pH, the multiarm block copolymer was fully extended to yield a unimeric threedimensional hydrophilic PEO with negatively charged carboxylate groups. By decreasing the pH, the star polymer forms micelles, whose size decreased with pH. However, at very low degree of neutralization (R), hydrogen bonds between carboxylic acids and ethylene-oxide segments, and hydrophobic interaction of methyl groups induced further (22) Alvarez-Lorenzo, C.; Barreiro-Iglesias, R.; Concheiro, A.; Iourtchenko, L.; Alakhov, V.; Bromberg, L.; Temchenko, M.; Deshmukh, S.; Hatton, T. A. Langmuir 2005, 21, 5142–5148. (23) Nguyen, H. K.; Lemieux, P.; Vinogradov, S. V.; Gebhart, C. L.; Guerin, N.; Paradis, G.; Bronich, T. K.; Alakhov, V. Y.; Kabanov, A. V. Gene Ther. 2000, 7, 126–138. (24) Malmsten, M.; Muller, D. J. Biomater. Sci.: Polym. Ed. 1999, 10, 1075–1087. (25) Sanabria-DeLong, N.; Agrawal, S. K.; Bhatia, S. R.; Tew, G. N. Macromolecules 2007, 40, 7864–7873. (26) Agrawal, S. K.; Sanabria-DeLong, N.; Jemian, P. R.; Tew, G. N.; Bhatia, S. R. Langmuir 2007, 23, 5039–5044. (27) Cai, S. S.; Vijayan, K.; Cheng, D.; Lima, E. M.; Discher, D. E. Pharm. Res. 2007, 24, 2099–2109. (28) Heath, F.; Haria, P.; Alexander, C. AAPS J. 2007, 9, E235–E240. (29) Taton, D.; Gnanou, Y.; Matmour, R.; Angot, S.; Hou, S.; Francis, R.; Lepoittevin, B.; Moinard, D.; Babin, J. Polym. Int. 2006, 55, 1138–1145. (30) Tezuka, Y.; Oike, H. Prog. Polym. Sci. 2002, 27, 1069–1122. (31) He, E.; Ravi, P.; Tam, K. C. Langmuir 2007, 23, 2382–2388.

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aggregation to produce larger nanostructures that were not observed in the association behaviors of polybase (four-arm PEO-b-PDEAEMA). The addition of a neutral salt significantly altered the electrostatic interaction between the macroions, counterions, and solvent molecules, resulting in changes in the polymer aggregation behavior.

Experimental Details Materials. The four-arm hydroxy-end-capped PEOs [degree

of polymerization (DP) = 4
56; Mw/Mn = 1.08] were purchased from NOF (Tokyo, Japan). tert-Butyl methacrylate (tBMA, Aldrich, 98%) was purified by passing it through a basic alumina column to remove any acidic contaminants, stirred over calcium hydride, and subsequently distilled under reduced pressure prior to use. 2-Bromoisobutyryl bromide, triethylamine, CuCl (99.99%), 1,1,4,7,10,10-hexamethyl triethylenetetramine (HMTETA, 97%,), anisole (anhydrous, 99%), tetrahydrofuran (THF, 99.9%) and hexane were purchased from Aldrich and used as received. Toluene (99.8%, Aldrich) was dried by heating with sodium strips and refluxing under argon environment at 110 °C for 3 days. Deionized (DI) water was produced by a Millipore Alpha-Q purification system.

Synthesis of the Block Copolymer Four-Arm PEO-bPMAA. Details of the synthesis for the PEO-b-PtBMA via ATRP and PEO-b-PMAA can be found in the Supporting Information. Gel Permeation Chromatography (GPC). Polymer molecular weights and polydispersity were determined using an Agilent 1100 series GPC system equipped with a liquid chromatography pump, photoluminescence gel 5 μm MIXED-C column, and refractive index detector. The column was calibrated with narrow molecular weight polystyrene standards. The highperformance liquid chromatography (HPLC)-grade THF stabilized with butylated hydroxytoluene was used as a mobile phase at a flow rate of 1.0 mL/min. Nuclear Magnetic Resonance Spectroscopy. The 1H NMR spectrum for the block copolymer was recorded using a Bruker DRX400 instrument. Chemical shifts (δ) were given in parts per million using tetramethylsilane (TMS) as an internal reference. The polymer sample was dissolved in deuterochloroform (CDCl3) for measurement, and the concentration used was about 10 mg/mL. The polymerization degree of four-arm PEO-b-PtBMA block copolymer copolymers was calculated from the peak intensity ratio of 3.58 ppm (-CH2CH2O- of the PEO block) and 1.40 ppm (-C(CH3)3 of the tBMA group). Potentiometric Titration. The potentiometric and conductometric titrations were conducted using the ABU93 Triburette Titration System equipped with a Radiometer pHG201 pH glass and REF201 reference electrodes and a CDM83 conductivity electrode system. The water jacketed titration vessel was maintained at a constant temperature of 25 °C using a circulating water bath. In the titration vessel, 50 mL of 0.1 wt % four-arm PEO-b-PMAA copolymer in various aqueous salt solutions were continuously stirred. The titrant used was 1 M standard NaOH solution (Merck), and sufficient lag time between two dosages was allowed to ensure that the reaction had reached equilibrium. Critical Micelle Concentration. All CMC measurements were conducted using a Dataphysics DCAT 21 tensiometer equipped with a Wilhelmy plate. The concentration of the four-arm PEO-b-PMAA block copolymer was 0.1 wt %, and the solution was titrated to 50 mL of various NaCl solutions at pH of 4. The data was analyzed using the SCAT program, where the surface tension was plotted against the polymer concentration. Laser Light Scattering. Dynamic (DLS) and static light scattering (SLS) experiments were performed using the DOI: 10.1021/la804056p

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He et al. Scheme 1. Synthetic Scheme of the Four-Arm PEO-b-PtBMA Block Copolymer

Brookhaven BI-200SM goniometer system equipped with a BI9000AT digital correlator. The power adjustable vertically polarized argon ion laser with a wavelength of 488 nm was used as the light source. For dynamic light scattering, the inverse Laplace transform of REPES supplied with the GENDIST software package was used to analyze the time-correlation functions and the probability of reject was set to 0.5. The refractive index increment of 0.1 wt % polymer solution (dn/ dC) in DI water is 0.1673 mL/g, which was measured by a Brookhaven BI-DNDC differential refractometer. The polymer solutions were prepared at a concentration of 0.1 wt % in aqueous medium, and a 0.45 μm PTFE filter was used to remove dust prior to the light scattering experiments. Transmission Electron Microscopy (TEM). The TEM micrographs of polyplexes were observed by a JEOL JEM-2010 electron microscope at an acceleration voltage of 200 kV. TEM specimens were prepared by placing a drop of polymer solution onto 200-mesh copper grids precoated with Formvar and stained by osmium tetroxide (OsO4). The copper grids were dried in a freeze-dryer and then kept in a desiccator overnight at room temperature prior to measurement. Isothermal Titration Calorimetry. A Microcal ITC system (Northampton, MA) was used to measure the enthalpy changes during the neutralization process of the star block copolymers. Details of the instrument and the experimental setup can be found in the Supporting Information.

Results and Discussion Polymer Synthesis. ATRP technique was used to synthesize the well-defined four-arm PEO-b-PtBMA using protected group chemistry as described in Scheme 1. Initially, four-arm hydroxy-end-capped PEO was coupled with the initiator, 2-bromoisobutyryl bromide to produce Br-terminated four-arm PEO macroinitiator with Mn = 10750 Da and Mw/Mn = 1.14. The resulting macroinitiator was copolymerized with tBMA monomers using CuCl complexed by HMTETA catalyst in anisole at 90 °C. Figure 1 shows the GPC traces of a four-arm PEO macroinitiator and four-arm PEO-b-PtBMA block copolymer. From the narrow and symmetric single peak, we confirmed that the copolymerization was well-controlled. The number average molecular weight (Mn) of the four-arm PEO-b-PtBMA block copolymer was 60 300 Da, which is consistent with the theoretical molecular mass (initiator-to-monomer molar ratio). The relative molecular mass distribution was narrow, with a polydispersity index (PDI) of 1.23. Figure 2 shows the 1H NMR spectrum of four-arm PEO-b-PtBMA block copolymer in CDCl3, 1H NMR (400 MHz, CDCl3, TMS), δ (ppm): 3.58 (-OCH2CH2O-), 1.75 (-CCH2C-), 1.58 (H2O), 1.40 (-C(CH3)3), 1.02 (-CCH3). The polymerization degree of four-arm PEO-b-PtBMA block copolymers was calculated 4894

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Figure 1. GPC traces of four-arm PEO-b-PtBMA block copolymer and Br-terminated four-arm PEO macroinitiator. from the peak intensity ratio of 3.58 ppm (-OCH2CH2O-) of the PEO block and 1.40 ppm (-C(CH3)3) of the tBMA block. The block lengths of the copolymer calculated from 1 H NMR spectrum were 56 and 88 for PEO and tBMA, respectively. Potentiometric and Conductometric Titrations. The pHresponsive four-arm PEO-b-PMAA block copolymer was studied by potentiometric and conductometric titrations, which provided information on the conformation and distribution of ions in solution. Details on the dissociation equilibria, and the utility of the Henderson-Hasselbalch equation to treat the data can be found in the Supporting Information. When NaOH was titrated to the four-arm PEO-b-PMAA block copolymer solution, both the conductivity and pH were measured simultaneously, as shown in Figure 3a. These two curves revealed changes in the concentrations of conducting ions, which are H+, Na+, OH-, Cl-, and macroion (p). The conductivity (Λ) can be expressed as follows: Λ ¼ CNa þ λNa þ þ CH þ λH þ þ COH - λOH - þ CCl - λCl - þ Cp λp

ð1Þ

where, Ci is the concentration of free ion in solution, and λi is the molar conductivity of the corresponding ion. Since the concentration of Cl- ion remained constant throughout the titration, the conductivity curve reflects the concentration changes of H+, Na+, OH-, and macroions. The figure was delineated into three regions with two transition points (point A, pH ∼ 4.3, and point B, pH ∼ 9.7). In region 1, addition of NaOH to the polymer solution caused the reduction of H+ and increase of Na+ concentrations. Since the mobility of H+ (λ0H+ = 350 S cm2/mol) is much larger Langmuir 2009, 25(9), 4892–4899

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Figure 2. 1H NMR spectrum of four-arm PEO-b-PtBMA block copolymer in CDCl3.

than Na+ (λ0Na+ = 50.5 S cm2/mol), the conductivity of polymer solution decreased to the first transition point A, where the pH displayed a sharp increase. Since the concentration changes of H+ and OH- are very small, the conductivity curve increased marginally in region 2 as a result of the addition of Na+ ions. The increase in the pH corresponded to the neutralization of carboxylic groups on the polymeric chains as more NaOH was added. 32 When the titration reached the second transition point B, the four-arm PEO-b-PMAA became fully neutralized, as reflected by the sharp increase in pH and conductivity curves. The increase in the conductivity and pH in region 3 was contributed by the Na+ and OH- ions from the addition of excess NaOH. The addition of a neutral salt significantly alters the electrostatic interaction between the macroions, counterions, and solvent molecules.33 To achieve a molecular-level understanding on the association mechanism of the four-arm PEO-b-PMAA, a series of titration experiments were conducted by titrating 1 M NaOH to 0.085 wt % of four arm PEO-b-PMAA block copolymer in 0.01, 0.02, 0.05, 0.1, 0.15 and 0.2 M NaCl solutions. Figure 3b shows the dependence of pH on the degree of neutralization (R), which was calculated on the basis of the moles of NaOH titrated to the polyelectrolyte solution. At a fixed R, the pH decreased with increasing sodium chloride concentrations. The positively charged sodium ions formed an ionic atmosphere in the vicinity of negatively charged carboxylate groups that screened the electrostatic interaction between macroions.34 The polyelectrolyte dissociation was favorable, and the acidic property of the polyacid was enhanced, as indicated by the reduced pH value.35 Identical pH curves observed for (32) Schilli, C. M.; Zhang, M. F.; Rizzardo, E.; Thang, S. H.; Chong, Y. K.; Edwards, K.; Karlsson, G.; Muller, A. H. E. Macromolecules 2004, 37, 7861–7866. (33) Choi, Y. W.; Lee, S.; Kim, K.; Russo, P. S.; Sohn, D. J. Colloid Interface Sci. 2007, 313, 469–475. (34) Smalley, M. V.; Schartl, W.; Hashimoto, T. Langmuir 1996, 12, 2340– 2347. (35) Bajpai, A. K.; Shukla, S. K.; Bhanu, S.; Kankane, S. Prog. Polym. Sci. 2008, 33, 1088–1118.

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Figure 3. (a) Potentiometric and conductometric titration curves of 0.085 wt % four-arm PEO-b-PMAA in 0.01 M NaCl solution: (0) pH; (9) conductivity. (b) Comparison of pH curves of four-arm PEO-b-PMAA in various concentrations of NaCl: (b) 0.01 M; (9) 0.02 M; (2) 0.05 M; (O) 0.1 M; (0) 0.15 M; (Δ) 0.2 M. (c) Comparison of pK(R) curves of four-arm PEO-b-PMAA in various concentrations of NaCl: (b) 0.01 M; (9) 0.02 M; (2) 0.05 M; (O) 0.1 M; (0) 0.15 M; (Δ) 0.2 M. 0.15 and 0.2 M NaCl solution suggested that complete shielding of polyions by small electrolyte ions had been reached. The pKa was plotted against the degree of neutralization (R) for the titration of the four-arm PEO-b-PMAA in different NaCl solutions as shown in Figure 3c. The pK(R) decreased at the initial titration stage and reached a minimum at R ∼ 0.05 (low cs) and R ∼ 0.1 (high cs). Thereafter, they increased to R ∼ 0.2 and exhibited a plateau between R ∼ 0.2 to R ∼ 0.4, subsequently it increased progressively for R greater than 0.4. At R ∼ 0, the MAA groups were DOI: 10.1021/la804056p

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Table 1. Summary of Potentiometric Characterization of Four-Arm PEO-b-PMAA Polymer in Various Salt Concentrations salt concentration (M)

pK(1/2)

ΔGel (kJ/mol)

0.01 0.02 0.05 0.1 0.15 0.2

6.7 6.5 6.2 6.0 5.8 5.7

0.37 0.32 0.25 0.20 0.18 0.10

nonionized and formed a core surrounded by hydrophilic PEO chains. The MAA groups on the polymeric chains and the hydrophobic interactions between nonionized MAA groups produced a compact chain configuration at low ionization degree. With small amounts of NaOH, the pKa decreased slightly, corresponding to the charge density of compact structure of the aggregates. As more COOH groups on the polymer chain were deprotonated, the electrostatic repulsion between charged groups increased, making the hypercoiled methacrylic acid segments more accessible. At the plateau region (R between 0.2 and 0.4), the charge density was relatively stable, resulting from the rearrangement of COO- groups on the polymer chain, which maintained a constant acidic environment. Further neutralization with the base (R > 0.4) enhanced the charge density of polymer chains, and the chain conformation became more extended as a result of the electrostatic repulsion between charged groups. The hydrophobic forces could not balance the electrostatic interaction to maintain the structure, resulting in the destruction of the compact structure. As depicted by the pKa curves for the four-arm PEO-bPMAA in various salt solutions, the pKa was lower at higher salt concentrations, and the transition region shifted to higher ionization degree. In the presence of small electrolytes, the electrostatic repulsion was screened by positively charged sodium ions, resulting in a more flexible polymer chain. The degree of swelling became lower with increasing ionization, yielding a more compact structure that shifted the transition region to a higher ionization degree. The attraction between COO- and H+ was also screened by salt ions, which increased the acidity, resulting in a lower pKa value in high salt solutions. The electrostatic Gibbs free energy needed to extract a proton from a charged polyion can be obtained by integrating the area under the pK(R) versus R curves. ΔGel can be derived from graphical integration based on the following equation: Z ΔGel ¼ 2:30RT 0

1

½pKðRÞ -pK0 dR

ð2Þ

where R is the gas constant, T is the absolute temperature, pK0 is the negative logarithm of the intrinsic dissociation constant that is independent of R and was obtained by linearly extrapolating the pK(a) curve to R ∼ 0, pK(R) is the negative logarithm of the dissociation constant at any given R values, and ΔGel is the electrostatic Gibbs free energy.36 The data of pK(1/2), the apparent dissociation constant at R ∼ 0.5 and the ΔGel in various salt solutions are summarized in Table 1. pK(1/2) and ΔGel decreased with increasing salt (36) Hirose, Y.; Sakamoto, Y.; Tajima, H.; Kawaguchi, S.; Ito, K. J. Phys. Chem. 1996, 100, 4612–4617.

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Figure 4. Surface tension versus concentration of four-arm PEO-bPMAA polymer at pH 4 in (() 0.001 M, (9) 0.002 M, (Δ) 0.004 M, ()) 0.01 M, and (0) 0.05 M NaCl solutions. concentrations, and ΔGel approached a constant value at NaCl concentration exceeding 0.2 M. As discussed earlier, the addition of salt favors the dissociation of carboxylic groups, which enhances the dissociation constant Ka, resulting in a lower pK(1/2). Since the Coulombic attraction between H+ and RCOO- was screened by the addition of salt, the energy required to extract protons from the polyacid was decreased. With further addition of NaCl, the polymer particles were destabilized, and they flocculated as a result of charged shielding effects. The addition of salt promoted and controlled the formation of larger aggregates, which coexisted with individual micelles.37 Critical Micelle Concentration. The CMC is an important parameter that describes the self-assembling behavior of block copolymers in solution. The surface tension method was used to determine the CMC values of the four-arm PEOb-PMAA block copolymer, where the surface tension variations with polymer concentrations were monitored. Figure 4 shows the surface tension versus polymer concentration in 0.01-0.15 M NaCl at pH of 4. The CMC value was determined to be 65 ppm (w/w) for the four-arm PEO-bPMAA block copolymer in 0.001 M NaCl aqueous solutions. It then decreased to 53, 34, and 28 ppm in 0.002, 0.004, and 0.01 M NaCl, respectively. Further increase in the salt concentration to 0.05 M did not result in any significant change, and the CMC remained constant. It is evident that the CMC decreased with increasing salt concentration,38 which was mainly attributed to the reduction in the solubility of PEO chains in salt solutions. The extremely low CMC values of the four-arm PEO-b-PMAA block copolymer suggested that the micelles were stable and they could be potential candidates for application in enhanced drug delivery. In addition, delivery systems with smaller aggregates may prolong the circulation time in the blood and facilitate their bioavailability.39 Light Scattering. The mathematical basis for determining the translation diffusion coefficient and the hydrodynamic radius is documented in the Supporting Information. Figure 5 shows the decay time distributions of the four-arm (37) Strandman, S.; Hietala, S.; Aseyev, V.; Koli, B.; Butcher, S. J.; Tenhu, H. Polymer 2006, 47, 6524–6535. (38) Mata, J. P.; Majhi, P. R.; Guo, C.; Liu, H. Z.; Bahadur, P. J. Colloid Interface Sci. 2005, 292, 548–556. (39) Kuskov, A. N.; Shtilman, M. I.; Goryachaya, A. V.; Tashmuhamedov, R. I.; Yaroslavov, A. A.; Torchilin, V. P.; Tsatsakis, A. M.; Rizos, A. K. J. Non-Cryst. Solids 2007, 353, 3969–3975.

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Debye equation:   KC 1 1 ¼ 1 þ q2 Rg 2 þ 2A2 C RðqÞ Mw 3

Figure 5. Relaxation time distribution functions at a scattering angle of 90° for four-arm PEO-b-PMAA in 0.1 M NaCl at different pH values. PEO-b-PMAA at different pHs. At high pH environment (R = 1; pH = 8.9), the carboxylic groups on the polymer chains were completely neutralized, resulting in highly charged MAA segments. The repulsive electrostatic interaction between polymer chains yielded unimers in aqueous solution with a hydrodynamic radius of about 12 nm and PDI of about 0.13. By adding HCl, the charged carboxylate groups were transformed to carboxylic acids, and hydrophobic interaction between carboxylic acid groups produced micelles at R less than 0.3. The apparent hydrodynamic radius (Rh) of the aggregates was determined to be about 84 nm. With further addition of HCl, more carboxylate groups were protonated, resulting in a reduction of negative charges on the polymer chains. The repulsive electrostatic interactions between ionized carboxylate groups were reduced, causing the aggregates to shrink. The relaxation time shifted to a lower value with a larger amplitude, where the Rh was found to be 63 and 46 nm at R ∼ 0.2 and 0.1, respectively. The PDI of the micelles at low R of 0.1 ∼ 0.3 was less than 0.06, indicating that the size distribution of the micelles are narrow. The starshaped polymer is more favorable to form monodispersed supramolecular rather than linear structures.40 When R reached approximately 0.05, another relaxation peak appeared, possibly resulting from the aggregation of micelles induced by hydrogen bonding between MAA groups to form larger aggregates of Rh of about 120 nm.41 The polymer solution was transparent, but it turned opaque at R < 0.05, where the scattering intensity increased sharply by 3 orders of magnitude. Larger aggregates of ∼410 nm were detected with the corresponding disappearance of the smaller micelles. The fast and slow modes corresponded to the small and large aggregates respectively. The interesting morphological transformation was observed using the TEM, and the results corresponded well with the trend of the negative logarithm of the apparent dissociation constant (pKa) during the course of potentiometric titration. SLS was used to measure time-average scattered intensities and analyzed microscopic properties of the aggregates, such as the z-average radius of gyration (Rg) according to the (40) Lambeth, R. H.; Ramakrishnan; Mueller, S.; Poziemski, R.; Miguel, G. S.; Markoski, L. J.; Zukoski, C. F.; Moore, J. S. Langmuir 2006, 22, 6352– 6360. (41) Tang, H. D.; Radosz, M.; Shen, Y. Q. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 5995–6006.

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ð3Þ

where K is an optical constant (K = 4π2n2(dn/dC)2/NAλ4) where n is the refractive index of the solvent, dn/dC is the the refractive index increment of the polyplexes solution, NA is Avogadro’s constant, λ is the wavelength of laser light, R(q) is the Rayleigh ratio, q is the scattering vector, and C is the concentration of the polymer solution.42 Various concentrations (0.02, 0.04, 0.06, 0.08 and 0.1 wt %) of four-arm PEOb-PMAA block copolymer in 0.1 M NaCl were prepared. Each of the polymer samples was measured at a scattering angle of 90° by SLS. The SLS software generated the Debye plot, where the weighted average molecular weights (Mapp w ) of the polymer at R ∼ 0.2 was determined to be 7.94 ( 0.52
105 g/mol. Since the molar mass of the single star polymer determined from GPC was 4.96
104 g/mol, the aggregation number Nagg was calculated to be approximately 16, consisting of 64 PEO-MAA chains. The radius of gyration (Rg) of the polymeric aggregates in solution was measured to be 68 nm at scattering angles ranging from 50° to 110°. The parameter F (Rg/Rh) was calculated to examine the morphology of the aggregates, and found to be about 1.1, suggesting that the particles possessed a spherical structure with a hydrophobic core and hydrophilic corona shell. Transmission Electron Microscopic Studies. A series of TEM images was evaluated to investigate the entanglement effects of MAA segments at low ionization degree. A 0.1 wt % four-arm PEO-b-PMAA block polymer was dissolved in 0.1 M NaCl solution. Figure 6a shows the spherical micelles consisting of a compact MAA core and four-arm PEO corona chains at R ∼ 0.1. The radius of the aggregates was in good agreement with the light scattering data of approximately 46 nm. At R ∼ 0.05, the hydrogen bond interactions between MAA segments induced the micelles to form larger aggregates.43 As shown by Figure 6b, two different sizes of aggregate were observed, which were in agreement with the results obtained from dynamic light scattering (DLS). With further reduction in R to 0.02, the large micelles associate to form a larger aggregate induced by hydrogen bonding between the “palm-shaped” PEO polymeric chains at the outer shell of the particles (Figure 6c). Figure 6d shows the presence of large compound aggregates that corresponded to the slow mode as detected by DLS at R close to zero. The association mechanism of four-arm PEO-b-PMAA block polymer during the ionization process in aqueous solution is depicted in Figure 7. At high ionization degree, the star polymer exists as negatively charged unimers. When the carboxylic acid groups are partially neutralized at R ∼ 0.3, the simple core-shell shape micelle is formed driven by the hydrophobic MAA segments. The nonionized PMAA chains are nonpolar, and they tend to form a compact coiled structure to minimize their contact with water molecules due to the hydrophobic effect. When R reaches 0.1, reduced ionization produces weaker electrostatic repulsion, which causes the micelles to shrink into a smaller size while maintaining the core-shell structure. The multiarm structure of (42) Khougaz, K.; Gao, Z. S.; Eisenberg, A. Macromolecules 1994, 27, 6341–6346. (43) Binder, W. H. Monatsch. Chem. 2005, 136, 1–19.

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Figure 6. TEM micrographs of aggregates formed from four-arm PEO-b-PMAA block polymer at R of (a) 0.1, (b) 0.05, (c) 0.02, and (d) 0.

Figure 7. Proposed microstructure of the four-arm PEO-b-PMAA block polymer in 0.1 M NaCl solution at R of (a) 1, (b) 0.3, (c) 0.1, and (d) 0.02.

copolymer contributed to the formation of compact aggregates, which was reported for the linear and star-shaped poly (N-isopropyl acrylamide)-b-poly(N,N-diethylacrylamide) (PNIPAM-b-PDEA) copolymer.42 However, the micelles tend to aggregate to form larger particles induced by hydrogen bonding between MAA groups, as shown in Figure 7d at very low degrees of neutralization. Isothermal Titration Calorimetric Study. The thermodynamics of the dissociation of four-arm PEO-b-PMAA block polymer were investigated by ITC via the titration of 30 mM NaOH solution to 0.05 wt % of the block polymer in various NaCl solutions. A thermogram showing the raw cell feedback (CFB) heat signal for the step-by-step injections of 4898

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NaOH to the polymer in 0.15 M NaCl solution can be found from the Supporting Information. Differential enthalpy curves for polymer solutions at different NaCl concentrations were obtained by integrating the area under the raw signal curve at each injection, and the results are shown in Figure 8. The conformational transition of the polymer chains in salt solutions can be deduced from the differential ITC data.44 At the early stage of neutralization, most of the carboxylic groups were nonionized and they were buried inside the large aggregates resulting in fairly low enthalpy. With the addition of NaOH, the OH- ions began to pene(44) Hoare, T.; Pelton, R. Langmuir 2006, 22, 7342–7350.

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high salt solution, where the range was from 0.1 to 0.9. The progressively less sensitive titration curves indicated that the interaction between polymer and sodium hydroxide was more cooperative than in the absence of salt, thus the addition of salt favored the neutralization process of the aggregates.

Conclusions

Figure 8. Differential enthalpy curves for titrating 30 mM NaOH solution into 0.05 wt % four-arm PEO-b-PMAA block polymer in (0) 0.001 M, (9) 0.005 M, (Δ) 0.02 M, and (2) 0.15 M NaCl solution. trate into the core of the micelles and neutralized the carboxylic acid groups. The large aggregates expanded, where the enthalpy increased drastically to a maximum at R of about 0.05. Continuous addition of NaOH resulted in a drop in the enthalpy at R of between 0.05 and 0.1, which might indicate that the large aggregates had dissociated into micelles. For R ranging from 0.1 to 0.3, the enthalpy curves approached a plateau, and this corresponded to the progressive neutralization of the swollen micelles. The enthalpy then decreased from R of approximately 0.3 in low salt environment (open and filled squares) and R of approximately 0.9 in high salt environment (open and filled triangles). Since the pronounced exothermic heat was observed, titrations in other salt solutions revealed similar enthalpy curves. However, the shape of the ITC titration curve was affected considerably by the addition of salt. The flat region in the enthalpy curve was observed over a narrower R range of 0.1 to 0.3 for polymer in low salt solution than for polymer in

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The novel four-arm PEO-b-PMAA synthesized by ATRP formed well-defined micelles or large spherical aggregates in aqueous solution, depending on the pH. The conformational transition during the process of neutralization was confirmed by the negative logarithm dissociation constant (pKa) curves of potentiometric titration, SLS, DLS, and TEM studies. The average Gibbs free energy to extract a proton from a charged polyion decreased with increasing salt content, which suggested that the addition of electrolyte favored the neutralization process. The size of spherical core-shell micelles consisting of a partially ionized PMAA core surrounded by a hydrophilic PEO corona could be reversibly manipulated by changing the pH. ITC results showed that the neutralization of polyacids with a strong base is an exothermic process dominated by enthalpy. The extremely low CMC suggested that the polymer may be suitable for use in the delivery of cationic biomolecules, such as peptides or cationic drugs. Acknowledgment. We wish to acknowledge the assistance of Dr. P. Ravi in the synthesis of the block copolymer using the ATRP technique. E.H. would like to thank Nanyang Technological University for providing the financial support for her graduate study. Supporting Information Available: Details on the polymer synthesis, potentiometric and conductometric titrations, and light scattering are provided in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

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