Hydrogels with β-Cyclodextrin Moi - American Chemical Society

Jan 25, 2007 - β-Cyclodextrin-incorporated poly(N-isopropylacrylamide) hydrogels ...... (13) Marshall, A. J.; Blyth, J.; Davidson, C. A. B.; Lowe, C...
0 downloads 0 Views 440KB Size
Download PDF
Ind. Eng. Chem. Res. 2007, 46, 1511-1518

1511

Thermosensitive Affinity Behavior of Poly(N-isopropylacrylamide) Hydrogels with β-Cyclodextrin Moieties Hai-Dong Wang,† Liang-Yin Chu,*,† Xiao-Qi Yu,‡ Rui Xie,† Mei Yang,† Dan Xu,† Jie Zhang,† and Lin Hu† Schools of Chemical Engineering, and Chemistry, Sichuan UniVersity, Chengdu, Sichuan 610065, China

β-Cyclodextrin-incorporated poly(N-isopropylacrylamide) hydrogels [P(NIPAM-co-CD) gel] were prepared via copolymerization of N-isopropylacrylamide (NIPAM) with mono-(6-N-allylamino-6-deoxy)-β-cyclodextrin (ACD). Using 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) as the guest molecule, an experimental study on the temperature dependence of affinity behavior of P(NIPAM-co-CD) gels and PNIPAM gels with ANS was carried out within a wide range of temperatures (21-60 °C). When the temperature increased from below the lower critical solution temperature (LCST) to above the LCST, both P(NIPAMco-CD) and PNIPAM gels exhibited much more enhanced association constants with ANS than they did at temperatures below the LCST. Moreover, P(NIPAM-co-CD) gels showed greater affinity toward ANS than PNIPAM gels due to the presence of β-cyclodextrin moieties in the polymeric network. Compared with the PNIPAM gel, the P(NIPAM-co-CD) gel exhibited an increased LCST in deionized water and a decreased LCST in dilute aqueous ANS solution. Introduction Environmental stimuli-sensitive hydrogels, with unique and desirable sensitivity to temperature,1-10 pH,11-13 magnetic field,14 as well as light,15 etc., have been extensively studied. Among the thermosensitive smart gels, the poly(N-isopropylacrylamide) hydrogel has been receiving the most attention due to its excellent thermosensitivity, which has a wide application in the fields of controlled drug delivery,1,16-18 separations,2,19,20 enzyme immobilizations,21-23 sensors,24,25 catalysis,26-28 tunable optical systems,29 colloidal arrays,30 and so forth. Moreover, it is an effective approach to fabricate multi-stimuli-sensitive or multifunctional hydrogels by means of introducing functional groups to the polymeric network of poly(N-isopropylacrylamide) hydrogels. Accordingly, combination of N-isopropylacrylamide and other functional groups can bring corresponding functions and/or stimuli-sensitive properties to poly(N-isopropylacrylamide) hydrogel in addition to the traditional thermosensitivity. By virtue of multi-stimuli-sensitivity, extended and even novel applications, which are very promising and of potential use in the future,2,5,31-35 come into being. Cyclodextrins, torus-shaped molecules composed of cyclic R-1,4-oligoglucopyranosides, are host molecules possessing hydrophobic cavities and hydrophilic external surfaces. On account of a series of weak intermolecular forces, such as hydrophobic, dipole-dipole, electrostatic, van der Waals, and hydrogen-bonding interactions, cyclodextrin is able to selectively associate with guest molecules having the similar size with its cavity. Such a unique property renders cyclodextrins as well as its derivatives of valuable use.36-38 Due to the unique molecular recognition ability of cyclodextrin and the interesting solution behavior of PNIPAM which exhibits a drastic reversible phase transition around its lower critical solution temperature (LCST),39 the combination of poly(N-isopropylacrylamide) with cyclodextrin, by means of either * Corresponding author. Tel: +86-28-8546-0682. Fax: +86-288540-4976. E-mail: [email protected]. † School of Chemical Engineering. ‡ School of Chemistry.

covalent bond32,33,40,41 or noncovalent bond,42,43 is becoming increasingly attractive. Some previous studies on cyclodextrincontaining PNIPAM hydrogels mainly dealt with the controlled drug release and/or the temperature/pH sensitivity of the gel.40,41,43 Also, researchers have cast attention on polymer acquired by the combination of NIPAM and cyclodextrin, among which Nozaki et al.32,33 investigated the temperature dependence of PNIPAM-modified β-cyclodextrin in terms of association constant with guest molecules, and found that PNIPAM chain attached to the side arm of cyclodextrin had considerable influence on the association constant of cyclodextrin toward guest molecule, which was ascribed to the steric hindrance that caused by PNIPAM chain. However, to the best of our knowledge, previous investigations on the temperature dependence of affinity behavior of cyclodextrin-containing PNIPAM hydrogels have not been reported, which naturally gave rise to the motif of our work. In the present research, we have successfully prepared β-cyclodextrin-incorporated poly(N-isopropylacrylamide) hydrogel [P(NIPAM-co-CD) gel]. Quantitative study on the thermosensitive inclusion behavior of P(NIPAM-co-CD) gel from the viewpoint of host/guest interaction was one of our main concerns. Meanwhile, the swelling behavior of P(NIPAM-coCD) and PNIPAM gels was investigated in both deionized water and dilute aqueous ANS solution. An apparent temperaturedependent affinity behavior was observed, and evident LCST shift occurred in both deionized water and dilute aqueous ANS solution with regard to P(NIPAM-co-CD) gel. The results obtained might be conducive to the design and construction of a temperature-controlled affinity separation system. Experimental Section Materials. N-Isopropylacrylamide (NIPAM) was kindly provided by Kohjin Co., Ltd., Japan, and purified by recrystallization from a mixed solvent of hexane/acetone and then dried in vacuo at room temperature. β-Cyclodextrin (β-CD) was purchased from Tianjin Bodi Chemicals Co., Ltd., and recrystallized twice from deionized water and then dried at 110 °C for 12 h before use. Mono-6-deoxy-6-(p-tolylsulfonyl)-β-cyclodex-

10.1021/ie061265a CCC: $37.00 © 2007 American Chemical Society Published on Web 01/25/2007

1512

Ind. Eng. Chem. Res., Vol. 46, No. 5, 2007

trin and mono-(6-N-allylamino-6-deoxy)-β-CD (ACD) were synthesized according to the previously reported methods, respectively.44,45 8-Anilino-1-naphthalenesulfonic acid ammonium salt (ANS) was from Aldrich Chemical Co., Inc. Allylamine, p-toluenesulfonyl chloride, ammonium persulphate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), N,N′methylenebis(acrylamide) (BIS), and other reagents were all of analytical grade, and used as received without further purification. Well-deionized water was used in the overall process of the present work. Preparation of P(NIPAM-co-CD) and PNIPAM Hydrogels. The preparation of poly[N-isopropylacrylamide-co-mono(6-N-allylamino-6-deoxy)-β-CD] hydrogel [P(NIPAM-co-CD) gel] and poly(N-isopropylacrylamide) hydrogel (PNIPAM gel) were carried out by means of free radical polymerization. Detailed process was as follows: NIPAM (1000 mg, 8.85 mmol), mono-(6-N-allylamino-6-deoxy)-β-CD (500 mg, 0.43 mmol), and BIS (20 mg, 1.30 × 10-1 mmol) were dissolved in 10 mL of DMF/H2O mixed solvent (1:1, v/v). The solution was then degassed by bubbling nitrogen through it for 10 min. After the addition of APS (50 mg) and TEMED (20 µL) as a redox pair to initiate free radical polymerization, the solution was swiftly transferred to a glass pipe with an inner diameter of 10 mm, the ends of which were then blocked with stoppers. The polymerization proceeded in a water bath thermostated at a temperature of 20 °C for 24 h. The prepared gel was then removed from the glass pipe and immersed in a large quantity of deionized water which was replaced every 8 h for 5 days to thoroughly leach out unreacted monomers and other impurities. The process and feed composition for preparing the PNIPAM gel was exactly the same as that for preparing P(NIPAM-co-CD) gel except that NIPAM was used as the only monomer. The monomers used in the polymerization process and the proposed molecular structure of P(NIPAM-co-CD) and PNIPAM gels are schematically illustrated in Figure 1. Gel specimens were sliced into pieces, freeze-dried, and then subjected to FT-IR characterization and elemental analysis to demonstrate the introduction of ACD onto the poly(N-isopropylacrylamide) polymeric network of the gel and determine the weight content of ACD in the gel, respectively. The FT-IR spectrum was followed on a NICOLET-560 Fourier transform infrared spectrometer using the KBr method. Elemental analysis was operated on a CARLO ERBA 1106 elemental analysis apparatus. Preparation of PNIPAM Linear Polymers. NIPAM (750 mg, 6.64 mmol) was dissolved in 15 mL of tetrahydrofuran (THF). The solution was then heated to 65 °C under a nitrogen atomsphere. After AIBN (15 mg, mmol) was added as initiator, the free radical polymerization was carried out under nitrogen atmosphere at 65 °C for 4 h. The resulting PNIPAM linear polymer was obtained via precipitating the reaction mixture in a large amount of petroleum ether. The precipitate obtained was dissolved in THF and precipitated from petroleum ether again. The purification process was repeated for several times to obtain pure product. The precipitate thus obtained was dried at 40 °C in vacuo for 8 h to give final product (Mn ) 1.1 × 103, Mw/Mn ) 1.26). Gel permeation chromatography (GPC) measurement was carried out on an Agilent 1100 apparatus with a refractive index detector thermal-stated at 35 °C employing tetrahydrofuran (THF) as eluent and polystyrene as standard. The flow rate of THF was 1 mL/min. Morphological Analysis. Freeze-dried specimens of P(NIPAMco-CD) and PNIPAM gels were subjected to morphological

Figure 1. Schematic illustration of molecular structure of monomers and gels: (a) monomers for the preparation of gels; (b) proposed molecular structure of P(NIPAM-co-CD) (x > 0, x′ > 0) and PNIPAM gels (x ) x′ ) 0).

analysis on a scanning electron microscope (JSM-5900LV, JEOL Technics Ltd., Tokyo, Japan). All specimens for SEM observations were sputtered with gold under fixed conditions. Determination of the Temperature Dependence of Equilibrium Swelling Ratio (ESR) of Hydrogels. The equilibrium swelling ratio (ESR) was defined as the ratio of Vt to V20, where Vt and V20 denoted the volume of gel specimen at a given temperature within the temperature range from 20 to 50 °C and that at 20 °C, respectively. The method for the determination of Vt was as follows: the gel sample was immersed in sufficient deionized water and thermostated in water bath at a given temperature to reach swelling/deswelling equilibrium. The changes in dimensions of the hydrogel with temperature were captured using a digital camera which was placed normal to the gel with fixed position and angle. Photos of the gel were then analyzed by graph-processing software to determine their lengths and diameters. Consequently, Vt, the volume of gel at a given temperature, was determined. The ESR measurements were carried out with gel samples immersed in deionized water and dilute aqueous ANS solution ([ANS] ) 1.0 mmol/L), respectively, in order to investigate the influence of β-cyclodextrin/ANS complexation on the LCST behavior of P(NIPAMco-CD) and PNIPAM gels. Differential Scanning Calorimetry (DSC) Measurements. In order to exactly ascertain the LCST behavior of gels, differential scanning calorimetry (DSC) measurements were carried out on a DSC apparatus (model DSC 200 PC, NETZSCH) within the temperature range 20-50 °C with a scan rate of 2 K‚min-1. DSC measurements for all the gel specimens were conducted under identical conditions to guarantee the comparability among different gel samples.

Ind. Eng. Chem. Res., Vol. 46, No. 5, 2007 1513

Figure 2. FT-IR spectra of P(NIPAM-co-CD) and PNIPAM gels: (a) PNIPAM gel; (b) P(NIPAM-co-CD) gel.

Determination of Host/Guest Association Constants as a Function of Temperature. The Benesi-Hildebrand equation was employed to quantitatively describe the interaction between gel sample and ANS at different temperatures. Gel samples were freeze-dried and cut into pieces (10 mm in diameter and 1.5 mm in thickness) which were then immersed in 30 mL of ANS solution with different ANS concentrations (concentration range from 5 × 10-5 to 5 × 10-4 mol/L), each holding the same amount of gel. Afterward, gel samples were left to swell/deswell sufficiently until an equilibrium state while thermostated in a water bath at a given temperature. Variations thus occurred in UV absorbance of ANS solution with different concentrations were monitored on a UV-vis spectrophotometer with wavelength fixed at 360 nm. Then, association constant Ks (L/mol) could be readily obtained from a linearized Benesi-Hildebrand equation. The process mentioned above was repeated at each given temperature to determine the temperature dependence of Ks. To ascertain the effect of temperature on the association constant of PNIPAM and native β-CD with guest molecule ANS, respectively, fluorimetric titration experiments were employed. The fluorescence intensity of ANS in the presence of PNIPAM polymer and native β-CD was followed on a fluorescence spectrophotometer (Hitachi 850 fluorescence spectrophotometer: excitation wavelength, 350 nm; emission wavelength, 420-620 nm). The quartz cell was thermostated by a temperature controller at each given temperature. Results and Discussion Chemical and Morphological Analysis of Hydrogels. Figure 2 exhibits the comparison between the FT-IR spectra of P(NIPAM-co-CD) and PNIPAM hydrogels. Pure PNIPAM hydrogel was found to be characteristic of CdO stretching vibration at the wavelength of 1646 cm-1 and N-H deformation vibration at 1551 cm-1; meanwhile, evident N-H stretching vibration at around 3440 cm-1 was also observed. With regard to P(NIPAM-co-CD) gel, a typical C-O-C stretching vibration at around 1050 cm-1 as well as a conspicuously broadened peak at around 3450 cm-1 (suggesting the introduction of a large amount of hydroxyl groups) was observed, indicating the introduction of β-cyclodextrin. The weight content of ACD in the P(NIPAM-co-CD) gel was determined to be 25.66% through C and N content by means of elemental analysis. Moreover, the structural differences between the two gels were evident through the comparison between SEM images. As shown in

Figure 3, SEM images of P(NIPAM-co-CD) and PNIPAM gels, both cross-section and surface, exhibited porous structures. However, it was conspicuous to note that PNIPAM gel exhibited net-shaped structure while P(NIPAM-co-CD) gel had evident flakelike structure attached to the polymeric network of the hydrogel which might be the result of the introduction of β-cyclodextrin. The existence of β-cyclodextrin moieties on the polymeric network of P(NIPAM-co-CD) gel could be further clearly demonstrated indirectly through ESR and DSC measurements and investigation on the affinity of gel toward guest ANS during the following discussion. Determination of the Temperature Dependence of Equilibrium Swelling Ratio (ESR) of Hydrogels. Poly(N-isopropylacrylamide) is characteristic of its thermosensitive behavior with the LCST around 32 °C. The temperature dependence of ESR of P(NIPAM-co-CD) gel as well as PNIPAM gel in deionized water and dilute aqueous ANS solution ([ANS] ) 1.0 mmol/L) is schematically illustrated in Figure 4. The LCST of gels could be determined by the temperature where a dramatic volume change (a dramatic ESR decrease in Figure 4) began to occur. It was noteworthy and interesting to give a special attention to the LCST behavior of the two gel samples in deionized water and dilute aqueous ANS solution, respectively. Park and Hoffman46 reported a sharp sodium-chloride-induced decrease in the LCST of neutral poly(N-isopropylacrylamide) gel. The influences affecting the LCST behavior of charged poly(N-isopropylacrylamide) gels were also covered by previous researchers attributing such influences to Donnan osmotic pressure,47,48 counterion condensation,49 ionic strength, as well as ionization/deionization of polyelectrolete,50 and so forth. In our work, nearly no variation in LCST occurred with respect to PNIPAM gel either in deionized water or dilute aqueous ANS solution, implying no apparent influence of dilute aqueous ANS solution on the hydrophilic/hydrophobic balance of neutral poly(N-isopropylacrylamide) polymeric network in the present research. In regard to P(NIPAM-co-CD) gel, both deionized water and dilute aqueous ANS solution could give rise to a conspicuous shift in LCST. As presented in Figure 4, an upshift in LCST occurred when deionized water was used, enhancing LCST to the temperature of up to around 35 °C. Not surprisingly, it was attributable to the introduction of β-cyclodextrin. β-Cyclodextrin, with a hydrophilic external surface, was liable to increase the hydrophilicity of the poly(N-isopropylacrylamide) network. Consequently, P(NIPAM-co-CD) gel displayed an uplifted LCST which resulted from the incorporation of ACD. In the case of dilute aqueous ANS solution, it was interesting to notice that the LCST of P(NIPAM-co-CD) gel descended from nearly 35 °C observed in deionized water to around 30 °C, which indicated an augment of hydrophobicity of gel polymeric network. It might be the association of β-cyclodextrin and guest molecule ANS that accounted for the LCST descent. β-Cyclodextrin incorporated in the gel, once exposed to dilute aqueous ANS solution, would associate with ANS to form a host/guest complex. Due to its large size, an ANS molecule could only be partially inserted into the cavity of β-cyclodextrin, with the naphthalene group residing outside. (Owing to large steric hindrance, it is difficult for ANS to complex with β-cyclodextrin through the naphthalene group, which accounts for a much lower association constant compared with that of other guest molecules.51) Therefore, the naphthalene group, which was relatively hydrophobic, was subject to increase the hydrophobicity of the gel network. Accordingly, a more hydrophobic polymeric network would necessarily result in a LCST decrease. The

1514

Ind. Eng. Chem. Res., Vol. 46, No. 5, 2007

Figure 3. SEM images of cross-sections and surfaces of (a, b) P(NIPAM-co-CD) and (c, d) PNIPAM gels.

Figure 5. DSC thermograms of P(NIPAM-co-CD) and PNIPAM gels: (a) P(NIPAM-co-CD) gel in deionized water; (b) PNIPAM gel in deionized water; (c) P(NIPAM-co-CD) gel in dilute aqueous ANS solution; (d) PNIPAM gel in dilute aqueous ANS solution ([ANS] ) 1.0 mmol/L).

Figure 4. Temperature dependence of swelling behaviors of (a) P(NIPAMco-CD) and (b) PNIPAM gels in deionized water (n) and in dilute aqueous ANS solution ([ANS] ) 1.0 mmol/L) (b), respectively.

thermosensitive behavior of the P(NIPAM-co-CD) gel described above indicated that β-cyclodextrin, as well as its complexation with guest molecule ANS, played an important role in the hydrophilic/hydrophobic balance of gel in the solution, which in turn affected the LCST behavior. Differential Scanning Calorimetry (DSC) Measurements. DSC thermograms of gels under investigation were schematically illustrated in Figure 5 to exhibit the endothermic behavior

of gels on heating, which were in agreement with the ESR results in Figure 4. The exact LCST of gels could be determined by the intersection of the baseline and leading edge of the endothermic peak. As can be seen from Figure 5, P(NIPAMco-CD) gel showed increased and decreased LCSTs in deionized water (around 34.4 °C) and dilute aqueous ANS solution (around 30.2 °C), respectively. While the LCST of PNIPAM gel nearly remained the same (32.6 °C in deionized water and 32.2 °C in dilute aqueous ANS solution). The LCST shift of P(NIPAMco-CD) gel was discussed in detail in the above-mentioned ESR measurements, and was attributable to the introduction of ACD and host/guest association. Determination of Host/Guest Association Constants as a Function of Temperature. Taking host/guest association into consideration, the Benesi-Hildebrand equation52 is utilized to determine the association constants of P(NIPAM-co-CD) and PNIPAM hydrogels with ANS at each given temperature through UV spectrophotometry. Assuming a 1:1 stoichiometry, the inclusion complexation of β-cyclodextrin (H) with ANS (G) could be expressed via eq 1, and the association constant Ks is given by eq 2.

Ind. Eng. Chem. Res., Vol. 46, No. 5, 2007 1515 Ks

H + G y\z H‚G Ks )

[H‚G] [H][G]

(1)

P(NIPAM-co-CD) gel

(2)

Under the conditions of the present work, the initial concentration of guest ANS was much greater than that of β-cyclodextrin incorporated in the gel (β-cyclodextrin content was previously given by elemental analysis), i.e., [G]0 . [H]0. In addition, no free β-cyclodextrin molecule existed in ANS solution since β-cyclodextrin was chemically bound to the polymeric network of gel. Therefore, ANS solution was the only contribution to UV absorbance. Thus, any decrease in UV absorbance could be presumably ascribed to the complexation of β-cyclodextrin and ANS. Upon reaching complexation equilibrium, association constant could be determined via eq 2a.

Ks )

[H‚G] [H‚G] ) [H][G] ([H]0 - [H‚G])‚([G]0 - [H‚G])

(2a)

According to the hypothesis made before that [G]0 . [H]0, eq 2a could be further written as the following eq 2b.

Ks )

[H‚G] [H‚G] ) [H][G] ([H]0 - [H‚G])‚[G]0

Table 1. Association Constants for P(NIPAM-co-CD) and PNIPAM Hydrogels with ANS at Different Temperaturesa PNIPAM gel

T (°C)

Ks (M-1)

R2

Ks (M-1)

R2

21 28 30 35 40 50 60

10350 8123 10100 39680 39630 53800c 55000c

0.9915 0.9943 0.9951 0.9852 0.9743 0.9498 0.9367

b b 440 507 822 2650c 3214c

0.9889 0.9807 0.9714 0.9403 0.9278

a Measured by UV spectrophotometry. b No apparent UV variations observed to determine Ks accurately. c Ks cannot be accurately determined due to relatively poor correlation coefficient.

Table 2. Association Constants for PNIPAM Linear Polymer and β-Cyclodextrin with ANS at Different Temperaturesa PNIPAM polymer T (°C)

Ks (M-1)

20 28 35 45 55

b b 35 84 126

β-cyclodextrin

R2

Ks (M-1)

R2

0.9983 0.9976 0.9998

102 112 108 86 61

0.9914 0.9956 0.9996 0.9930 0.9959

a Measured by fluorimetry. b No apparent UV spectrum changes observed to determine Ks accurately.

(2b)

On the basis of the discussion above, the expression shown by eq 3 holds

∆A ) [H‚G]

(3)

where  and ∆A denote molar extinction coefficient of ANS and the change in absorbance of ANS solution upon the complexation with host β-cyclodextrin, respectively. Then, a substitution of eq 3 into eq 2b gives the form of the extended Benesi-Hildebrand equation as shown by eq 4 which could be expressed in another form by eq 5.

[G]0 [H]0[G]0 1 ) + ∆A Ks‚ 

(4)

1 1 1 1 + ‚ ) ∆A Ks[H]0 [G]0 [H]0

(5)

The association constant Ks is readily calculated by the intercept and slope of plot 1/∆A versus 1/[G]0. With regard to the determination of association constants of PNIPAM/ANS as well as β-cyclodextrin/ANS by fluorescence spectrometry, the process is similar to that of the gel. Then, an extended form of the Benesi-Hildebrand equation is presented via eq 6

1 1 1 1 ) + ‚ ∆F [G]0KsR [H]0 [G]0R

(6)

where R signifies the proportionality coefficient, which may be taken as a sensitivity factor for the fluorescence change upon complexation, and ∆F denotes the change in fluorescence intensity of ANS upon the complexation with host molecule (PNIPAM and β-cyclodextrin), respectively. Similarly, the association constant (Ks) is readily determined by the intercept and slope of plot 1/∆F versus 1/[H]0.

Figure 6. Double-reciprocal plots of ∆A vs [ANS] for P(NIPAM-co-CD) gel at temperatures below LCST (21 °C) and above (40 °C).

Equations 5 and 6 are the final forms of equations used in the calculation process, from which association constant Ks could be determined and listed in Tables 1 and 2, respectively. Figure 6 illustrates the linear regression of experimental data by means of eq 5 (plots of 1/∆A vs 1/[G]0) at temperatures below and above LCST (21 and 40 °C), respectively. An evident temperature-sensitive affinity behavior could be readily observed through the comparison between high and low temperatures. Using hydrophobic molecule ANS as fluorescence probe, which is highly sensitive to environmental changes, the variations in fluorescence intensity of ANS in the presence of PNIPAM and natural β-cyclodextrin at 20 and 45 °C during

1516

Ind. Eng. Chem. Res., Vol. 46, No. 5, 2007

Figure 7. Fluorescence spectral changes of ANS (15 µM) upon the regular addition of PNIPAM polymer at (a) 20 °C and (b) 45 °C. [PNIPAM polymer]: 0, 300, 450, 600, 750, and 900 (µM, from the bottom to top). Excitation wavelength: 350 nm.

Figure 8. Fluorescence spectral changes of ANS (15 µM) upon the regular addition of β-cyclodextrin at (a) 20 °C and (b) 45 °C. [β-cyclodextrin]: 0, 300, 450, 600, 750, and 900 (µM, from the bottom to top). Excitation wavelength: 350 nm.

the fluorimetric titration experiments are schematically illustrated in Figures 7 and 8, respectively. Temperature Dependence of Affinity Behavior and Temperature-Induced Shift of Association Sites. With regard to P(NIPAM-co-CD) gel, as shown in Table 1, association constant Ks was relatively low at lower temperatures (below LCST), and rose dramatically when temperature increased above 35 °C. As for PNIPAM gel, association constant Ks was difficult to be accurately determined at low temperatures due to rather weak interaction between gel and ANS molecule. However, similar

to P(NIPAM-co-CD) gel, a marked increase in Ks was observed when temperature rose above LCST. As temperature goes up from below LCST to above, the polymeric network of poly(N-isopropylacrylamide) undergoes a coil-globule transition,53-57 shifting from a hydrophilic state to a hydrophobic state. Therefore, in view of the hydrophobicity of the guest molecule ANS, we made a hypothesis that the hydrophilic/hydrophobic balance of polymeric network of the gel played an important role in the affinity behavior of P(NIPAM-co-CD) and PNIPAM hydrogels. Thereby, a satisfactory elucidation might be reached. Accordingly, it was necessary to ascertain the temperature dependence of the association constants of PNIPAM polymer and β-cyclodextrin toward ANS, respectively. As shown in Figure 7, the increase in fluorescence intensity of ANS upon the regular addition of PNIPAM aqueous solution was nearly undetectable at 20 °C but was considerably enhanced with a blue shift by 40 nm at 45 °C, indicating a sharp hydrophilic/hydrophobic transition with a marked temperature increase across LCST. In contrast, Figure 8 exhibits that the fluorescence intensity of ANS in the presence of β-cyclodextrin was much higher at 20 °C compared with that observed at 45 °C, suggesting that many more ANS molecules resided in the hydrophobic cavities of β-cyclodextrins at 20 °C than at 45 °C. Though utterly opposite in tendency, they were identical in themselves: high fluorescence intensity implied a more hydrophobic environment, and vice versa. As can be seen from Table 2, association constants for PNIPAM/ANS cannot be accurately calculated at temperatures below LCST due to no apparent spectral variations in fluorimetric titration experiments, while when temperature increased above LCST the association constant kept increasing noticeably. At temperatures below the LCST, the hydrophilic PNIPAM had little influence on the fluorescence intensity of probe ANS, while hydrophobic PNIPAM at high temperatures greatly impacted the surroundings of ANS, leading to a considerable increase in fluorescence intensity. The association constants of β-cyclodextrin/ANS maintained a decrease with a temperature increase due to the fact that the β-cyclodextrin/ANS complex was more thermodynamically stable at low temperatures than at high temperatures. Considering the data of association constants listed in Table 2, the hypothesis made previously held that hydrophilic/ hydrophobic balance of polymeric network of the gel played an important role in the affinity behavior. At temperatures below the LCST, the network of P(NIPAM-co-CD) and PNIPAM gels was highly hydrophilic. Therefore, the inclusion toward guest ANS was mostly attributed to β-cyclodextrin with respect to P(NIPAM-co-CD) gel. As temperature went up across LCST, the increase in the hydrophobicity of PNIPAM network gave rise to a much more enhanced affinity toward ANS; meanwhile, with regard to β-cyclodextrin, there was a decrease in association ability with ANS. Thus, at temperatures above LCST, the enhanced affinity toward ANS was attributable to the combination of host/guest complexation and hydrophobic interaction. The results obtained from the current work were contrary to those of Nozaki et al.32,33 In Nozaki’s work, β-cyclodextrin modified with the PNIPAM chain exhibited much higher association constant at low temperatures than at high temperatures, which was attributable to steric hindrance caused by PNIPAM at high temperatures. Current work mainly dealt with hydrogels prepared by copolymerization of poly(N-isopropylacrylamide) and modified β-cyclodextrin. Due to the densely organized polymeric network, it might be the hydrophobic

Ind. Eng. Chem. Res., Vol. 46, No. 5, 2007 1517

interaction and not the steric hindrance that accounted for the temperature dependence of the affinity toward guest molecules. Compared with Nozaki’s work, the density of PNIPAM in the gel might be much higher than that of PNIPAM chains attached to the side arm of β-cyclodextrin. Accordingly, the hydrophilic/ hydrophobic transition of PNIPAM in the gel dominated, and thus, the temperature-dependent affinity of gel toward ANS was, to a large extent, determined by hydrophilic/hydrophobic transition induced by the temperature variation. Along with Ks listed in Table 1 is the correlation coefficient of linear regression of experimental data. As for P(NIPAM-coCD) gel, it was very noteworthy that an excellent curve fitting with correlation coefficient greater than 0.99 was observed at temperatures below LCST, compared with a relatively poor fitting when temperature rose above LCST. The derivation process of the Benesi-Hildebrand equation was based on the 1:1 stoichiometry of host/guest complexation. In our work, at low temperatures β-cyclodextrin made the greatest contribution to the inclusion of ANS and made a 1:1 complexation (a 1:1 stoichiometry was evidenced by excellent linearity shown in Table 2 and previous report58) which met the precondition of the Benesi-Hildebrand equation quite well. Accordingly, a desirable curve fitting was acquired. At high temperatures, hydrophobicity was predominant, and thus, a combination of host/guest association as well as hydrophobic interaction accounted for the increased affinity toward ANS. Therefore, there was a shift of association sites from original β-cyclodextrin to a combination of β-cyclodextrin and PNIPAM polymeric network, which accordingly led to a less desirable curve fitting. As the temperature increased above 50 °C, a further decrease in association ability of β-cyclodextrin with ANS and a further considerable increase in hydrophobicity of PNIPAM network eventually resulted in a poor curve fitting; thus, accurate association constants were not available. Conclusions In the present study, we have successfully prepared P(NIPAMco-CD) hydrogel employing free radical copolymerization of NIPAM and ACD in a mixed solvent of DMF/H2O. Quantitative research on the thermosensitive affinity behavior toward ANS was carried out in terms of the Benesi-Hildebrand equation, revealing that a considerable increase in the affinity of P(NIPAMco-CD) gel was attributable to a drastic increase in the hydrophobicity of gel network with temperature rising across the LCST. Meanwhile, the association site was demonstrated to shift from original β-cyclodextrin at low temperatures to a combination of β-cyclodextrin and the PNIPAM network at high temperatures. Also, it was noteworthy to draw attention to an upshift in the LCST of P(NIPAM-co-CD) gel in deionized water and a downshift in dilute aqueous ANS solution. The former was ascribed to the result of the incorporation of β-cyclodextrin onto the gel network, and the latter to the complexation of β-cyclodextrin and ANS. Such a P(NIPAM-co-CD) system has potential to be applied to the temperature-controlled affinity separation system. Acknowledgment This research was financed by the National Natural Science Foundation of China (50373029), the Fok Ying Tung Education Foundation (91070), and the Specialized Research Fund for the Doctoral Program of Higher Education by the Ministry of Education of China (20040610042). We are also grateful to Kohjin Co., Ltd., Japan, for kindly supplying N-isopropylacryl-

amide. Meanwhile, many thanks go to Mrs. Ping Zou at the Analytic and Testing Center of Sichuan University for her assistance in fluorimetric titration experiments. Literature Cited (1) Xiao, X. C.; Chu, L. Y.; Chen, W. M.; Wang, S.; Xie, R. Preparation of submicrometer-sized monodispersed thermoresponsive core-shell hydrogel microspheres. Langmuir 2004, 20, 5247. (2) Chu, L. Y.; Li, Y.; Zhu, J. H.; Chen, W. M. Negatively thermoresponsive membranes with functional gates driven by zipper-type hydrogenbonding interactions. Angew. Chem., Int. Ed. 2005, 44, 2124. (3) Xiao, X. C.; Chu, L. Y.; Chen, W. M.; Wang, S.; Li, Y. Positively thermo-sensitive monodisperse core-shell microspheres. AdV. Funct. Mater. 2003, 13, 847. (4) Yoshida, R.; Uchida, K.; Kaneko, Y.; Sakai, K.; Kikuchi, A.; Sakurai, Y.; Okano, T. Comb-type grafted hydrogels with rapid de-swelling response to temperature-changes. Nature 1995, 374, 240. (5) Haraguchi, K.; Takehisa, T. Nanocomposite hydrogels: A unique organic-inorganic network structure with extraordinary mechanical, optical, and swelling/de-swelling properties. AdV. Mater. 2002, 14, 1120. (6) Kato, N.; Sakai, Y.; Shibata, S. Wide-range control of deswelling time for thermosensitive poly(N-isopropylacrylamide) gel treated by freezedrying. Macromolecules 2003, 36, 961. (7) Annaka, M.; Tanaka, C.; Nakahira, T.; Sugiyama, M.; Aoyagi, T.; Okano, T. Fluorescence study on the swelling behavior of comb-type grafted poly(N-isopropylacrylamide) hydrogels. Macromolecules 2002, 35, 8173. (8) Zhang, X. Z.; Yang, Y. Y.; Chung, T. S. Effect of mixed solvents on characteristics of poly(N-isopropylacrylamide) gels. Langmuir 2002, 18, 2538. (9) Yoshinari, E.; Furukawa, H.; Horie, K. Fluorescence study on the mechanism of rapid shrinking of grafted poly(N-isopropylacrylamide) gels and semi-IPN gels. Polymer 2005, 46, 7741. (10) Chern, J. M.; Lee, W. F.; Hsieh, M. Y. Preparation and swelling characterization of poly (N-isopropylacrylamide)-based porous hydrogels. J. Appl. Polym. Sci. 2004, 92, 3651. (11) Kokufuta, E.; Wang, B.; Yoshida, R.; Khokhlov, A. R.; Hirata, M. Volume phase transition of polyelectrolyte gels with different charge distributions. Macromolecules 1998, 31, 6878. (12) Chu, L. Y.; Li, Y.; Zhu, J. H.; Wang, H. D.; Liang, Y. J. Control of pore size and permeability of a glucose-responsive gating membrane for insulin delivery. J. Controlled Release 2004, 97, 43. (13) Marshall, A. J.; Blyth, J.; Davidson, C. A. B.; Lowe, C. R. pHsensitive holographic sensors. Anal. Chem. 2003, 75, 4423. (14) Szabo, D.; Szeghy, G.; Zrinyi, M. Shape transition of magnetic field sensitive polymer gels. Macromolecules 1998, 31, 6541. (15) Irie, M.; Miyatake, O.; Uchida, K. Blocked photochromism of diarylethenes. J. Am. Chem. Soc. 1992, 114, 8715. (16) Chu, L. Y.; Park, S. H.; Yamaguchi, T.; Nakao, S. Preparation of micron-sized monodispersed thermoresponsive core-shell microcapsules. Langmuir 2002, 18, 1856. (17) Chu, L. Y.; Park, S. H.; Yamaguchi, T.; Nakao, S. Preparation of thermo-responsive core-shell microcapsules with a porous membrane and poly(N-isopropylacrylamide) gates. J. Membrane Sci. 2001, 192, 27. (18) Chu, L. Y.; Niitsuma, T.; Yamaguchi, T.; Nakao, S. Thermoresponsive transport through porous membranes with grafted PNIPAM gates. AIChE J. 2003, 49, 896. (19) Kanazawa, H.; Kashiwase, Y.; Yamamoto, K.; Matsushima, Y.; Kikuchi, A.; Sakurai, Y.; Okano, T. Temperature-responsive liquid chromatography. 2. Effects of hydrophobic groups in N-isopropylacrylamide copolymer-modified silica. Anal. Chem. 1997, 69, 823. (20) Choi, Y. J.; Yamaguchi, T.; Nakao, S. A novel separation system using porous thermosensitive membranes. Ind. Eng. Chem. Res. 2000, 39, 2491. (21) Ogawa, K.; Wang, B.; Kokufuta, E. Enzyme-regulated microgel collapse for controlled membrane permeability. Langmuir 2001, 17, 4704. (22) Bayhan, M.; Tuncel, A. Uniform poly(N-isopropylacrylamide) gel beads for immobilization of alpha-chymotrypsin. J. Appl. Polym. Sci. 1998, 67, 1127. (23) Shiroya, T.; Tamura, N.; Yasui, M.; Fujimoto, K.; Kawaguchi, H. Enzyme immobilization on thermosensitive hydrogel microspheres. Colloid. Surf., B 1995, 4, 267. (24) Komori, K.; Takada, K.; Tatsuma, T. Toward selectivity control of a heme peptide electrode by modification with a phase-transition polymer. Anal. Sci. 2005, 21, 351. (25) Tatsuma, T.; Saito, K.; Oyama, N. Enzyme electrodes mediated by a thermoshrinking redox polymer. Anal. Chem. 1994, 66, 1002.

1518

Ind. Eng. Chem. Res., Vol. 46, No. 5, 2007

(26) Yamada, Y. M. A.; Tabata, H.; Ichinohe, M.; Takahashi, H.; Ikegami, S. Oxidation of allylic alcohols, amines, and sulfides mediated by assembled triphase catalyst of phosphotungstate and non-cross-linked amphiphilic copolymer. Tetrahedron 2004, 60, 4087. (27) Bergbreiter, D. E.; Case, B. L.; Liu, Y. S.; Caraway, J. W. Poly(N-isopropylacrylamide) soluble polymer supports in catalysis and synthesis. Macromolecules 1998, 31, 6053. (28) Bergbreiter, D. E.; Osburn, P. L.; Wilson, A.; Sink, E. M. Palladiumcatalyzed C-C coupling under thermomorphic conditions. J. Am. Chem. Soc. 2000, 122, 9058. (29) Kim, J.; Serpe, M. J.; Lyon, L. A. Photoswitchable microlens arrays. Angew. Chem., Int. Ed. 2005, 44, 1333. (30) Tsuji, S.; Kawaguchi, H. Self-assembly of poly(N-isopropylacrylamide)-carrying microspheres into two-dimensional colloidal arrays. Langmuir 2005, 21, 2434. (31) Chu, L. Y.; Yamaguchi, T.; Nakao, S. A molecular-recognition microcapsule for environmental stimuli-responsive controlled release. AdV. Mater. 2002, 14, 386. (32) Nozaki, T.; Maeda, Y.; Ito, K.; Kitano, H. Cyclodextrins modified with polymer chains which are responsive to external stimuli. Macromolecules 1995, 28, 522. (33) Nozaki, T.; Maeda, Y.; Kitano, H. Cyclodextrin gels which have a temperature responsiveness. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 1535. (34) Holtz, J. H.; Asher, S. Polymerized colloidal crystal hydrogel films as intelligent chemical sensing materials. Nature 1997, 389, 829. (35) Suzuki, A.; Tanaka, T. Phase transition in polymer gels induced by visible light. Nature 1990, 346, 345. (36) Murakami, H.; Kawabuchi, A.; Kotoo, K.; Kunitake, M.; Nakashima, N. A light-driven molecular shuttle based on a rotaxane. J. Am. Chem. Soc. 1997, 119, 7605. (37) Breslow, R.; Zhang, B. L. Very fast ester hydrolysis by a cyclodextrin dimer with a catalytic linking group. J. Am. Chem. Soc. 1992, 114, 5882. (38) Loftsson, T.; Jarho, P.; Masson, M.; Jarvinen, T. Cyclodextrins in drug delivery. Expert Opin. Drug DeliVery 2005, 2, 335. (39) Heskins, M.; Guilleit, J. E. Solution properties of poly(N-isopropylacrylamide). J. Macromol. Sci., Chem. 1968, 28, 1441. (40) Liu, Y. Y.; Fan, X. D.; Hu, H.; Tang, Z. H. Release of chlorambucil from poly(N-isopropylacrylamide) hydrogels with β-cyclodextrin moieties. Macromol. Biosci. 2004, 4, 729. (41) Liu, Y. Y.; Fan, X. D. Synthesis and characterization of pH- and temperature-sensitive hydrogel of N-isopropylacrylamide/cyclodextrin based copolymer. Polymer 2002, 43, 4997. (42) Wei, H. L.; Yu, H. Q.; Zhang, A. Y.; Sun, L. G.; Hou, D. D.; Feng, Z. G. Synthesis and characterization of thermosensitive and supramolecular structured hydrogels. Macromolecules 2005, 38, 8833. (43) Zhang, J. T.; Huang, S. W.; Gao, F. Z.; Zhuo, R. X. Novel temperature-sensitive, β-cyclodextrin-incorporated poly(N-isopropylacrylamide) hydrogels for slow release of drug. Colloid Polym. Sci. 2005, 283, 461. (44) Petter, R. C.; Salek, J. S.; Sikorski, C. T.; Kumaravel, G.; Lin, F. T. Cooperative binding by aggregated mono-6-(alkylamino)-β-cyclodextrins. J. Am. Chem. Soc. 1990, 112, 3860.

(45) Lai, X. H.; Ng, S. C. Preparation and chiral recognition of a novel chiral stationary phase for high-performance liquid chromatography, based on mono(6A-N-allylamino-6A-deoxy)-perfunctionalized β-cyclodextrin and covalently bonded silica gel. J. Chromatogr., A 2004, 1031, 135. (46) Park, T. G.; Hoffman, A. S. Sodium chloride-induced phasetransition in nonionic poly(N-isopropylacrylamide) gel. Macromolecules 1993, 26, 5045. (47) Sasaki, S.; Kawasaki, S.; Maeda, H. Effect of donnan osmotic pressure on the volume phase transition of N-isopropylacrylamide gels. Langmuir 1999, 15, 4266. (48) Kawasaki, H.; Sasaki, S.; Maeda, H. Effect of introduced electric charge on the volume phase transition of N-isopropylacrylamide gels. J. Phys. Chem. B 1997, 101, 4184. (49) Liu, X. X.; Tong, Z.; Hu, Q. Swelling equilibria of hydrogels with sulfonate groups in water and in aqueous salt-solutions. Macromolecules 1995, 28, 3813. (50) Zhang, J.; Peppas, N. A. Synthesis and characterization of pH- and temperature-sensitive poly(methacrylic acid)/poly(N-isopropylacrylamide) interpenetrating polymeric networks. Macromolecules 2000, 33, 102. (51) Liu, Y.; You, C. C.; Chen, Y.; Wada, T.; Inoue, Y. Molecular recognition studies on supramolecular systems. 25. Inclusion complexation by organoselenium-bridged bis(β-cyclodextrin)s and their platinum(IV) complexes. J. Org. Chem. 1999, 64, 7781. (52) Benesi, H. A.; Hildebrand, J. H. A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons. J. Am. Chem. Soc. 1949, 71, 2703. (53) Maeda, Y.; Higuchi, T.; Ikeda, I. FTIR spectroscopic and calorimetric studies of the phase transitions of N-isopropylacrylamide copolymers in water. Langmuir 2001, 17, 7535. (54) Maeda, Y.; Higuchi, T.; Ikeda, I. Change in hydration state during the coil-globule transition of aqueous solutions of poly(N-isopropylacrylamide) as evidenced by FTIR spectroscopy. Langmuir 2000, 16, 7503. (55) Shan, J.; Chen, J.; Nuopponen, M.; Tenhu, H. Two phase transitions of poly(N-isopropylacrylamide) brushes bound to gold nanoparticles. Langmuir 2004, 20, 4671. (56) Ramon, O.; Kesselman, E.; Berkovici, R.; Cohen, Y.; Paz, Y. Attenuated total reflectance/Fourier transform infrared studies on the phaseseparation process of aqueous solutions of poly(N-isopropylacrylamide). J. Polym. Sci., Part B: Polym. Phys. 2001, 39, 1665. (57) Tiktopulo, E. I.; Uversky, V. N.; Lushchik, V. B.; Klenin, S. I.; Bychkova, V. E.; Ptitsyn, O. B. Domain coil-globule transition in homopolymers. Macromolecules 1995, 28, 7519. (58) Liu, Y.; Li, B.; Wada, T.; Inoue, Y. Novel o-phenylenediseleno bridged bis(β-cyclodextrin)s complexes with platinum(IV) and palladium(II) ions. Supramol. Chem. 1999, 10, 279.

ReceiVed for reView September 30, 2006 ReVised manuscript receiVed December 7, 2006 Accepted January 2, 2007 IE061265A