J. Phys. Chem. B 2009, 113, 14849–14853
14849
Investigation of Physical-Chemical Properties of Agarose Hydrogels with Embedded Emulsions Galina A. Komarova,*,†,‡ Sergey G. Starodubtsev,‡ and Alexei R. Khokhlov†,‡ Institute of Polymer Science, UniVersity of Ulm, Ulm D-89081, Germany, and Physics Department, Moscow State LomonosoV UniVersity, Moscow 119991, Russia ReceiVed: February 11, 2009; ReVised Manuscript ReceiVed: September 25, 2009
Composite agarose hydrogels with embedded tetradecane emulsions stabilized by cetylpyridinium chloride were studied. The absorption efficiency of 4-nitrophenyl ethers of carbonic acids by the composite agarose gels increases with the length of the hydrocarbon tail of the ester. The diffusion rate of amphiphilic substances in the composite gels was demonstrated to be much less that than in the standard agarose gels. The reaction kinetics between the esters and dodecylmercaptan dissolved in tetradecane droplets of composite hydrogel was studied. In the region of physiological pH, the reactivity of SH groups embedded in the composite agarose gel in the reaction with the esters is significantly higher than that in a homogeneous solution. Hydrogels with embedded emulsion droplets are of considerable practical importance as drug delivery systems, microreactors, and absorbers. Composite gels filled with emulsions incorporating lipophilic mercaptanes are effective absorbers of heavy metal ions. Introduction Oil-in-water emulsions have a great practical importance, for instance, in food industry, pharmaceutics, perfumery, etc. In particular, they have a high ability to adsorb and release amphiphilic substances. However, the use of the emulsions with the droplets moving freely in the aqueous medium is limited. Mixing of the droplets in contact with the surface of tissues, for example, with skin will significantly accelerate the transport of bioactive substances from the emulsions into them. Application of polymeric gels containing emulsions (or oil droplets) can help in solving this problem. Large interface area (or small oil droplet size) is the necessary condition of effective application of immobilized emulsions for adsorption and release of amphiphilic substances. On the other hand, the droplet size should be large enough in order to prevent droplet diffusion from the swollen gel network into the surrounding solution. The solubility of oil in water and the pressure of oil vapors should be negligibly small for the system to be stable. The hydrogels with embedded emulsion droplets are a new class of emulsion systems, and to date, only several works were dedicated to their study.1–6 The first aim of the present work is obtaining and studying the main features of absorption and transport properties of hydrogel films filled with lipophilic emulsion. Interfacial physical and chemical processes are known to have a number of specific features, for example, high ability to concentrate amphiphilic substances. Lipid membranes in live cells are responsible for the most important metabolic processes.7 Some of the membranes (e.g., chloroplasts and mitochondria) have a well-developed folded structure, which ensures the intensification of such processes. Active centers of many enzymes (e.g., peptidases) can be considered as interfaces with a complex shape and electronic configuration. The attempts of using the principles of enzyme catalysis in synthetic polymeric * To whom correspondence should
[email protected]. † University of Ulm. ‡ Moscow State Lomonosov University.
be
addressed.
E-mail:
catalysts8 and in micelle catalysis9–11 are well-known. However, the problem of removal of the catalyst or the products of reaction from the reaction medium is an obvious drawback of catalytic systems using soluble polymers and micelles. In many cases, a reactive chemical functional group must be attached to some carrier. Polymer resins, clays, zeolites, etc., are well-known examples of such functional materials. In this respect, polymeric gels (membranes, fibers, granules) with entrapped emulsions of lipophilic substances are of large practical importance. In the present study, we will demonstrate some features of the behavior of the chemical functional group, namely, the SH group of dodecylmercaptan (DDM) dissolved in tetradecan (TD) droplets of agarose composite hydrogel. The model reaction under the study was chosen as the reaction of DDM with the 4-nitrophenyl ethers of carbonic acids. The reaction can be described by the following equation:
R1S- + R2COOPh f R1SCOR2 + PhO-
(1)
Only a few papers have been published on the studies of the reaction ability of mercapto groups in the reaction with esters.12–15 Thus, Overberger et al. have studied the kinetics of thiol groups of 2,6-dimercaptohexane (DMH) and polyvinyl mercaptan (PVM) and copolymers of vinyl mercaptan and vinylimidazole with 4-nitrophenylacetate (4-NPhA), and also with anionic and cationic esters.13 The reaction of vinyl thiol groups in the copolymers of partially quarternarized poly-4vinyl pyridine (VPVM) with 4-NPhA and 3-nitro-4-acetobenzoic acid (NABA) was studied in refs 14 and 15. The transition of SH groups of propyl mercaptan (PM) and DMH into an active anionic phase in aqueous solution occurs in strongly alkaline media (pKa ) 10.5-11.0). Due to partial dehydration, the pKa value in PVM becomes even higher. At the same time, an essential shift of pKa toward physiological pH values can be observed for mercapto groups of vinylthiol monomer units surrounded by cationic groups of quaternized pyridine units in alkylated VPVM derivatives. In fact, the effect of the neighbor-
10.1021/jp901255c CCC: $40.75 2009 American Chemical Society Published on Web 10/16/2009
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J. Phys. Chem. B, Vol. 113, No. 45, 2009
ing groups in the synthetic polymer leads to the same shift of pKa of mercapto groups as that observed for SH groups in the active centers of some proteolytic enzymes, e.g., of papain.16 Considerable increase of the reaction ability of mercapto groups of VPVM copolymer in reaction with NABA was observed in the paper.14 It was explained by the shift of pKa caused by the effect of cationic neighboring groups, and also by accumulation of anionic ester in the vicinity of the thiol units of the cationic copolymer. It was noted besides that the treatment of reactionformed thioester with N-acetyl histidine leads to the regeneration of mercapto groups. Thus, reaction 1 can be considered as the first stage of a catalytic reaction similar to those observed in proteolytic enzymes with active centers containing SH groups. In our previous study of the reaction of DDM with the esters in cationic micelles, it was demonstrated that the cations of surfactants in micelles, as well as in cationic polymer VPVM, can induce an essential shift of pKa of mercapto groups from the alkaline region toward the physiological pH values (pKa ) 8.3-8.4).14 A kind of bifunctional catalysis is thus observed, so that neighboring groups activate the nucleophilic group, as is the case in the active centers of the enzymes. The reaction rate of 4-NPhA with mercapto groups of DDM incorporated in micelles was compared with that calculated for the SH group in DMH in the solution for the same pH (pH 8.0). The comparison shows that the reaction rate enhancement due to the effects of concentration increase of the ester and DDM in micelles and that of pKa shift exceeds as much as 3 × 103. Experimental Section 4-Nitrophenyl ethers of acetate (4-NPhA), butyrate (4-NPhB), valerate (4-NPhV), caproate (4-NPhC), and caprilate (4-NPhCC) as well as 4-nitrophenol (4-NPh), cetylpyridinium chloride (CPC), tetradecane (TD), dodecylmercaptane (DDM), and highmelting agarose for electrophoresis were obtained from Sigma. Hg acetate was of the “chemically pure” grade. For the preparation of composite agarose hydrogel films, the following mixture was used: 1.7% water solution of agarose 2.0 g, TD - 1.0 g, CPC - 0.045 g. In the kinetics experiments, the amount of DDM added was 0.06 g. The hot agarose solution was mixed with TD, CPC, and DDM in a plastic vial. After intensive shaking, the mixture was ultrasound treated using “Ultraschall” 450-D installation (Branson, Germany). An intermittent treatment regime was applied: 10 s long periods of exposure to the ultrasound were alternated with 5 s long cooling periods. The emulsion thus produced was formed into gel between two hot glasses with a 0.10 mm thick spacer between them. After cooling to room temperature, the film of composite agarose hydrogel with a thickness of 0.10-0.12 mm was obtained. The volume fraction of TD VTD in agarose hydrogel was 0.39. The kinetic experiments of the ester hydrolysis were performed under stirring of the solution (100 rpm) with a piece of the gel film in 0.01 M Tris-HCl buffer solution at pH 7.8 and T ) 25 °C. During the kinetics experiments, the volume ratio r of the solution and gel film was 160. The average density of the composite hydrogel is smaller, 1.0 g/mL, so the gels swim on the surface of aqueous solutions. The esters were introduced into aqueous solutions as 0.01 M solutions in isopropanol. The volume fraction of isopropanol in water was about 1%. During the reaction between an ester and DDM in the composite gel, 4-NPh yields both in the gel and in the solution. At the same time, a small amount of 4-NPh accumulates in the solution due to hydrolyses by the buffer. The quantity of 4-NPh released from the agarose gel film at a
Komarova et al. fixed time t into the solution (MS) plus the quantity of 4-NPh released in the solution due to the reaction with buffer (Mb) at a time t was determined using an UV-spectrophotometer. Then, the gel was removed from buffer solution and placed into a large excess of water acidified with HCl to pH 4.0-4.2. This caused termination of the hydrolysis reaction of esters. Then, 4-NPh was extracted in an acidic environment for 12 h. After the extract was poured into a cuvette, the pH was increased to 8.0 and the molar quantity (ME) of 4-NPh extracted from the film was determined. Adding the quantity of 4-NPh accumulated into the solution due to release from the film and due to spontaneous hydrolyses until time t to the quantity of 4-NPh extracted from the composite hydrogel film after termination of the reaction, we can calculate the averaged rate of the reaction (νa) between the ester and DDM using the formula:
νa ) (MS + ME)/(VSt) - νb
(2)
where VS is the volume of the system and νb is the reaction rate of the spontaneous ester hydrolyses in the absence of gel film. The latter term was determined in a separate experiment before the gel was added in the system. In fact, the term νb does not play any significant role because the values of νb are as much as 102 smaller than that of νa. The values of the effective constants of the second order k2 for the reaction between DDM and esters were calculated by the formula:
k2 ) νa[S]-1[DDM]-1
(3)
where [S] and [DDM] are averaged concentrations of an ester and DDM in the volume of the system. The distribution constants KTD of the esters and CPC between TD and water were determined at pH 5.0 by UV spectrophotometry. Here, KTD ) CTD/CW, where CTD and CW are concentrations of ester in TD and water, respectively. Equal volumes of 0.01 M ester solution in TD and water or 0.01 M solution of CPC in water and TD were equilibrated for this end. After isolation of a known amount of aqueous phase, the concentration of the ester in it was determined after hydrolysis of the ester by alkali. In the case of CPC, its concentration was determined in the phase of TD. The distribution constants KCG of the esters between composite gel film and water were determined by a similar procedure where the gel of known volume was used instead of the TD phase. Here, KCG ) CCG/CW, where CCG and CW are the concentrations of the esters in composite gel and water, respectively. The diffusion coefficients of the esters in the gels were determined on the basis of the measurements of the depth L of the ester penetration in the gel during the known time t. The cylindrical sample of the gel with diameter ∼1 cm was immersed in the solution under study. After a definite time interval, the gel was taken from the solution and cut by the blade. The area containing the substance was visualized using alkali solution. The Deff values were calculated using the formula:
Deff ) L2 /6t
(4)
The size of the emulsion droplets was determined via DLS using the ALV/DLS/SLS-5000 system. The calculations were performed with the system programs. The optical density of
Agarose Hydrogels with Embedded Emulsions
J. Phys. Chem. B, Vol. 113, No. 45, 2009 14851
∆G ) -RT ln K
Figure 1. DLS curves obtained from initial aqueous emulsion diluted with hot 10-4 M solution of CPC (1), emulsion prepared by dilution of hot agarose TD-CPC mixture with hot 10-4 M CPC solution (2), and the emulsion (3) released from the composite gel after its mechanical destruction by a spatula in 10-3 M CPC solution at room temperature. VTD ) 0.39.
TABLE 1: Distribution Constants KTD and KCG and the Changes in Free Energy for the Esters and CPC at 20 °C substance
KTD
∆GTD, kJ/mol
KCGa
∆GCG, kJ/mol
4-NPhA 4-NPhB 4-NPhV 4-NPhC CPC
4.5 78 415 >700 0.018
-3.75 -10.8 -14.9 < -16.2 9.95
19.5 120 490 1900
-7.35 -11.9 -15.4 -18.7
a
r ) 160, VTD ) 0.39.
the solutions was measured using the “HELIOS R” spectrophotometer produced by UNICAM. Results and Discussion The DSL measurements were performed with three kinds of emulsions. The first one was an initial TD-in-water emulsion (VTD ) 0.39) diluted with hot 10-4 M solution of CPC; the second was prepared by dilution of a hot agarose TD-CPC mixture with hot 10-4 M CPC solution; and the third was released from the composite gel after its mechanical destruction by a spatula in 10-3 M CPC solution at room temperature. Figure 1 shows normalized DLS curves measured from the emulsions at 90° and their parameters. The narrowest peak with a maximum at Rh ) 94.9 ( 0.44 nm is obtained from the emulsion obtained in hot agarose solution (Figure 1, curve 2). This is due to the presence of viscous polymer that probably facilitates the formation of the droplets. The droplets of the emulsion obtained in aqueous solution without agarose have the same dimensions (Rh ) 94.6 ( 0.50 nm). The emulsion droplets released from the cooled agarose gel after mechanical treatment have slightly larger dimensions: Rh ) 119.6 ( 0.43 nm. However, the increase in the values of Rh is rather small. The obtained data suggest that the droplet emulsion sizes do not vary much before and after gelation. Taking the last value of Rh, one can calculate that the total square of TD droplets in the composite gel with volume fraction of TD VTD ) 0.39 is about 104 cm2/1 mL of the gel. The distribution constants KTD of the esters and CPC between TD and water were determined at pH 5.0. The distribution constants KCG of the same substances between the composite agarose gel film and water have also been measured. KTD and KCG values are given in Table 1. The free energy values of substance transport from water in TD and from water in the composite agarose gel were calculated from obtained data with the formula:
(5)
Both distribution constants increase markedly with the length of the hydrocarbon residue of the ester. The comparison of the values of the distribution constants shows that the KCG values are larger than those of KTD. Thus, some fraction of esters absorbed by the composite gel film not only is located in dissolved form in TD droplets but probably is adsorbed by agarose helixes or unfolded sections. Direct measurements of the distribution constants of the esters between water and conventional agarose hygrogel film supported this assumption; actually, their values are between 15 and 25. The calculations show that at volume ratio of the solution and gel film r ) 160, the greater part of 4-NPhA is located in the aqueous phase; the amounts of 4-NPhB in water and in the gel phases are close and in the case of 4-NPhV and 4-NPhC the larger part of the ester is located in the TD droplets in the gel. For CPC, the ∆G value is positive and KTD is much lower than unity; i.e., in spite of the long hydrophobic residue, the surfactant is preferentially dissolved in water. The KCG value is a function of the size of oil particles and CPC concentration because the binding of the cationic surfactant depends strongly on the degree of infill of the surface. Here, we will give only one example. If r ) 500 and the surfactant concentration is 8 × 10-6 at equilibrium, just ∼5% of CPC will diffuse out of the gel. It is obvious that the surfactant ions are mostly located on the surface of the TD droplets. It should be noted that the chemical nature of oil plays an essential role in the stability of the composite agarose hydrogel film. If the analogous gel film is prepared using benzene, practically all of the surfactant will diffuse out of it within several minutes (halftime of diffusion is 180 s). UV measurements show that the diffusion of CPC out of the film is accompanied by the diffusion of benzene. Agarose hydrogel used as an emulsion carrier was chosen, in particular, due to its very high water content (98.3%). The diffusion rates of low molecular weight substances in agarose gels are close to that in water.6,17–19 For instance, the time of extraction of half the quantity of 4-NPh anions and CPC cations from a thin agarose film with 0.1 mm thickness into the solution is ∼0.6 and ∼3.0 s, respectively.6 The presence of TD droplets in the composite agarose gel film could be expected to decrease the rate of diffusion of amphiphilic molecules due to their absorption by the droplets and also due to adsorption at their surface. The influence of the hydrophobic residue length of the esters on their penetration rate in the composite gel was studied. The values of effective diffusion coefficient Deff are listed in Table 2. From these data, it follows that amphiphilic molecules of the esters diffuse much slower in the composite gel than in the TD-free agarose gel. The decrease of Deff values is more pronounced for the esters with a longer hydrophobic residue. It is explained by the adsorption of the lipophilic substances on the TD-water interphase and by their parallel dissolution in TD droplets. It can be expected that for more hydrophobic substances the diffusion rate in the composite gel will be negligibly small because practically all substance will be dissolved in the immobilized emulsion droplets near the gel surface. The volume fraction of TD in the emulsion droplets is another factor that influences the diffusion rate of the organic substances in the composite gels. The data listed in Table 2 show that the Deff values decrease in the range VTD ) 0-39.0. This effect is explained by the decrease of the fraction of free molecules of amphiphilic esters in aqueous agarose phase in the gel. For 4-NPhA, the decrease of the Deff value in the composite gels in comparison with agarose is not large.
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Komarova et al.
TABLE 2: Effective Diffusion Coefficients of Esters Deff × 10-10 m2/s inside the Composite Gels with Different Volume Fractions of TD VTDa VTD 4-NPhA 4-NPhB 4-NPhV 4-NPhC a
0
0.051
0.12
0.19
0.26
0.39
6.6 5.3 5.6 6.3
4.2 1.3 0.4 0.2
3.0 0.6 0.2 0.08
2.8 0.7 0.2 0.01
3.2 0.7 0.18 0.01
2.6 0.5 0.13 0.01
The experimental error was about 20%.
TABLE 3: Apparent νapp and True νa Values of Average Rates, Average Flux j of the Esters in the Composite Agarose Gel, the Decrease of the Ester Concentration in the Solution ∆c/∆t and Effective Values of the Second-Order Rate Constants k2 of the Reactions between DDM and the Esters with Different Lengths of Hydrocarbon Residuesa ester 4-NPhA 4-NPhB 4-NPhV 4-NPhC a
However, this difference increases sharply with the elongation of the hydrophobic residue of the ester. Together with the diffusion of the amphiphilic esters in the phase of the composite gel, we have studied the absorption rate of the esters by 0.1 mm composite gel films with a known square. Figure 2 shows the uptake rates of the esters with a concentration of 10-4 M by the composite gel film at the ratio r ) 160. After the initial jump, the concentrations of the esters monotonically decrease. The uptake rate of the esters increases with the length of the hydrophobic residue. The exclusion is 4-NPhA whose concentration only slightly changes during the observation time. From the data listed in Figure 2, the average flux j of the esters into the film in the definite time interval can be calculated. The j values are listed in Table 3. These values are the threshold of the rates of the chemical reactions of the esters with the substances entrapped into the gel. Figure 3 shows the kinetic curves of 4-NPh accumulation in the ester-containing buffer solutions in the absence (clear signs) and in the presence (filled signs) of the composite gel in the system. The observed rates in the absence of the gel νb are approximately 10-10 M/s, while the initial rates averaged over the total volume of the system νapp are approximately 10-8 M/s. However, we have found that the larger part of 4-NPh ME (formula 1) yielded during the reaction remains in the gel film due to interactions with the cationic surfactant. The measurements have shown that the quantity of 4-NPh remaining in the composite agarose film after 20 min of reaction is 2.5-3.0 times higher than that exuded into the external solution. This input in the overall reaction rate was accounted in the calculations of the total values of the averaged reaction rates νa. Table 3 shows the νa values and effective constants of the second order k2 for the reactions of DDM with the esters differing in hydrophobicity (formulas 2 and 3). The k2 value for 4-NPhA obtained in this work is as much as 60 times higher than the calculated value of the second order rate constant of the reaction between DMH and 4-NPhA at pH 8.0.13
Figure 2. Dependences of the concentrations of 4-NPhA (1), 4-NPhB (2), 4-NPhV (3), and 4-NPhC (4) in buffer solution on time in the presence of composite gel film (thickness is 0.1 mm). VTD ) 0.39, T ) 25 °C, pH 4.1, r ) 160. The initial concentration of the esters was 1 × 10-4 M.
k2, j × 10-11, ∆c/∆t × 10-8, νapp × 10-8, νa × 10-8, mol/L · s mol/L · s mol/L · s mol/s · cm2 mol/L · s 1.15 1.15 0.8 0.9
4.3 3.8 3.2 2.15
0.65 0.60 0.50 0.35
2.0 2.7 3.0
0.18 2.65 3.55 4.00
The time of observation was 20 min.
The data listed in Table 3 shows that the values of the reaction rates νa and of the amounts of the esters absorbed by the composite gels during the same time interval ∆c/∆t are rather close (Table 3). This result can be explained by the fact that in the region of low concentrations of the esters (low because of their poor solubility in water, ∼10-4 M) the chemical reaction rate νa is limited by the flux j of the esters into the composite agarose gel film. The exclusion is 4-NPhA for which the absorption is small and the ester preferentially remains in the aqueous phase. In spite of this, the reaction rate for 4-NPhA is higher than that for the other esters. The explanation of this result is that the reaction on the surface of the gel film gives a significant input in overall rate. In the case of micelle catalysis, the increase of the ester hydrophobicity leads to the double increase of the reaction rate between solubilized DDM and the esters.6 For the composite agarose gel, the elongation of the hydrocarbon residue leads to a certain decrease of the reaction rate. The latter effect can be attributed to the increase of the fraction of nonreactive ester hidden in the oil droplets with the functional groups exposed in the TD phase. When the solution is diluted, weakly hydrophobic esters shall predominantly remain in the solution and the relative rate of their conversion should become lower. In fact, when the external solution of the ester is diluted by a factor of 3, the rate of ester molecule conversion in the system is decreased stronger for 4-NPhA than for 4-NPhC. In the latter case, the reaction rate for 4-NPhC is almost twice as high as that for 4-NPhA. It is well-known that mercapto groups bind effectively heavy metal ions, e.g., Hg2+ ions. If the composite hydrogel incorporating DDM is immersed in Hg2+ solution, then it absorbs mercury ions. Mercury ion absorption by composite gel film was detected through the kinetics method, namely, by measuring the decomposition rate of 4-NPhC in the presence of the gel film in the solution containing Hg2+ ions and in the control
Figure 3. Kinetics of 4-NPh-anion release into DDM-free buffer solution in the absence (clear signs) and in the presence (filled signs) of the DDM containing composite gel for 4-NPhA (squares), 4-NPhB (circles), 4-NPhV (triangles), and 4-NPhC (diamonds).
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J. Phys. Chem. B, Vol. 113, No. 45, 2009 14853 apparently initially rich in surfactant cations and DDM anions. At the same time, the greater part of the ester and DDM molecules are hidden in the TD bulk and cannot take part in the reaction. Detailed study of such objects can be performed using theoretical approaches, and also via computer simulation, where important results have been achieved recently.20,21
Figure 4. The release kinetics of 4-NPh anions from the composite gel film in the presence (1) and in the absence (2) of Hg2+ ions. Averaged concentrations of mercury ions, DDM, and 4-NPhC in the system were 3.45 × 10-4, 2 × 10-4, and 10-4 M, respectively. VTD ) 0.39, T ) 25 °C, pH 8.0, r ) 160. D400 ) optical density of 4-NPh anions at 400 nm.
solution free of mercury ions. Figure 4 shows the dependencies of 4-NPh anions released into the solution during the reaction of 4-NPhC in the composite gel film in the cases of the presence (curve 1) and absence (curve 2) of Hg2+. As may be seen from Figure 4, the hydrolysis rate of 4-NPhC in the presence of Hg2+ ions is negligibly small in comparison with mercury-free solution. This is due to the fact that Hg2+ ions bind with SH groups of DDM. Thus, the composite systems obtained are of undoubted practical interest as adsorbents of heavy metal ions, e.g., for their rapid removal in the case of acute poisoning or for their accumulation from water solutions. Conclusions Hydrogels with embedded emulsions are new perspective systems that can be used for concentrating and release various organic compounds, carry chemical, in particular, catalytic functions, and realize chemical syntheses at the oil-water interface. The ease of gel separation from the solutions is an important feature of such systems. Application of physical gels, which can be transferred into polymeric solutions (the similar agarose gels used in the present study), provides additional possibilities for separating the reaction products after synthesis or catalysis performed with the use of amphiphilic compounds. The composite hydrogels filled with emulsions stabilized by surfactants and containing reagents are complex, multifunctional dynamic systems. Controlling the composition of the nearsurface layer of the droplets can be one of the ways to enhance the efficiency of the micro- and nanoreactors based on composite gels. This layer has a complex composition and in our case includes surfactant ions, their counterions, DDM in anionic and neutral forms, 4-NPh, the polar groups of amphiphilic ester, and the anions of the yielded acids. The surface layer is
Acknowledgment. We would like to thank Dr. Inessa V. Blagodatskikh for carrying out DLS experiments. The work was supported by the Russian Foundation for Basic Research (Project No. 07-03-00396), Federal Target-Oriented Program “Research and scientific-pedagogical staff of innovational Russia” (Government contract No. 02.740.11.5078) and in the framework of the German Research Society (DFG) within the Program SPP 1259 “Intelligent Hydrogels.” References and Notes (1) Komarova, G. A.; Starodubtsev, S. G.; Khokhlov, A. R. Dokl. Phys. Chem. 2007, 416, 253. (2) Starodoubtsev, S. G.; Khokhlov, A. R. Macromolecules 2004, 37, 2004. (3) Komarova, G. A.; Starodoubtsev, S. G.; Khokhlov, A. R. Macromol. Chem. Phys. 2005, 206, 1752. (4) Nagao, M.; Okabe, S.; Shibayama, M. J. Chem. Phys. 2005, 123, 144909. (5) Tokuyama, H.; Kanehara, A. Langmuir 2007, 23, 11246. (6) Komarova, G. A.; Starodubtsev, S. G.; Lozinsky, V. I.; Kalinina, E. V.; Landfester, E.; Khokhlov, A. R. Langmuir 2008, 24, 4467. (7) Voet, D.; Voet, J. G.; Pratt, C. W. Fundamentals of Biochemistry: Life at the Molecular LeVel, 2nd ed.; John Wiley & Sons: New York, 2006; p 284. (8) Klotz, I. M.; Suh, J. In EVolution of Synthetic Polymers with Enzyme-like Catalytic ActiVities in Artificial Enzymes; Breslow, R., Ed.; Wiley: Weinheim, Germany, 2005; p 63. (9) Fendler, E. J.; Fendler, J. H. AdV. Phys. Org. Chem. Res. 1970, 8, 271. (10) Berezin, I. V.; Martinek, K.; Yatsimirskii, A. K. Russ. Chem. ReV. 1973, 42, 787. (11) Martinek, K.; Yatsimirsky, A. K.; Levashov, A. V.; Berezin, I. V. In Micellization, Solubilization and Microemulsions; Mittal, K. L., Eds.; Plenum: New York, 1977; Vol. 2, p 489. (12) Murakami, Y.; Nakano, A.; Matsumoto, K. Bull. Chem. Soc. Jpn. 1979, 52, 2996. (13) Overberger, C. G.; Pacansky, T. J.; Lee, J.; Pierre, T.St.; Yaroslavsky, S. J. Polym. Sci., Polym. Symp. 1974, 46, 209. (14) Starodubtsev, S. G.; Kirsh, Yu.E.; Kabanov, V. A. Dokl. Akad. Nauk SSSR 1976, 227, 156. (15) Starodubtsev, S. G.; Kabanov, V. A. Vysokomol. Soedin. 1977, A19, 1948. (16) Torchinsky, Y. M. Sulfur in Proteins; Pergamon: Oxford, U.K., 1981; p 11. (17) Longsworth, L. J. Phys. Chem. 1954, 58, 770. (18) Gosting, L. J.; Morris, M. S. J. Am. Chem. Soc. 1949, 71, 1998. (19) White, M. L.; Dorion, G. H. J. Polym. Sci. 1961, 55, 731. (20) Vasilevskaya, V. V.; Aerov, A. A.; Khokhlov, A. R. Dokl. Phys. Chem. 2004, 398, 258. (21) Vasilevskaya, V. V.; Aerov, F. A.; Khokhlov, A. R. Colloid Polym. Sci. 2006, 284, 459.
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