Thermogelling Chitosan-g-(PAF-PEG) Aqueous Solution As an

May 20, 2012 - ... grafted chitosan (CS-g-(PAF-PEG)) system, focusing on phase diagram, .... Real-time and non-invasive fluorescence tracking of in vi...
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Thermogelling Chitosan-g-(PAF-PEG) Aqueous Solution As an Injectable Scaffold Eun Young Kang, Hyo Jung Moon, Min Kyung Joo, and Byeongmoon Jeong* Department of Bioinspired Science (WCU), Department of Chemistry and Nano Science, Ewha Womans University, 52, Ewhayeodae-gil, Seodaemun-gu, Seoul, 120-750, Korea ABSTRACT: The present study reports on a thermogelling poly(ethylene glycol)-poly(L-alanine-co-L-phenyl alanine) grafted chitosan (CS-g-(PAF-PEG)) system, focusing on phase diagram, transition mechanism, and in vivo gel duration. The sol-to-gel transition temperature decreased from 27 to 11 °C as the concentration increased from 4.0 wt % to 9.0 wt %. The polymer formed micelles with 10−50 nm in diameter at 10 °C and formed large aggregates ranging from hundreds to thousands of nanometers in size as the temperature increased from 10 to 35 °C, suggesting that an extensive molecular aggregation might be involved in the sol-to-gel transition. To study the transition mechanism on a molecular level, we investigated pH, circular dichroism spectra, and 13C NMR spectra of the CS-g(PAF-PEG) aqueous solution as a function of temperature. As the temperature increased, deprotonation of the chitosan and dehydration of the PEG were suggested, whereas the α-helical secondary structure of PAF was slightly changed in the sol-to-gel transition temperature range of 10−50 °C. A gel was formed in situ after injecting the CS-g-(PAF-PEG) aqueous solution into the subcutaneous layer of rats. About 60−70% of the gel was eliminated in 1 week, and the remaining gel was completely cleared from the implant site in 14 days. The results indicate the potential of CS-g-(PAF-PEG) as a promising short-term carrier for pharmaceutical agents.

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polypeptide, and stereochemistry of the amino acids.17−20 The self-assembly, the sol−gel phase diagram, and the biodegradation of the polymer could be controlled by varying these molecular parameters. In particular, in vivo gel duration of the implant could be modulated over 1 week to 1 month. Our recent study on PEG-polyalanine grafted chitosan (CS-g-(PAPEG)) aqueous solutions focused on a pH/temperature dual sensitivity and reported the pH-dependent gelation and nanoassemblies at a specific concentration.7 As a continuation of the research on chitosan grafted polypeptide-based thermogelling systems, the present study describes a poly(ethylene glycol)-poly(L-alanine-co-L-phenyl alanine) grafted chitosan (CS-g-(PAF-PEG)) aqueous solution. In this study, phenyl alanine was incorporated in the polypeptide to strengthen the hydrophobicity of the polypeptide. The sol−gel phase diagram over whole concentration ranges, molecular assemblies, and the mechanism of thermogelation were investigated using dynamic mechanical analysis, 1H and 13C NMR spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy. To assess the feasibility as an injectable in situ gelling biomaterial, we also studied the gel duration and tissue compatibility of the system after subcutaneous injection of the CS-g-(PAF-PEG) aqueous solution into rats.

INTRODUCTION Chitosan is widely used as dietary supplements, bandage materials, food additives, and base materials in cosmetics. Chitosan has also been investigated as a biomaterial due to its (1) natural abundance as a chitin in crustacean shells, (2) ease of modification on the pendant amino groups, and (3) biological activities including antifungal, antimicrobial, and antidiabetic activities.1−4 However, its parenteral applications as a pharmaceutical excipient are not approved by the Food and Drug Administration and are still under investigation.1 Since the pioneering research on a thermogelling chitosan/ glycerol phosphate aqueous system, poly(ethylene glycol) (PEG) grafted chitosan, PEG-polyalanine grafted chitosan, and PEG-poly(propylene glycol)-PEG (Poloxamer) grafted chitosan have been developed as a thermogelling chitosan derivatives.5−8 Thermogelling systems are low viscous polymer aqueous solutions at low temperatures; however, they undergo sol-to-gel transition as the temperature increases.9−13 The gel depot can be formed in situ by injection of the polymer aqueous solution into a target site, typically in the subcutaneous layer of a warm-blooded mammal, where the pharmaceutical agents-containing gel depot acts as a reservoir of drugs or a cells. In addition to sustained drug delivery, thermogelling systems have begun to be applied for injectable tissue engineering, 3-D cell culture, and prevention of adhesion after surgical operation.14−16 We reported a series of thermogelling PEG-polypeptide systems by varying the composition of the polypeptide, molecular weights of the PEG, molecular weights of the © 2012 American Chemical Society

Received: January 17, 2012 Revised: May 14, 2012 Published: May 20, 2012 1750

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dyn/cm2) and a frequency of 1.0 rad/s. The heating rate was 0.5 °C/ min. The frequency dependency of the modulus of a polymer aqueous solution (6.0 wt %) was investigated by the frequency sweep test in sol (10 °C) and gel (37 °C) states. The sample was equilibrated for 20 min at each temperature in a water-saturated chamber before measurements. The frequency was varied from 0.1 to 10 rad/s. Strain amplitude of 0.2% was used for the dynamic mechanical analysis. Transmission Electron Microscopy. The CS-g-(PAF-PEG) aqueous solution (0.1 wt %, 10 μL) was placed on the carbon grid, and the excess solution was blotted with filter paper. The grids were air-dried at room temperature for 24 h. The microscopy image of the polymer was obtained by a JEM-2100F microscope (JEOL, Japan) with an accelerating voltage of 200 kV. Dynamic Light Scattering. The apparent size of CS-g-(PAFPEG) in water (0.01 wt %) was studied by a DLS instrument (ALV 5000−60 × 0) as a function of temperature. The polymer aqueous solution was equilibrated for 20 min at each temperature. A YAG DPSS-200 laser (Langen, Germany) operating at 532 nm was used as a light source. The results of DLS were analyzed by the regularized CONTIN method. The decay rate distributions were transformed to an apparent diffusion coefficient. From the diffusion coefficient, the apparent hydrodynamic size of the polymer could be obtained by the Stokes−Einstein equation. Change in pH of CS-g-(PAF-PEG) Aqueous Solution. Changes in pH of CS-g-(PAF-PEG) aqueous solutions (1.0 wt %) were investigated as a function of temperature after adjusting the pH to about 7.3 at 10 °C. A pH meter was calibrated by standard solutions at pH 4.0, 7.0, and 10.0. Nitrogen gas was purged for 20 min, and the pH of polymer aqueous solution was measured with increasing and decreasing temperature at an increment/decrement of 1 °C under the nitrogen atmosphere, whereas the pH meter was being immersed. As a comparison, the change in pH of phosphate buffer (10 mM) (control) was also investigated as a function of temperature. Circular Dichroism Spectroscopy. A CD spectrophotometer (J810, JASCO) was used to study the ellipticity of the CS-g-(PAF-PEG) aqueous solution as a function of polymer concentration at a fixed temperature of 15 °C in a polymer concentration range of 0.01 to 0.10 wt %. In addition, ellipticity of the PEG-PAF aqueous solution was obtained as a function of temperature at a fixed concentration of 0.01 wt % in a range of 10−50 °C at increments of 10 °C per step. The aqueous solution was equilibrated for 20 min at each temperature. In Situ Gel Formation and Gel Duration. Aqueous solutions (6.0 wt %) of CS-g-(PAF-PEG) (0.5 mL/rat) in prefilled syringes were injected into the subcutaneous layer of rats using a 25 gauge syringe needle. Rats were sacrificed 6 h (0 day), 1 day, 7 days, and 14 days postinjection to study the in vivo duration of the gel. For the in vitro study, water (3.0 mL; 37 °C) was added on top of the preformed gel (0.5 mL) in a vial with an inner diameter of 11 mm, and the vial was incubated in a shaking bath with 90 strokes/minute at 37 °C for 14 days. Tissue Compatibility. The histology around the implant site was investigated 7 days after implantation of the CS-g-(PAF-PEG) gel. Neutral buffered formalin (NBF) solution was prepared by mixing formaldehyde solution (3 −40%; 100 mL), sodium phosphate monobasic (4.0 g), sodium phosphate dibasic (6.5 g), and deionized water (900 mL). The tissue surrounding the gel was stored in NBF solution at −20 °C. After 24 h, the tissue was embedded in a frozen section compound (Leica, USA) and stored at −80 °C. Microcryotomed sections of tissue with a thickness of ∼8 μm were stained with hematoxylin and eosin (H&E) and examined using a microscope (Olympus lX71-F22PM, Japan). Animal Procedures. All experimental procedures using animals were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Committee of Ewha Womans University.

EXPERIMENTAL SECTION

Materials. α-Amino-ω-methoxy-poly(ethylene glycol) (PEG) (Mn ≈ 2000 Da; ID Bio, Korea), N-carboxy anhydrides of L-alanine, Ncarboxy anhydrides of L-phenyl alanine (KPX Life Science, Korea), and chitosan (MW ∼ 7500 Da purified by dialysis membranes with cutoff molecular weights of 5000 and 10 000 Da; Kitto Life, Korea) were used as received. Succinic anhydride, succinic acid, N-hydroxy succinic imide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carboimide hydrochloride (EDC), anhydrous N,N-dimethyl formamide, and anhydrous dimethyl sulfoxide were used as received from Sigma-Aldrich. Toluene and chloroform were dried before use. Synthesis. PEG-PAF diblock copolymers were synthesized by ringopening copolymerization of the N-carboxy anhydrides of L-alanine and the N-carboxy anhydrides of L-phenyl alanine in the presence of αamino-ω-methoxy-poly(ethylene glycol) (PEG).17 The polymer was purified by fractional precipitation into diethyl ether, followed by evaporation of the residual solvent under vacuum. The yield was ∼75%. The amino end group of PEG-PAF was converted to carboxylic acid by reacting with succinic anhydride. The PEG-PAF (2000−1430 Da in each block, 2.60 g; 0.76 mmol) and anhydrous chloroform (40 mL) were added to the flask. Succinic anhydride (0.25 g; 2.50 mmol) were added to the flask, and the reaction mixtures were stirred for 24 h at room temperature. The reaction product was precipitated into diethyl ether. The residual solvent was removed under vacuum. The carboxylic acid-terminated PEG-PAF was dialyzed using a membrane with a cut off molecular weight of 2000 Da, followed by lyophilization. The yield was ∼82%. NHS (0.113 g, 0.98 mmol) was added to the carboxylic acidterminated PEG-PAF (2.15 g, 0.61 mmol) aqueous solution (40 mL) and stirred for 15 min at 0 °C. EDC (0.152 g, 0.98 mmol) was added to the reaction flask and stirred for 4 h at 0 °C. Then, chitosan (0.141 g, 0.019 mmol) dissolved in deionized water (20 mL) was dropped into the reaction flask over 1 h at 0 °C. The reaction mixtures were stirred for 72 h at room temperature (20 °C) and were then dialyzed using a membrane with a cut off molecular weight of 8,000 Da to separate the CS-g-(PAF-PEG), followed by lyophilzation to a powder form. The yield was ∼61%. 1 H- and 13C NMR Spectroscopy. 1H NMR spectra of polymers were obtained using a 500 MHz NMR spectrophotometer (Varian, USA) to determine the composition or molecular weight of chitosan (D2O), PEG-PAF (CF3COOD), and CS-g-(PAF-PEG) (CF3COOD). In addition, to confirm the core−shell structure of CS-g-(PAF-PEG) in water, we compared 1H NMR spectra of the polymer in D2O and CF3COOD at 15 °C. 13C NMR spectra of a CS-g-(PAF-PEG) aqueous solution (6.0 wt % in D2O) were studied as a function of temperature to investigate conformational changes of the polymer during the solto-gel transition. The samples were equilibrated for 20 min at each temperature. Gel Permeation Chromatography (GPC). A Waters 515 GPC system with a Waters 410 refractive index detector was used to obtain the molecular weights and molecular weight distributions of the polymers. A water/acetonitrile (80/20 v/v) cosolvent was used as an eluting solvent and GPC columns of Ultrahydrogel linear and Ultrahydrogel 120 (Waters, USA) were used in series. Phase Diagram. The sol−gel transition of the polymer aqueous solution was investigated by the test tube inverting method. The aqueous polymer solution (1.0 mL) was added to a vial with an inner diameter of 11 mm. The transition temperature was determined by the flow (sol)-non flow (gel) criterion with a temperature increment of 1 °C per step. Each data point is an average of three measurements. Dynamic Mechanical Analysis. Changes in modulus of the CS-g(PAF-PEG) aqueous solutions (6.0 wt %) were investigated by dynamic rheometry (Thermo Haake, Rheometer RS 1). The aqueous polymer solution was placed between parallel plates of 25 mm diameter with a gap of 0.5 mm. To minimize water evaporation during the experiment, we enclosed the plates in a water-saturated chamber. The data were collected under conditions of controlled stress (4.0 1751

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Scheme 1. Synthesis of CS-g-PAF-PEG. x + y = m, p = 45

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RESULTS AND DISCUSSION The synthetic procedures of CS-g-(PAF-PEG) are presented in Scheme 1. First, the PEG-PAF diblock copolymer was prepared by the ring-opening copolymerization of N-carboxy anhydrides of L-alanine and N-carboxy anhydrides of L-phenyl alanine on the α-amino-ω-methoxy-poly(ethylene glycol) (PEG).17 Then, PEG-PAF was reacted with succinic anhydrides to form carboxylic acid terminated PEG-PAF (PEG-PAF-COOH). Finally, CS-g-(PAF-PEG) was synthesized by a coupling reaction between chitosan and carboxylic acid-terminated PEG-PAF using NHS and EDC as coupling agents. 1 H NMR spectra of chitosan (in D2O), PEG-PAF (in CF3COOD), and CS-g-(PAF-PEG) (in CF3COOD) showed the progress of the reaction (Figure 1a). The composition of chitosan was calculated by 1H NMR spectra of chitosan. The peak at 2.0 to 2.2 ppm came from the acetyl group of chitosan. The peaks at 3.0 to 3.2 ppm came from C5 (1H) of both sugar rings and C3 (1H) of the deacetylated sugar-ring. The peaks at 3.3 to 4.2 ppm came from protons of C3 (1H), C4 (1H), C6 (1H), and C7 (2H) of the acetylated sugar-ring and C4 (1H), C6 (1H) and C7 (2H) of the deacetylated sugar-ring.21 The C2 (1H) peak of acetylated sugar-ring appeared at 5.4 to 5.5 ppm and the C2 (1H) peak of deacetylated sugar-ring overlapped

with the solvent peak at 4.8 to 5.0 ppm. The numbering of C1− C7 in the repeating unit of chitosan is shown in Scheme 1. The degree of deacetylation of chitosan (m/(n + m) in Scheme 1 calculated by the following equation was 90%. 3n/(5m + 4n) = A 2.0 − 2.2 /A3.3 − 4.2

(1)

where A2.0−2.2 and A3.2−4.2 are the areas under the peak at 2.0 to 2.2 ppm and 3.3 to 4.2 ppm of chitosan, respectively. The molecular weight of each block of PEG-PAF calculated by the peaks at 1.4 to 1.9 ppm (CH3CONH− of PAF), 3.6 to 3.7 ppm (−OCH3 of PEG end group), 3.8 − 4.2 ppm (−OCH2CH2− of PEG), and 7.1 to 7.5 ppm (C6H5CH2CONH− of PAF) was 2000−1430 Da with the structure of (ethylene glycol)45-[(L-alanine)12(L-phenyl alanine)4]. Interestingly, the end groups of alanine (1.7 to 1.9 ppm) and phenyl alanine (7.4 to 7.5 ppm) of PEG-PAF could be distinguished from internal alanine (1.4 to 1.7 ppm) and phenyl alanine (7.1 to 7.4 ppm) in the 1H NMR spectra. The end-group peaks were incorporated into internal alanine (1.4 to 1.7 ppm) and phenyl alanine (7.1 to 7.4 ppm) peaks after conjugating the PEG-PAF to chitosan, as shown in the 1H NMR spectra of CS-g-(PAF-PEG). 1 H NMR spectra of CS-g-(PAF-PEG) revealed the acetyl group of chitosan (2.0 to 2.2 ppm), PEG (3.8 to 4.2 ppm), 1752

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weight of the sphere (γs), the specific weight of the fluid (γf), the diameter of the sphere (D), and the velocity of the sphere (υ) by the following equation26−28 μ = (γs − γf )D2 /(18υ)

where γs = ρsg, γf = ρfg, and υ is the travel distance/travel time. g is a gravitational acceleration of 980 cm/s2. ρs and ρf are the density of sphere and fluid, respectively. As described in our previous paper,29 the sol-to-gel transition temperature was defined by a temperature at which a steep increase in dynamic viscosity was observed. Dynamic mechanical analysis measures storage modulus (G′) and loss modulus (G″) of a system as a function of temperature at a fixed frequency (stress-controlled test) or as a function of oscillation frequency at a fixed temperature (frequency sweep test). The geometry and size of the plate should be carefully selected in the dynamic mechanical analysis for non-Newtonian fluids. A slip film can be formed for coarse slurries at the rotor face if parallel plates are used. In our system, the polymer aqueous solution was placed between parallel plates of 25 mm diameter with a gap of 0.5 mm. The slip film was not formed during the measurement of our system. G′ and G″ are measures of an elastic component and a viscous component of the system. G′ increased significantly (>10 000 times) during the sol-to-gel transition. Therefore, the transition temperature was reproducibly measured by the dynamic mechanical analysis. In addition, G′ is smaller than G″ in a sol state, whereas G′ is greater than G″ in the gel state.30,31 The crossing point of G′ over G″ was suggested as the sol-to-gel transition temperature.32,33 The solto-gel transition temperatures determined by the above three methods fell within ±2 °C for thermogelling PEG/PLGA aqueous systems.29 Therefore, the phase diagram of the thermogelling CS-g-(PAF-PEG) aqueous system was determined by the test tube inverting method by the flow (sol) or nonflow (gel) criterion in a reproducible manner.34,35 The solto-gel transition temperature decreased from 27 to 11 °C as the polymer concentration increased from 4.0 to 9.0 wt % (Figure 2a). Below 4.0 wt %, an increase in viscosity was observed as the temperature increased; however, the gel was not rigid enough to resist the flow when the test tube was inverted. Above 9.0 wt %, the system existed as a gel in the investigated temperature range of 0−70 °C. The sol-to-gel transition was clearly demonstrated by changes in modulus of the system (Figure 2b). The modulus of a CS-g-(PAF-PEG) aqueous solution (6.0 wt %) increased from 3.0 × 10−3 to 128 Pa as the temperature increased from 10 to 37 °C. In addition, the crossing of storage modulus (G′) over loss modulus (G″) was observed as the temperature increased.36,37 As a comparison, an aqueous solution of PEG (MW = 2000 Da) with the same concentration (6.0 wt %) did not show significant changes in G′ and G″. In addition, G′ was smaller than G″, indicating a sol state of the PEG aqueous solution over the experimental temperature range. The frequency sweep test showed that the sol and gel phases of the CS-g-(PAF-PEG) aqueous solution were characterized by fluid-like behavior and solid-like behavior, respectively (Figure 2c). At 10 °C (sol), G′ and G″ of the polymer aqueous solution (6.0 wt %) were proportional to ω2.1 and ω1.1, respectively, indicating a typical viscoelastic fluid-like phase of the sol.23,38,39 In addition, G″ was greater than G′ at 10 °C; therefore, the viscous component overwhelmed the elastic component in the sol phase. G′ was greater than G″ by one

Figure 1. (a) 1H NMR spectra of chitosan (D2O), PEG-PAF (CF3 COOD), and CS-g-(PAF-PEG) (CF3 COOD). (b) GPC chromatogram of chitosan, PEG-PAF and CS-g-(PAF-PEG). Acetonitrile/water (80/20) (v/v) was used as an eluting solvent.

alanine (1.4 to 1.9 ppm), and phenyl alanine (7.1 to 7.5 ppm) moieties. The succinate linked group also appeared at 2.8 to 3.0 ppm in the 1H NMR spectra of CS-g-(PAF-PEG). On the basis of the above assignment, the grafting ratio defined by y/(x + y + n) of CS-g-(PAF-PEG) in Scheme 1 was calculated by the acetyl group of chitosan (2.0 to 2.2 ppm) and methoxy end group of (3.6 to 3.7 ppm) originating from PEG. y/n = A3.6 − 3.7 /A 2.0 − 2.2

(2)

where A2.0−2.2 and A3.6−3.7 are the areas under the peak at 2.0 to 2.2 ppm and 3.6 to 3.7 ppm, respectively. m/(m + n) = 0.90, as already calculated for neat chitosan, and m = x + y in Scheme 1. On the basis of the above analysis, the ratio of each block (x/y/ n) of CS-g-(PAF-PEG) in Scheme 1 was 9.5/3.4/1.0, and the grafting ratio was defined by y/(x + y + n) of CS-g-(PAF-PEG) in the Scheme 1 was 24.5%. On the basis of the 7500 Da of chitosan, the final structure of the CS-g-(PAF-PEG) in the Scheme 1 was calculated to be x = 29, y = 10, and n = 3. The GPC of CS-g-(PAF-PEG) in a water/acetonitrile (80/ 20) cosolvent system showed a unimodal distribution of the molecular weight. The interactions between column-packing agents and chitosan delayed the retention time of neat chitosan. The retention time of CS-g-(PAF-PEG) decreased to 8 min after the coupling reaction between PEG-PAF (10 min) and chitosan (10.4 min) (Figure 1b). Thermogelation of a polymer aqueous solution accompanied a significant change in flow properties. Several methods have been suggested to determine the sol−gel transition temperature. The most convenient and widely used method for the determination of the sol-to-gel transition temperature is the test tube inverting method.15,22−25 Sol and gel phases are decided by the flow (sol)-no flow (gel) criterion when a vial containing polymer aqueous solution is inverted. This method is particularly useful when the sol-to-gel transition involves an abrupt change in modulus or viscosity in several orders of magnitude as in the current system. In the falling ball method, dynamic viscosity (μ) can be calculated using the specific 1753

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To show the sol−gel transition of the polymer aqueous solution (6.0 wt %), photos were taken for sol (10 °C) and gel (37 °C) phases (Figure 2d). In addition, to confirm the robustness of the gel for a long period of time, we took photos for the gel incubated in an excess amount of water at 37 °C for 0, 7, and 14 days, where the gel kept its integrity for >14 days (Figure 2e). CS-g-(PAF-PEG) consists of hydrophilic PEG, chitosan, and hydrophobic PAF. The amphiphilic nature of the CS-g-(PAFPEG) could be demonstrated by comparing the 1H NMR spectra in water (D2O) and organic solvent (CF3COOD).43 In D2O, the PEG appeared as a sharp peak at 3.2 to 4.0 ppm, whereas the PAF peak was collapsed and appeared as a shoulder in the enlarged spectra at 7.0 to 7.5 ppm (phenylalanine of PAF) and a small peak at 1.2 to 1.5 ppm (alanine of PAF) (Figure 3a). All of the components consisting CS-g-(PAF-PEG) appeared as sharp peaks in CF3COOD, indicating that CF3COOD is a good solvent for PEG, PAF, and chitosan (CS). Therefore, CF3COOD was used for composi-

Figure 2. (a) Phase diagram of CS-g-(PAF-PEG) aqueous solutions. The sol-to-gel transition temperatures were determined by the test tube inverting method (n = 3). (b) Changes in modulus of the CS-g(PAF-PEG) aqueous solutions (6.0 wt %) as a function of temperature. An aqueous solution (6.0 wt %) of PEG (M.W = 2000 Da) was compared. (c) Frequency sweep test in the sol (10 °C) and gel (37 °C) phases of the polymer aqueous solution (6.0 wt %). (d) Photos showing sol (10 °C) and gel (37 °C) phases of the polymer aqueous solution (6.0 wt %). (e) Photos of the gels incubated in an excess amount of water at 37 °C for 0 (0th D), 7 (7th D), and 14 days (14th D).

order of magnitude at 37 °C (gel). At 37 °C, G′ was nearly independent of frequency, whereas G″ slightly decreased as the frequency increased in the investigated frequency range of 0.1− 10 rad/s. A similar frequency dependence was reported for a series of thermogelling aqueous systems of chitosan/glycerol phosphate, poly(ethylene oxide)-poly(ethoxy tri(ethylene glycol) acrylate-co-nitrobenzyl acrylate), poly(N-isopropyl acrylamide)-polystyrene-poly(ethylene oxide)-polystyrenepoly(N-isopropyl acrylamide), poly(methoxy diethylene glycol methacrylate-co-methacrylic acid)-poly(ethylene oxide)-poly(methoxy diethylene glycol methacrylate-co-methacrylic acid), and organogel nanoemulsion.38,40−42 In the sol state, all of the above systems showed viscous fluid-like behavior with G″ > G′ and a frequency-dependent modulus, whereas in the gel state, G′ > G″ and G′ was independent of the frequency.

Figure 3. (a) Comparison of 1H NMR spectra of CS-g-(PAF-PEG) in D2O and CF3COOD at 15 °C. The spectra were normalized by the PEG peak. (b) TEM images of the CS-g-(PAF-PEG) developed from polymer aqueous solution (0.1 wt %) at 15 °C. (c) Apparent size distribution of CS-g-(PAF-PEG) in water as a function of temperature determined by the dynamic light scattering of CS-g-(PAF-PEG) aqueous solution (0.01 wt %). (d) Enlarged for the small size region (