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Preparation and Characterization of Thermosensitive Polyampholyte Nanogels Kazuyoshi Ogawa, Atsushi Nakayama, and Etsuo Kokufuta* Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8565, Japan Received October 18, 2002. In Final Form: February 3, 2003 Nanosized polyampholyte gel particles were synthesized by aqueous redox polymerization in the presence of sodium dodecylbenzene sulfonate as a surfactant. Acrylic acid (AAc) and 1-vinylimidazole (VI) were respectively used as an anionic and a cationic monomer, both of which were incorporated into the network of N-isopropylacrylamide (NIPA) cross-linked with N,N′-methylenebisacrylamide. Potentiometric titration (0.05-0.5 M KCl solutions as a solvent) gave the following contents (in mol %) of anions (A) and cations (C) in the nanogels: A ) 3 and C ) 13 for nanogel G(1/4); A ) 14 and C ) 12 for nanogel G(1/1). It is worthy of notice that G(1/1) is insoluble in pure water but soluble in KCl solutions over the concentrations (CS) > 0.01 M; this phenomenon is the so-called “antipolyelectrolyte” behavior. Both nanogels were characterized in detail by a combination of static and dynamic light scattering, electrophoretic light scattering, and turbidity measurements. The pH dependence of the gel size exhibited a characteristic pattern, from which an isoelectric point (pI) was observed: pH 6.5 for G(1/4) and pH 5.3 for G(1/1). At pI, at which the negative and positive charges are fully balanced, the electrophoretic mobilities became zero. From the light scattering of nanogels in 0.1 M KCl solution at pH ) pI, the following data were obtained: radius of gyration (Rg) ) 107 nm for G(1/4) and 81 nm for G(1/1); hydrodynamic radius (Rh) ) 132 nm for G(1/4) and 87 nm for G(1/1). Thus, the Rg/Rh ratio was within 0.85 ( 0.05, suggesting a spherical conformation of the nanogel particles. From the study of the effects of CS and temperature, the following characteristics were obtained: (i) Rh of G(1/4) varies little depending on KCl concentration, while G(1/1) shows a slight increase in Rh with CS from 0.01 to 0.5 M and a marked decrease at CS ∼ 2 M without accompanying aggregation. (ii) G(1/1) undergoes a shape phase separation at CS ∼ 0.008 when decreasing CS, whereas G(1/4) is dispersible both in pure water and in KCl solutions at CS < 1 M, the concentration at which aggregation takes place, as observed in the pure NIPA nanogel system. (iii) Both G(1/4) and G(1/1) shrink up to 41 and 51 °C, respectively, and around these temperatures the gel suspensions undergo a shape phase separation. The results obtained were compared with those of terpolymers as a control sample and discussed in terms of the intra- and interparticle interactions, in which hydrogen bonding and/or hydrophobic association, other than the usual electrostatic attraction, were found to play an important role.
Introduction Microgels with diameters in the range of tens to hundreds of nanometers, often referred to as “nanogels,” are of wide interest in the fields of colloids and polymers. In particular, much attention is being given to temperature-responsive nanogels consisting of a lightly crosslinked polymer chain of N-isopropylacrylamide (NIPA). A number of studies have dealt with NIPA-based nanogel systems in connection with syntheses, properties, and applications, most of which were summarized in a recent review by Pelton.1 Copolymerization of NIPA with either anionic or cationic monomers gives rise to temperature-responsive polyelectrolyte nanogels,2 the size of which varies depending not only upon temperature but also upon pH and salt concentration. For example, an elimination of network charges by controlling pH lowers the temperature at which the gel particle undergoes an abrupt shrinking change without aggregation. In cases of polyelectrolyte nanogels with both anionic and cationic charges,3-7 that is, polyampholyte nanogels, an increase in salt concentration causes the gel to swell when the negative and positive charges are balanced; this phenomenon is analogous to “antipolyelectrolyte” behavior observed in aqueous polyampholyte * To whom correspondence should be addressed. Fax: 81-29853-4605. E-mail:
[email protected]. (1) Pelton, R. Adv. Colloid Interface Sci. 2000, 85, 1. (2) (a) Ito, S.; Ogawa, K.; Suzuki, H.; Wang, B.; Yoshida, R.; Kokufuta, E. Langmuir 1999, 15, 4289. (b) Ogawa, K.; Wang, B.; Kokufuta, E. Langmuir 2001, 17, 4704.
solutions. In the previous studies dealing with polyampholyte nanogels, however, only one paper7 focused on a NIPA-based system without paying attention to the effect of temperature on the particle size and the colloidal stability. Therefore, it would be of interest to study NIPAbased polyampholyte nanogels in connection with the swelling mechanism as well as with the colloidal stability. To understand the volume phase transition in “bulk” gels at the molecular level, we have proposed a simple model.8 The transition was then accounted for by hypothesizing a balance between the repulsion and attraction among functional groups attached to the network which arise from a combination of four intermolecular forces: ionic, hydrophobic, van der Waals, and hydrogen bonding. When a repulsive force, usually electrostatic in nature, overcomes an attractive force such as hydrogen bonding or hydrophobic interaction, the gel volume should increase discontinuously in some cases and continuously in others. The variables that trigger the transition influence these intermolecular forces and thereby the balanced state of the attractive and repulsive forces. (3) Kashiwabara, M.; Fujimoto, K.; Kawaguchi, H. Colloid Polym. Sci. 1995, 273, 339. (4) Kudaibergenov, S. E. Ber. Bunsen-Ges. Phys. Chem., Chem. Phys. 1996, 100, 1079. (5) Neyret, S.; Vincent, B. Polymer 1997, 38, 6129. (6) Hampton, K. W.; Ford, W. T. Macromolecules 2000, 33, 7292. (7) Braun, O.; Selb, J.; Candau, F. Polymer 2001, 42, 8499. (8) (a) Ilmain, F.; Tanaka, T.; Kokufuta, E. Nature 1991, 349, 400. (b) Kokufuta, E. In Physical Chemistry of Polyelectrolytes; Radeva, T., Ed.; Marcel Dekker: New York, Basel, 2000; Chapter 17.
10.1021/la0267185 CCC: $25.00 © 2003 American Chemical Society Published on Web 03/19/2003
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Table 1. Compositions of Monomer Feeds Used in Preparation and Several Characteristics (at 25 °C) of the Ampholytic Nanogels and Terpolymers Obtained sample G(1/4)a G(1/1)b P(1/4)a P(1/1)b
monomer feed composition (mol %) charge density (mmol/g dry weight) 106A2 NIPA AAc VI Bis carboxylc,d imidazolec 10-5Mw (g/mol) Rg (nm) Rh (nm) ((cm3mol)/g2) 69.3 60.3 70.0 60.9
14.9 25.9 15.0 26.1
14.9 12.9 15.0 13.0
0.9 0.9 0.0 0.0
0.31 1.36 0.31 1.40
(3) (14) (3) (15)
1.15 1.12 1.16 1.16
(13) (12) (13) (12)
524 149 1.92 2.97
107 81 28 45
132 87 20 23
1.1 1.0 98.6 247
pI 6.50 5.30 6.50 5.30
a Prepared at 60 °C. b Prepared at 80 °C. c Denotes mole % in parentheses. d Determined by ignoring the sulfonate groups which might be bound to the network in the polymerization with APS (see ref 1), because the calculated sulfonate amount was less than 1.8% of all the anions titrated.
To understand the colloid stability, on the other hand, the balance of van der Waals attraction and steric or electrostatic repulsion is generally assumed (e.g., see ref 1). In cases of amphoteric colloids such as proteins, however, the electrostatic attraction between opposite charges should become the chief factor governing the colloidal stability. This can be seen from the aggregation of proteins in aqueous solutions at an isoelectric point (pI). In addition, hydrophobic association as a key interaction in the temperature-induced NIPA gel collapse9 is also believed to play an important role in protein aggregation, in particular in biochemical fields. Therefore, it is expected that study of intra- and interparticle interactions using NIPA-based amphoteric nanogels makes it possible to discuss the molecular mechanism in gel collapse and colloidal aggregation on the same grounds. Use of the polyampholyte with the same chemical composition, other than the absence of cross-linker, should help us to promote better understanding of this molecular mechanism. Taking the above into account, we attempted to prepare temperature-responsive amphoteric nanogels consisting of a cross-linked terpolymer of NIPA, acrylic acid (AAc), and 1-vinylimidazole (VI). Also prepared as the control sample were linear terpolymers with the same composition. The characterization was carried out in detail by examining molecular weights, charge densities, and pI values. The stimuli-responsive characteristics due to the intraparticle interaction were studied by measuring hydrodynamic diameter (Rh) as a function of pH, ionic strength, and temperature. Turbidity measurements were also employed to study the interparticle interaction that affects colloid stability. The results obtained in both polymer and gel systems have demonstrated that both intra- and interparticle interactions are explainable in terms of the electrostatic repulsion or attraction, as well as of both hydrophobic interaction and hydrogen bonding as the attractive force. Experimental Section Materials. All chemicals were obtained from commercial sources: NIPA from Kojin Chemical Co. (Tokyo, Japan); AAc, N,N′-methylenebisacrylamide (Bis, cross-linker), and ammonium persulfate (APS; initiator) from Wako Pure Chemical Co. (Osaka, Japan); VI and sodium dodecylbenzene sulfonate (NaDBS; surfactant) from Tokyo Chemical Industry Co. (Tokyo, Japan). The monomers (NIPA, AAc, and VI) were purified according to the usual methods. All pregel solutions were prepared with distilled water passed through a Milli-Q filter. Polymerization. A microemulsion polymerization technique using oil (Isopar M) has been employed in the preparation of NIPA-based polyampholyte nanogels,7 but we adopted here an oil-free aqueous redox polymerization initiated by APS because this way enabled us to prepare NIPA-based anionic and cationic polyelectrolyte nanogels (see ref 2). (9) For example: Schild, H. G. Prog. Polym. Sci. 1992, 17, 163.
The polymerization was carried out using an aqueous 0.01 M NaDBS solution (300 mL) containing the desired quantities of the monomers. (Details of monomer compositions and reaction conditions will be described in the Results and Discussion section, together with characterization data.) The O2-free monomer solution was placed in the usual separable flask (500 mL) equipped with a cooler and a magnetic stirrer, and maintained at a desired temperature with stirring (200 rpm). To remove oxygen well, nitrogen gas was continuously supplied above the surface of the solution for 1 h before the reaction. The polymerization reaction was initiated by adding 1 mL of an aqueous O2-free solution of APS (7.5 w/w %), allowed to continue for 2 h, and terminated by blowing oxygen through the reactor. After that, residual monomers and NaDBS were removed from the resulting reaction mixture by an alkaline dialyzing method2 using a Spectra/Por CLC500 tube with a molecular weight cutoff of 100 000. The further purification was carried out by passing the dialyzed solution through a mixed bed of anion- and cationexchange resins. The purified nanogel suspension and polymer solution were then lyophilized for 3 days. The yield was more than 85% in all the preparations. It is quite important to examine the existence of un-crosslinked and dissolved polymers in nanogel dispersion. However, this is a rather difficult problem with respect to experimental techniques and is usually ignored in previous studies. To overcome this difficulty, we employed here an analytical method based on a combination of dialysis and colloid titration because (i) the terpolymer can permeate into the outer solution through a dialyzing membrane with a molecular weight cutoff of 1 000 000 and (ii) a very slight amount (less than 0.005 w/v %) of permeated terpolymers is detectable by colloid titration.10 As a result, it was found that our nanogel samples are free of un-cross-linked and dissolved terpolymers after the purification. By colloid titration, it was confirmed that our terpolymers are not a mixture of NIPA copolymers of AAc and VI. No existence of NIPA homopolymer in our terpolymer system was also confirmed by investigations of temperature change of turbidity for supernatant liquids from which the terpolymer was separated by forming a water-insoluble complex with colloid titrants. Measurements. The overall contents of acidic and basic groups were determined by pH titration at 25 °C and in 0.5 M KCl solution with HCl and KOH (0.1 M each) as the standard titrant. Hydrodynamic diameter (dh) and weight-average molecular weight (Mw) were determined by a laser light scattering technique (Otsuka DLS-7000 apparatus) with a 75 mW argon ion laser (NEC model GLG-3112). Electrophoretic mobility was measured by electrophoretic light scattering (ELS) using an Otsuka ELS-6000 apparatus. Turbidity was measured at 500 nm with a Hitachi spectrophotometer (model U-2001).
Results and Discussion Characterizations. Table 1 shows several characteristics of the NIPA-based ampholyte nanogels and terpolymers, together with the compositions of monomer feeds used in the preparation. The contents of AAc and VI monomer residues were determined by potentiometric titration. The fully deionized G(1/1) and P(1/1) samples were insoluble in pure water; therefore, an aqueous 0.5 (10) Kokufuta, E. Macromolecules 1979, 12, 350.
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Figure 2. Dependence of electrophoretic mobility (U) on pH for nanogel G(1/4) and terpolymer P(1/4) in 0.01 M KCl solution at 25 °C. Polymer concentration ∼ 1 mg/mL for both samples. Figure 1. Potentiometric titration curves of nanogels G(1/4) and G(1/1) at 25 °C. The sample solution (50 mL) was prepared by dissolving the following quantity of fully deionized and lyophilized polymers into 0.5 M KCl solution: 81.5 mg for G(1/ 4) and 100 mg for G(1/1). ∆[H+] was determined by subtracting the moles of ions bound to the polymer from those added into the sample solution.
M KCl solution was used as a solvent for the titration. As can be seen from Figure 1, the titration curve with KOH of thes-COOH plus sCOO- +NHt groups in the nanogel shows a clear pH jump at the end point. In contrast, a broad pH change around the end point appears in the titration of the sCOO- +NHt plus dNs groups in G(1/1) with HCl. Thus, a difference (∆[H+]) in molarity of the H+ ions added and bound was calculated in the titration with HCl. Then, the end point was determinable by a break point of the plots of ∆[H+] against HCl volume, as reported by Nagasawa and Noda.11 By comparing the AAc or VI contents before and after the polymerization, it is found that the reactivity of NIPA with AAc is lower than that with VI. This is the reason we used a large AAc/VI monomer ratio and a high temperature for obtaining G(1/1) and P(1/1) in comparison with the reaction conditions for G(1/4) and P(1/4). In practice, we carried out several of preliminary experiments and succeeded in synthesizing a balanced nanogel G(1/1) and terpolymer P(1/1) with a total of 26 mol % of the carboxyl plus imidazole groups in their ratio ∼ 1:1. Also prepared in this study were an unbalanced nanogel G(1/ 4) and terpolymer P(1/4), both of which contain 3 mol % carboxyl groups and 13 mol % imidazole groups (carboxyl/ imidazole ratio ∼ 1:4). ELS was employed to accurately determine pI, that is, the pH at which the mobility of the ampholyte nanogel or terpolymer becomes zero. Typical mobility curves for G(1/ 4) and P(1/4) are shown in Figure 2. No difference is observed in the mobility curves between the nanogel and terpolymer; consequently, both pI values are found to be the same. Also found from the mobility data is that over a wide pH range the ionization behavior of the nanogel is very close to that of the terpolymer. The same conclusion was obtained by comparing G(1/1) with P(1/1) (data not shown). As a result, the terpolymer seems to be the precursor of the corresponding nanogel. The values of Mw, root-mean-square of gyration (Rg), and second virial coefficient (A2) for the nanogels and terpolymers were determined by static light scattering (11) Nagasawa, M.; Noda, I. J. Am. Chem. Soc. 1968, 90, 7200.
(SLS) using the following equation:
(
)
1 16π2 2 Kc θ ) 1+ Rg sin2 + 2A2c 2 Rθ Mw 2 3λ
(1)
Here, c denotes the weight concentration of the gel or polymer, Rθ is the Rayleigh ratio, θ is the scattering angle, λ is the wavelength of light in the medium, and K is given as12
K ) (4π2/λ40NA)n20(dn/dc)2
(2)
where λ0 is the wavelength of light in a vacuum, NA is Avogadro’s number, n0 is the refractive index of the medium, and (dn/dc) (in mL/g) is the change in refractive index with the concentration of nanogels or terpolymers. We performed SLS measurements at pH ) pI and at ionic strength ) 0.1 (KCl). Zimm plots were then obtained at the scattering angle (θ) ) 45-135° (see Figure 3). For both the gel and the polymer, the double extrapolations of Kc/Rθ vs (sin2(θ/2) + kc) plots to θ f 0 and c f 0 gave two straight lines with correlation coefficients > 0.99, cointersecting the Y axis, enabling us to determine Mw. The A2 values were negligibly small for both the samples, in particular, on the order of 10-6 for the nanogels. In addition to SLS, we also performed dynamic light scattering (DLS), from which the hydrodynamic radius (Rh) was estimated at θ ) 90° by analyzing the homodyne intensity-intensity correlation function with the cumulants method. It has been well-known that the Rg/Rh ratio changes from infinity to 0.775 when the polymer structure changes from a long rod to a sphere, with values from 1.3 to 1.5 for random coils. Taking this into account, our results indicate that both nanogels G(1/4) and G(1/1) are spherical at pI. However, the Rg/Rh ratio is 1.40 for P(1/4) and 1.96 for P(1/1), suggesting a random coil or a slightly extended conformation of the terpolymer chain. From DLS and SLS data, we may estimate the polymer density (F) using the relation F ) (f/fmin)(3/4π)(Mw/NA)(1/ Rh3), where f represents the actual frictional coefficient and fmin is the minimum frictional coefficient for a hypothetical sphere. Because our nanogels are spherical, it would be assumed that f/fmin ∼ 1. Then, we obtain F ) ∼0.01 g/cm3 (i.e., 1 w/v %). This value is much smaller than the F values (∼1.4 g/cm3) of proteins (partial specific (12) It should be noted that the optical constant K in eq 2 for vertically polarized light is given by (4π2/λ40NA)n20(dn/dc)2, instead of (2π2/λ40NA) 2 n0(dn/dc)2.
Thermosensitive Polyampholyte Nanogels
Figure 3. Zimm plots for nanogel G(1/1) and terpolymer P(1/ 1) in 0. 1 M KCl solution at pI and at 25 °C. Polymer concentration (c), 0.5-4 (mg/mL); θ, 45-135 °. Values of (dn/ dc) (in mL/g) were 0.1722 for G(1/1) and 0.1784 for P(1/1), as determined using vertically polarized light from an iodine arc with a spectrum filter (wavelength ) 488 nm).
volume ) 1/F ∼ 0.70-0.75)13 but close to the F value (0.008 g/cm3) of a γ-ray-cross-linked poly(vinyl alcohol) gel particle (Mw ) 1.0 × 108 g/mol; Rh ) 195 nm; Rg/Rh ) 0.98).14 Also, a similar F value (0.026 g/cm3) at 25 °C has been obtained from Bis-cross-linked NIPA homopolymer nanogel in water (Mw ) 1.5 × 108 g/mol; Rh ) 132 nm; Rg/Rh ) 0.66).15 From these results, the morphological characteristic of our nanogels seems to be different from that of spherical “hard” colloids such as poly(styrene) latex and proteins. Instead, we may see a morphological similarity with microgel particles14 which are formed via intrapolymer cross-linking and have an obscure surface due to a lot of dangling chains:
Effects of pH. ELS and DLS have suggested that the ionization behavior of our nanogels is similar to that of the corresponding terpolymers over a wide pH range, although at pH ) pI there is a marked difference in the conformations of nanogel and terpolymer. Taking this into account, we studied first the pH dependence of dh for the (13) Tsuboi, A.; Izumi, T.; Hirata, M.; Xia, J.; Dubin, P. L.; Kokufuta, E. Langmuir 1996, 12, 6295. (14) Wang, B.; Mukataka, S.; Kodama, M.; Kokufuta, E. Langmuir 1997, 13, 6108. (15) Gao, J.; Hu, Z. Langmuir 2002, 18, 1360.
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Figure 4. Dependence of normalized hydrodynamic diameter (dh/d0) on pH for nanogels (a) and terpolymers (b) in salt-free and KCl solutions at 25 °C. KCl concentration (mol/L): 0 (O, b); 0.01 (9, 0); 0.1 (2, 4). Polymer concentration: 0.1 mg/mL for nanogels and 1 mg/mL for terpolymers. The normalization was made by each diameter (d0) at KCl concentration ) 0.1 M and at pH ) pI.
nanogels and terpolymers. The measurements for the nanogels were mainly carried out in salt-free solutions at different pH levels, other than a narrow pH range around pI. For the terpolymers, most of the measurements were made in 0.01 M KCl solution, since it is well-known that many effects such as small ion-polyion and polyionpolyion interactions must be taken into consideration when making sense of DLS data of polyelectrolytes in pure water as well as in solutions with very low concentrations of salts. The dh values as a function of pH were then normalized by a minimum diameter (d0) at pI and at ionic strength 0.1 (see Table 1), because there was a difference of the size at pI between G(1/4) and G(1/1) as well as between P(1/4) and P(1/1). From Figure 4, we may see the same trend in the dh/d0 versus pH curves for the nanogel and the terpolymer; that is, at the pH ranges below and above pI the nanogel is in a swollen state and the terpolymer adopts an extended conformation. These can be understood as a result of the electrostatic intrachain repulsion due to the “net” charges of either negative or positive sign. Thus, the dh/d0 values at pH > pI for G(1/1), whose COOH content is 4.7 times that for G(1/4), are larger than those for G(1/4). The same conclusion can be obtained by comparison of P(1/1) with P(1/4). At pH < pI, however, G(1/4) is more swollen as compared with G(1/1), even though there is little difference in the imidazole content between both nanogels. This trend is also observed in the terpolymers. In general, ions are completely protonated at pH < 3, but the resulting COOH has a tendency to form the hydrogen bonding with the NIPA residue in the linear or cross-linked polymer.16 Taking this into account, one may assume that the hydrogen bonding acts as a cross-linking point, thereby causing shrinkage of the linear or cross-linked polymer. As another explanation, one may consider the formation of a stable ion pair between COO- and dNH+s which also (16) Kokufuta, E.; Wang, B.; Yoshida, R.; Khokhlov, A. R.; Hirata, M. Macromolecules 1998, 31, 6878.
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0.001
0.01
0.1
0.5
1.0
2.0
G(1/4) G(1/1) P(1/4) P(1/1) neutral NIPA nanogel
272 ag 44 ag 75
269 162 37 ag 72
264 174 40 46 70
244 168 41 47 69
ag
ag 131 ag ag ag
38 42 ag
a Shown as ag is the aggregation of nanogels or terpolymer through which phase separation takes place.
Figure 5. Changes in transmittance (T %) with pH for saltfree solutions containing nanogel G(1/1) and terpolymer P(1/1) at 25 °C. Polymer concentration: 1 mg/mL for both samples.
acts as a cross-linking point. However, this would be contradicted by the following ELS results: (i) At pH 3, a few differences were observed in the mobilities (U in cm2/ s‚V) between G(1/4) (U ) 1.92 × 10-4) and G(1/1) (U ) 1.70 × 10-4) because the imidazole contents were 13 mol % for G(1/4) and 12 mol % for G(1/1). (ii) At pH 10 the values were -1.96 × 10-4 for G(1/1) and -0.65 × 10-4 for G(1/4), since the COOH content (14 mol %) of G(1/1) was larger than that (3 mol %) of G(1/4). To estimate the net charges per nanogel particle from the mobility, a more detailed discussion should be required, as reported in our previous paper (see ref 2a). Nevertheless, from the fact that there is little difference in the pH versus mobility curve between the nanogel and the terpolymer (see Figure 2), we may say that the imidazole groups are fully ionized at pH < 3. The advantage of the study of nanogels is not only to be able to investigate the intraparticle interaction by such a way as DLS but also to be able to examine the interaction among the particles (i.e., interparticle interaction) in connection with aqueous colloid stability. Thus, we investigated the pH change of transmittance (T %) of saltfree 0.1% (w/v) solutions of G(1/1) and P(1/1) (see Figure 5). Both in the nanogel system and in the terpolymer system, an abrupt fall of T % is observed at a very narrow pH range when the solution pH comes close to pI from either the acidic side or the alkaline side, indicating a phase separation around pI. In general, the phase separation of this sort in polyampholyte systems is considered to take place when the net charge becomes zero at pI by balancing the positive charges with the negative charges (or vice versa). This situation should be conceivable for the results of G(1/1) and P(1/1). However, in both G(1/4) and P(1/4) systems whose pH was accurately adjusted to pI, no phase separation was observed, even at high polymer concentrations (i.e., 1 w/v %). Thus, the phase separation of G(1/1) and P(1/1) may not be interpreted by a simple consideration that at pI the net charge becomes zero. Of course, it is hard to consider the effect of COOH as mentioned above, because the phase separation is observed at pH 5.0-5.5. Neyret et al.17 have reported that the charge distribution along the polyampholyte copolymer considerably affects its conformation in aqueous solutions. Taking this into account, it would be reasonable to assume that the difference in the interparticle interaction between G(1/1) and G(1/4) or in the interpolymer interaction between P(1/ 1) and P(1/4) is due to an “inhomogeneous” distribution of the balanced positive and negative charges on the nanogel surface or along the terpolymer. In the case of (17) Neyret, S.; Baudouin, A.; Corpart, J. M.; Candau, F. Nuovo Cimento D 1994, 16, 669.
nanogel particles, this inhomogeneous distribution seems to be like “charge patches” on the surface of proteins which are believed to play an important role in their aggregation at pI. Effect of Salt Concentration. It has been demonstrated that a polyampholyte bulk gel with the balanced positive and negative charges swells upon the addition of salts; for example, see ref 18. This antipolyelectrolyte behavior was then considered as a result of weakening of the electrostatic interchain attraction by screening with counterions, by which the network strands formed between oppositely charged chains become disentangled. Thus, it would be interesting to study how salt concentration affects the swelling of our nanogels. Table 2 shows the dependence of dh on KCl concentration for the nanogels and the terpolymers whose positive and negative charges are fully balanced by adjusting the pH to pI. Also shown in the table is the result for neutral NIPA nanogel particles, since it has been known that, with increasing concentration of salts such as NaCl, bulk gels or polymers of NIPA exhibit a lowering of Tv (LCST), accompanying a decrease in the swelling ratio.19 It is found that there are a few influences of salt concentration on the size of the nanogels and terpolymers; thus, the antipolyelectrolyte behavior is not revealed in the size change. The value of G(1/1) increased about 10 nm with increasing salt concentration from 0.01 to 0.1 M, but this increment is not enough to discuss the antipolyelectrolyte behavior. Thus, it seems that the salt effect observed in both the gel and the polymer system is due to formation of very stable ion pairs (sCOO- +NHt) at pI, most of which are not labile and prone to reconfiguration upon the addition of KCl. Such an ion pairing would be possible because our nanogels (and also terpolymers) contain more than 50 mol % of the neutral NIPA monomer. A marked salt effect appears in colloidal dispersibility or polymer solubility rather than in the size of the nanogel or the terpolymer. G(1/4) is dispersible in water but not in 1 M KCl solution, while G(1/1) shows the opposite trend (see Table 2). Since in 1 M KCl the neutral NIPA nanogels aggregate with one another and lose the colloidal stability, a similar molecular mechanism could be assumed for the aggregation of G(1/4) in 1 M KCl, that is, an increase in hydrophobicity due to a structural change of water molecules hydrated around the main and side chains of NIPA residues. This would cause shrinkage of G(1/1) in 2 M KCl without accompanying aggregation, because of a considerable amount of hydrophilic ion pairs bound to the surface of G(1/1) particles. However, the aggregation of G(1/1) in response to a very small change in KCl concentration around 0.01 M, which is accompanied by a (18) Nisato, G.; Munch, J. P.; Candau, S. J. Langmuir 1999, 15, 4236. (19) (a) For the polymer, see: Schild, H. G.; Tirrell, D. A. J. Phys. Chem. 1990, 94, 4352. (b) For the gel, see: Suzuki, A. Adv. Polym. Sci. 1993, 110, 199.
Thermosensitive Polyampholyte Nanogels
Figure 6. Changes in transmittance (T %) with concentration of KCl added into solutions (pH ) pI) of nanogel G(1/1) and terpolymer P(1/1) at 25 °C. Polymer concentration: 1 mg/mL for both samples.
Figure 7. Temperature dependence of hydrodynamic diameter (dh) of nanogels G(1/4) and G(1/1) in 0.1 M KCl solution at pI. Polymer concentration: 0.1 mg/mL for both samples.
shape phase separation (see Figure 6), cannot be explained in connection with the salt-induced change in the hydrophobicity. Then, let us notice the fact that terpolymer P(1/1) with the balanced charges becomes soluble upon the addition of electrolytes. This phenomenon (i.e., antipolyelectrolyte behavior) is explainable as a result of screening of the electrostatic interpolymer attraction. Similarly, in the case of G(1/1), it is reasonable to assume that the addition of salts weakens the electrostatic interparticle attraction due to inhomogeneously distributed but fully balanced positive and negative charges on the nanogel surface. As a result, the study of the salt effect clearly demonstrates that in our nanogels, in particular in G(1/1), the antipolyelectrolyte behavior appears in the interparticle interaction (turbidity measurements) but not in the intraparticle interaction (size measurements). Effect of Temperature. It is of particular importance to examine temperature-responsive property changes for our NIPA-based nanogels. Figure 7 shows the temperature dependence of dh for G(1/4) and G(1/1) at pI and at ionic strength 0.1. Under these conditions, the electrostatic interactions within the polyampholyte network are fully eliminated. However, a rise in temperature shrinks G(1/ 4) above 41 °C and G(1/1) above 52 °C without accompanying aggregation due to the interparticle interaction. The observed temperature-induced gel collapse is understood as a result of the intraparticle hydrophobic association between the cross-linked polyampholyte chains, in particular between segments consisting of NIPA monomer units. The presence of hydrophilic segments consisting of AAc and VI monomer units, most of which form the ion pairs, should cause a rise in Tv; therefore, Tv of G(1/1) is higher than that of G(1/4). In contrast, the magnitude of a size change for G(1/1) is smaller than that for G(1/4) over a wide temperature range, because the
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Figure 8. Temperature changes of transmittance (T %) of 0.1 M KCl solutions (pH ) pI) containing nanogels G(1/4) and G(1/ 1). Polymer concentration: 1 mg/mL for both samples.
content of the ion pairs is larger in G(1/1) than in G(1/4) (i.e., cross-linking effect). The temperature changes of T % are shown in Figure 8, from which we may estimate LCST: 40 °C for G(1/4) and 50 °C for G(1/1). These agree with the temperature at which the aggregation of gel particles takes place. Also, there was a good agreement of LCST between the nanogel and terpolymer systems. The detailed studies on the electrostatic interactions in our nanogel systems have demonstrated that G(1/1) is prone to aggregate at pH ) pI and at low ionic strengths. Under such conditions, nevertheless, the gel particle exhibits a high LCST. The reason is the presence of a large amount of hydrophilic segments consisting of ion-paired AAc and VI monomer units. Thus, we may assume that hydrophobic association plays a key role in the LCST behavior observed not only in the terpolymer system but also in the nanogel system. The temperature-induced phase separation of aqueous NIPA homopolymer solutions, in which aggregation of the polymer was observed at its usual concentration, was accounted for by considering the hydrophobic association (see refs 8 and 9). Instead, the van der Waals attraction was assumed to account for the temperature effect upon aqueous colloid stability of NIPA homopolymer gel particles having a slight amount of charge due to the binding of ionic free radical initiators such as APS (see ref 1). There are two different ways for illustrating the aggregation of materials with the morphological difference but with the same chemical composition. Just this is the case in our experimental results mentioned here. However, we have preferred the hydrophobic association to the van der Waals attraction. The reasons are as follows: (i) The temperature-induced aggregation was observed at pI as well as at a considerably high ionic strength. (ii) The thermally collapsed gel particle was still in a swollen state because its polymer content was about 3 w/v %, as estimated from F using Rh near LCST (or Tv); 83 nm for G(1/4) and 63 for G(1/1). (iii) Therefore, the collapsed gel particle has an obscure surface due to a large amount of hydrophilic segments. Conclusions Polyampholyte gels with a size of several hundred nanometers in diameter were prepared by aqueous redox polymerization in the presence of surfactant (NaDBS). The gel network was composed of cross-linked thermosensitive NIPA chains with bound anionic AAc and cationic VI units in the AAc/VI ratios ∼ 1:1 and 1:4. The diameter of nanogel particles varied depending on pH and showed a minimum value at the isoelectric point (pI); therefore, two pH regions bringing about swelling lay on either side of pI. The Rg/Rh ratios at pI were between 0.8
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and 0.9, suggesting that the nanogels with balanced negative and positive charges are spherical in shape. For the nanogel in the AAc/VI ratio ∼ 1:1, the antipolyelectrolyte behavior was observed at pH ) pI in colloidal dispersibility due to the interparticle interaction rather than in the size change due to the intraparticle interaction. An increase in temperature brought about both the volume collapse due to the intraparticle interaction and the phase separation (LCST behavior) due to the interparticle interaction. These phenomena caused by pH, salt concentration, and temperature were also observed in the terpolymer system. The results obtained suggest a simi-
Ogawa et al.
larity in the molecular mechanism between the stability of colloids and the solubility of polymer, both of which can be accounted for by considering hydrogen bonding and/or hydrophobic association, other than the usual electrostatic attraction. Acknowledgment. This work was supported in part by a Grant-in-Aid for Scientific Research to E.K. from the Ministry of Education, Japan (No. 08558092). LA0267185
