ph-thermoreversible hydrogels. I. Synthesis and characterization of poly(n-isopropylacrylamide/maleic acid) copolymeric hydrogels

Radiation Physics and Chemistry 69 (2004) 3 310 ph-thermoreversible hydrogels. I. Synthesis and characterization of poly(n-isopropylacrylamide/maleic acid) copolymeric hydrogels B. Ta-sdelen a, *, N. Kayaman-Apohan b,o.g.uven c, B.M. Baysal d,e a Chemistry Department, -Cekmece Nuclear Research and Training Center, P.O. Box 1, Istanbul 34831, Turkey b Department of Chemistry, Marmara University, 810 G.oztepe/ Istanbul, Turkey c Department of Chemistry, Hacettepe University, 06532 Beytepe/Ankara, Turkey d TUBITAK Marmara Research Center, Research Institute of Materials and Chemical Technologies, 41470 Gebze, Kocaeli, Turkey e Department of Chemical Engineering, Bo &gaziçi University, 80815 Istanbul, Turkey Received 3 April2003; accepted 18 July 2003 Abstract N-isopropylacrylamide (NIPAAM)/maleic acid (MA) copolymeric hydrogels were prepared by irradiating the ternary mixtures of NIPAAM/MA/Water by g-rays at ambient temperature. The influence of externalstimuli such as ph and temperature of the swelling media on the equilibrium swelling properties was investigated. The hydrogels showed both temperature and ph responses. The effect of comonomer concentration and irradiation dose on the swelling equilibria and phase transition was studied. For the characterization of these hydrogels, the diffusion behaviour and molecular weight between crosslinks were investigated. r 2003 Elsevier Ltd. All rights reserved. Keywords: Hydrogel; Copolymers; N-isopropylacrylamide; LCST; Maleic acid; g-rays 1. Introduction Hydrogels are crosslinked, three-dimensional hydrophilic polymer networks that swell but do not dissolve when brought into contact with water. Hydrogels sometimes undergo a volume change in response to a change in surrounding conditions, such as ph (-Sen et al., 1999; Lee and Shieh, 1999a,b), temperature (-Senelet al., 1997; Lee and Shieh, 1999a,b) and ionic strength (Akka-s et al., 1999). In particular, most of the research work has been centered on the temperature and ph effects due to the importance of these variables in typical physiological, biological and chemical systems. Temperature- and ph-sensitive hydrogels have been suggested for use in a variety of novel applications including controlled drug delivery (Lim et al., 1997; Safrany, 1997; Kaetsu, 1996), *Corresponding author. Fax: +90-212-54822. E-mail address: btasdelen2002@yahoo.com (B. Ta-sdelen). immobilization of enzymes and cells (Dong et al., 1986; T.umt.urk et al., 1999) and separation of the aqueous solution of proteins (Kayaman et al., 1998). Poly(N-isopropylacrylamide) (PNIPAAm) hydrogels are attracting more and more interest in biomedical applications because they exhibit a well-defined lower criticalsolution temperature (LCST) in water around 31 34 C which is close to the body temperature. PNIPAAm hydrogels swell when cooled below LCST, and they collapse when heated above the LCST. The temperature-sensitive networks containing ionizable functionalgroups exhibit ph sensitivity. With the increase of ionizable groups, the volume change at the transition increases because of increasing electrostatic interaction between the same charged groups and the transition temperature rises. In more recent years, a series of papers has been published by G.uven and coworkers who synthesized new hydrogels from the copolymers of acrylamide and diprotic itaconic and 0969-806X/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2003.07.004

4 B. Ta-sdelen et al. / Radiation Physics and Chemistry 69 (2004) 3 310 maleic acid (MA) and showed that the use of even very small quantities of diprotic acid proved to impart remarkable properties to the hydrogels of starting monomers and/or homopolymers (Saraydın et al., 1995; Karada&g et al., 1996; -Sen et al., 2000). There is renewed interest in radiation-induced polymerization and crosslinking in polymeric hydrogels. The advantages of radiation methods are that they are relatively simple and do not require addition of any extra materials for polymerization and crosslinking. Moreover, the degree of crosslinking, which strongly determines the extent of swelling in hydrogels, can be controlled easily by varying the irradiation dose. Therefore, these methods are found to be very usefulin preparing hydrogels for medical applications, where even a small contamination is undesirable. Nagaoka et al. have reported for the first time the synthesis of PNIPAAm hydrogelby the g-radiation technique (Nagaoka et al., 1993). The purpose of this study is to develop a temperature- and ph-reversible hydrogel. In this respect, N-isopropylacrylamide (NIPAAm)/MA copolymeric hydrogels were prepared by irradiating the ternary mixtures of (NIPAAm)/MA/water by g-rays at ambient temperature. The influence of comonomer concentrations (1, 2 and 3 mol%) and irradiation dose on equilibrium swelling behaviour of the hydrogels was investigated. The hydrogels thus prepared were characterized with respect to their swelling properties, network structures and diffusion behaviour. 2. Experimental 2.1. Materials NIPAAm was obtained from Aldrich Chemical Company. MA was purchased from Fluka Chemical Company. 2.2. Preparation of hydrogels The hydrophilic NIPAAm monomer was used as a base monomer in the synthesis of hydrogels. The comonomer was MA carrying diprotic acid groups. Aqueous solutions of NIPAAm (%10 w/w) were prepared in distilled water. Different amounts of MA were added to 1 mlof NIPAAm solution (NIPAAm/MA mole ratios, 100:0, 99:1, 98:2, 97:3). Monomer solutions thus prepared were placed in a glass tube of 5 mm inner diameter. All irradiations were carried out under air at C with a Gammacell 220-type gamma irradiator in the Ankara Nuclear Research and Training Centre. The dose range was between 48 and at a dose rate of 3 kgy/hour. Water was chosen as the extraction solvent for the crude hydrogels and employed at room temperature. After polymerization, crosslinked copolymers were removed from tubes and the hydrogels obtained in long cylindrical shapes were cut into pieces of approximately 1 cm length. Each sample was placed in an excess of water and the solvent was replaced every other day over a period of at least 1 week until no further extractable polymer could be detected. Uncrosslinked polymer and/ or residualmonomer were removed with this extraction from the gelstructure. Extracted gels were dried in a vacuum oven at C to constant weight and the gel fraction was calculated. The amount of uncrosslinked MA was determined by titration of the extract against NaOH to phenolphthalein end point (-Sen et al., 1999). The percentage gelation, W g, was calculated as W g ¼ðm ae =m be Þ100; ð1þ where m ae and m be are the weights of dry gels after and before extraction. 2.3. Swelling measurements Dried hydrogels (1 cm length, 5 mm diameter) were immersed in vials (100 ml) filled with distilled deionized water. The vials were set in a temperature-controlled bath at 70.1 C. In order to reach the equilibrium degree of swelling, the gels were immersed in distilled water for at least 1 week. For the measurement of the equilibrium weight swelling ratio, q w, the hydrogels were weighed in the swollen state and dried under vacuum to constant weight. The equilibrium weight swelling ratio q w was calculated as q w ¼ m s =m d ; ð2þ where m s and m d are the weights of the hydrogels in the swollen and dry states, respectively. The equilibrium swelling ratio, V/V o, was calculated as V=V o ¼ v 0 2 1 þ ðq w 1Þr ; ð3þ d 1 where v 0 2 is the volume fraction of the polymer network after preparation, q w is the ratio of the weights of the network in the swollen state and the dry state, r and d 1 are the densities of PNIPAAm (d=1.1 g/ml) and water, respectively (Erbilet al., 1999). The mass swelling and equilibrium mass swelling percentages were calculated from the following equations: Mass swelling ð%þ ¼½ðm t m 0 Þ=m 0 Š100; ð4þ Equilibrium mass swelling ð%þ ¼½ðm N m 0 Þ=m 0 Š100; where m 0 is the mass of the dry geland m t and m N are the masses of swollen gel at time t and at equilibrium, respectively. ð5þ

B. Ta-sdelen et al. / Radiation Physics and Chemistry 69 (2004) 3 310 5 3. Results and discussion 3.1. Composition of hydrogels When NIPAAm/water and NIPAAm/MA/water mixtures have been irradiated with gamma rays, polymerization and crosslinking reactions take place simultaneously. In this work, the total dose required for an approximately 100% gelation of NIPAAm/MA copolymeric hydrogels has been found to be when the comonomer (MA) was used in the range of 1.0 3.0% in the initialmixture. Mole percentages of MA in the feed and in the copolymeric gels and percentage gelation are summarized in Table 1. These results show that increasing the mole percentage of MA causes a decrease in the extent of gelation from monomer to gel. Moreover, as can be seen from Table 1, an increase in the irradiation dose reduces the amount of soluble fraction. Equilibrium degree of swelling 50 20 10 P(NIPAAm/MA)-1 P(NIPAAm/MA)-2 P(NIPAAm/MA)-3 PNIPAAm(1) 2 3 4 5 6 7 8 9 Fig. 1. Effect of ph on the equilibrium percentage mass swelling of NIPAAm/MA copolymeric hydrogels. ph 3.2. ph sensitivity of hydrogels Fig. 1 represents ph dependence of the equilibrium mass swelling percentage for NIPAAm/MA hydrogels at C in phosphate buffer solution from ph 2 to 8. Consistent with poly-electrolyte behaviour, the swelling of hydrogels was found to increase with ph. In all compositions, maximum extents of swelling were reached at ph 7, this being due to the complete dissociation of acidic groups of MA at this ph value. The first and second dissociation constants of MA are pk a1 =1.85, pk a2 =6.06, respectively (Weast, 1972). Due to large differences in dissociation constants for MA, swelling takes place in a stepwise manner, as shown in Fig. 1. The equilibrium mass swelling percentage for pure PNIPAAm is not affected by varying the ph of the swelling medium since PNIPAAm is non-ionic hydrogel and does not have any group that could be ionized in an Table 1 The characterization of P(NIPAAm/MA) hydrogels Gelname Mole% MA both in feed and in gel Irradiation dose (kgy) PNIPAAm(1) 48 95.0 P(NIPAAm/MA)-1 1.0 48 92.3 P(NIPAAm/MA)-2 2.0 48 91.1 P(NIPAAm/MA)-3 3.0 48 90.0 P(NIPAAm/MA)-4 1.0 82 94.5 P(NIPAAm/MA)-5 2.0 82 93.4 P(NIPAAm/MA)-6 3.0 82 92.9 P(NIPAAm/MA)-7 1.0 104 96.7 P(NIPAAm/MA)-8 2.0 104 95.4 P(NIPAAm/MA)-9 3.0 104 94.9 W g Fig. 2. Percentage mass swelling as a function of time for the series of NIPAAm/MA copolymeric hydrogels at C at different ph. aqueous solution. With the introduction of the MA groups into the main chain, ph of the solution becomes an even more important factor determining swelling kinetics and equilibrium swelling value. The percentage mass swelling, as a function of time for NIPAAm-MA copolymeric hydrogels in several ph buffer solutions is shown in Fig. 2. The results indicate that under acidic conditions, anionic carboxylate groups are protonated, and the copolymeric network collapsed. At high ph values, the concentration of anionic groups

6 B. Ta-sdelen et al. / Radiation Physics and Chemistry 69 (2004) 3 310 in the polymer network increases. This occurrence makes the percentage mass swelling of the hydrogels increase with an increase in the ionizable constituent. The maximum percentage mass swelling occurred at ph 7, indicating the complete neutralization of carboxylic acid groups. 3.3. Effect of comonomer concentration When weak acidic or basic groups are incorporated, then the gels should exhibit both reversible temperature and ph swelling and deswelling. The incorporation of acidic moieties into base polymeric structures for the synthesis of microspheres and hydrogels have been mostly carried out by using acrylic acid. The use of diprotic acids, however, has been shown to impart additionaladvantages over monoprotic acids such as acrylic and methacrylic acids (G.uven and -Sen, 1999). As shown in Fig. 3, equilibrium percentage mass swelling of NIPAAm/MA copolymeric hydrogels (at fixed irradiation dose) increases as the comonomer concentration increases because of increase in the electrostatic interactions of the neighbouring carboxylate groups in MA in the hydrogels. It can be seen that the percentage mass swelling of an ionic network very much depends on the concentration of ionizable groups in the network. An increase in the MA content from 0 to 3 mole% causes immense increases in water uptake in deionized water. Fig. 4 illustrates the temperature dependence of the equilibrium swelling ratio for series NIPAAm/MA copolymeric gels at different comonomer concentration at the same irradiation dose. The results clearly show that as the comonomer concentration increases, the swelling ratio increases. The higher MA content leads to the broader phase transition and a shift of the LCST to a higher temperature. It has been shown that the LCST of PNIPAAm copolymers is strongly influenced by the nature of the comonomer (Feilet al., 1993; Kuckling et al., 2000). Hydrophobic compounds lower the LCST and hydrophilic compounds raise it (Dong et al., 1999). Equilibrium mass swelling (%) 00 00 2000 1000 0 PNIPAAm(1) 0 0.5 1 1.5 2 2.5 3 3.5 Maleic acid content (mol %) Fig. 3. Equilibrium percentage mass swelling as a function of MA content (%) for NIPAAm/MA copolymeric hydrogels. 3.4. Effect of irradiation dose As shown in Fig. 5, equilibrium percentage mass swelling of PNIPAAm/MA copolymeric hydrogels (at fixed comonomer concentration) decreases as the irradiation dose increases because of increasing crosslinking percentage in the hydrogels. The effects of irradiation dose on the temperature dependence of equilibrium swelling ratios of NIPAAm/MA copolymeric hydrogels at the same comonomer concentration are shown in Fig. 6. The results clearly show that as the irradiation dose increases, the swelling ratio decreases. The results also indicate that the higher irradiation dose leads to the narrower phase transition and a shift of the LCST to a lower temperature. Previously, Ilavsky has reported similar behaviour detected for chemically crosslinked polyacrylamide gels (Ilavsky, 1993). With the lowest crosslinker concentration, a pronounced phase transition was observed. The increasing content of the crosslinker suppressed the collapse and with the highest content of crosslinker the phase transition was continuous. Increasing the crosslinking density (at a constant charge concentration on the chain) is therefore reflected in an opposite effect to that with a rise in the number of charges in hydrogels. 3.5. Determination of M c and n e values One of the basic parameters that describes the structure of electrolyte and non-electrolyte hydrogels is the average molecular weight between crosslinks (M c ). This describes the average molecular weight of polymer chains between two consecutive junctions. These junctions may be chemical crosslinks, physical entanglements, crystalline regions, or even polymer complexes. Several theories have been proposed to calculate the molecular weight between crosslinks in polymeric networks. The earliest theory to describe the equilibrium swelling characteristics of networks was developed by Flory and Rehner for a crosslinked polymer system where the polymer chains are reacted in the solid state, and the macromolecular chains exhibit a Gaussian distribution. The Flory Rehner theory is used to determine M c ; effective crosslinking densities of polymer networks (n e ) and polymer-solvent interaction parameter (w). The Flory Rehner theory consists of the elastic, mixing, and ion contributions. The analysis of the terms in the Flory Rehner equation shows that the influence of w becomes minor for charged hydrogels at high degrees of swelling (Flory, 1953). In this study, it was assumed that the fraction of charged structuralunits, i.e., weakly ionized MA in the networks is sufficiently low to have a negligible effect on the mixing term. The Flory Huggins theory with a Flory w parameter fitted to network swelling data was used in order to obtain a reasonable value of w (Flory, 1953).

B. Ta-sdelen et al. / Radiation Physics and Chemistry 69 (2004) 3 310 7 1% 2% 3% 1% 2% 3% 1% 2% 3% Fig. 4. Variation of temperature dependence of the equilibrium swelling ratios of P(NIPAAm/MA) with MA content. Equilibrium mass swelling% 5000 00 00 2000 1000 P(NIPAAm/MA)-1 P(NIPAAm/MA)-2 P(NIPAAm/MA)-3 PNIPAAm(1) 0 50 60 70 80 90 100 110 Dose (kgy) Fig. 5. Variation of equilibrium mass swelling % with irradiation dose.

8 B. Ta-sdelen et al. / Radiation Physics and Chemistry 69 (2004) 3 310 1 % 2 % Table 2 M c and n e values of NIPAAm/MA copolymeric hydrogels Gelname M c 10 3 ðg=molþ PNIPAAm(1) 36.8 3.0 P(NIPAAm/MA)-1 110.0 1.0 P(NIPAAm/MA)-2 126.9 0.7 P(NIPAAm/MA)-3 148.7 0.2 P(NIPAAm/MA)-4 96.6 1.1 P(NIPAAm/MA)-5 103.6 1.1 P(NIPAAm/MA)-6 1.5 0.8 P(NIPAAm/MA)-7 90.0 0.5 P(NIPAAm/MA)-8 97.0 1.1 P(NIPAAm/MA)-9 98.9 1.1 n e 10 5 ðmol=cm 3 Þ 3 % The w values were calculated by using the following equation: w ¼½ln ð1 n 2m Þþn 2m Š=n 2 2m ; Fig. 6. Variation of temperature dependence of the equilibrium swelling ratios of P(NIPAAm/MA) with irradiation dose. where n 2m is the volume fraction of the swollen gel in the equilibrium state. By applying the Flory Rehner equation to PNIPAAm gels, w was calculated as a constant 0.53 in water (Erbilet al., 1992). In this work, w values of homopolymer and copolymer gels of NIPAAm were found to be 0.53 and 0.51, respectively; w was held constant at 0.52. Equilibrium swelling values were used to calculate the effective crosslinking densities of polymer networks (n e ) by the following equation (Bae et al., 1990): v e ¼ ln ð1 v 2mÞþv 2m þ w v h 2 2m i; ð7þ V 1 v 0 2 ðv 2m=v 0 2 Þ1=3 0:5ðv 2m =v 0 2 Þ where v 2m is the volume fraction of the swollen gel in the equilibrium state and V 1 is the molar volume of the solvent. The effect of the presence of MA on the network properties of polymer-solvent (water) interaction parameter is obvious from Table 2. With increasing amount of ionizable constituent (MA) in the copolymer structure the average M c values increase, whereas the ð6þ effective crosslinking densities of polymer networks (n e ) decrease. This indicates that MA does not act as a crosslinking agent. 3.6. Diffusion To obtain a more quantitative understanding of the nature of the sorption kinetic in NIPAAm/MA copolymeric gels, the initial swelling data were fitted to the exponentialheuristic equation (8) (Peppas et al., 1983): F ¼ M t =M N ¼ kt n : ð8þ Here F is the fractionaluptake, M t =M N ; where M t is the amount of diffusant sorbed at time t, M N is the maximum amount absorbed, k is a constant incorporating characteristics of macromolecular network system and the penetrant, n is the diffusionalexponent, which is indicative of the transport mechanism. The exponents n and k values were calculated from the slope and intercept of the plots of ln F versus ln t for the series of pure PNIPAAm and P(NIPAAm/MA) copolymeric hydrogelat different MA contents. Eq. (8) is valid for the first 60% of the normalized solvent uptake. For Fickian kinetics in which the rate of penetrate diffusion is rate limiting, n ¼ 0:5; whereas values of n between 0.5 and 1 indicate the contribution of non-fickian processes such as polymer relaxation. The results in Table 3 indicate that as the MA content of the samples increases the water fractional uptake at the same absorption time increases. It is clear from the analysis that as the MA content in the gel structure increases the diffusionalrelease kinetic exponent n increases from 0.49 to 0.61 for P(NIPAAm/MA) hydrogels. This evidence shows that the swelling transport mechanism was transferred from Fickian to non-fickian transport with the increasing MA content in the gelstructure. Diffusion coefficients are important penetration parameters of some chemicalspecies to polymeric systems. Using n and k, the diffusion coefficient (D) of

B. Ta-sdelen et al. / Radiation Physics and Chemistry 69 (2004) 3 310 9 Table 3 The parameters of diffusion of water into the P(NIPAAm/MA) hydrogels Gelname k 100 n D 10 8 /cm 2 s 1 PNIPAAm(1) 3.2 0.49 3.2 P(NIPAAm/MA)-1 2.2 0.52 3.0 P(NIPAAm/MA)-2 1.9 0.53 3.4 P(NIPAAm/MA)-3 1.7 0.61 13.2 Acknowledgements This work was supported by Turkish-Macedonian Science and Technology Program for 2001 2003 and -Cekmece Nuclear Research and Training Center. B.M.B. acknowledges support from T.UBA Turkish Academy of Sciences. The authors thank Dr. A. Yılmaz Erkoland Ayhan Mesci for their technicalassistance. References solvent in the matrix could be calculated using the following equation (Korsmeyer and Peppas, 1983): k ¼ 4½D=pr 2 Š n ; 4D n ¼ kðpr 2 Þ n ; D n ¼ðk=4Þðpr 2 Þy; where D is the diffusion coefficient and r is the radius of geldisc. The D values are also presented in Table 3. The diffusion coefficients D increase with an increase in MA content in the present hydrogels. This is due to the hydrophilicity for these copolymeric hydrogels in the order of PNIPAAm(1)oP(NIPAAm/MA)-1oP (NIPAAm/MA)-2oP(NIPAAm/MA)-3, and the more hydrophilic groups in the gel, the easier the diffusion for water molecules. So, P(NIPAAm/MA)-3 has a higher D value. 4. Conclusion In this study, the influence of externalstimuli such as ph and temperature of the swelling media on the equilibrium swelling properties were investigated. The equilibrium percentage mass swelling of NIPAAm/MA copolymeric hydrogel increased from 1264 to 39 as the mole % of maleic acid (MA) content increased from 0 to 3. This has been explained due to the incorporation of more specific acidic groups into the network and consequent higher swelling capacity of the gels. The swelling studies of P(NIPAAm/MA) hydrogels showed that ph and temperature of swelling media are the basic parameters affecting the equilibrium degree of swelling of the hydrogels. In the diffusion transport mechanism study, the results indicate that the swelling exponents n for all NIPAAm/MA copolymeric gels at C are in the range from 0.49 to 0.61. This implies that the swelling transport mechanism is a non-fickian transport. The diffusion coefficients (D) for the copolymeric gels increase with an increase in MA content, so the water molecule easily infiltrates into hydrogels for gels containing higher MA content. ð9þ Akka-s, P., Sari, M., -Sen, M., G.uven, O., 1999. The effect of external stimuli on the Bovine Serum Albumin adsorption capacity of poly(acrylamide/maleic acid) hydrogels prepared by gamma rays. Radiat. Phys. Chem. 55, 717 721. Bae, Y.H., Okano, T., Kim, S.W., 1990. Temperature dependence of swelling of cross-linked poly(n, N 0 -alkyl substituted acrylamides) in water. J. Polym. Sci. Part B Polym. Phys. 27, 923 936. Dong, L.C., Hoffman, A.S., 1986. Thermally reversible hydrogels immobilization of enzymes feedback reaction control. J. Controlled Release 4, 223 227. Erbil, C., Aras, S., Uyanik, N., 1999. Investigation of the effect of type and concentration of ionizable comonomer on the collapse behavior of N-isopropylacrylamide copolymer gels in water. J. Polym. Sci. Polym. Chem. 37, 1847 1855. Feil, H., Bae, Y.H., Feijen, J., Kim, S.W., 1993. Effect of comonomer hydrophilicity and ionization on the lower criticalsolution temperature of N-isopropylacrylamide copolymers. Macromolecules 26, 2496 00. G.uven, O., -Sen, M., 1999. Radiation synthesis of poly(n-vinyl 2-pyrolidone/itaconic acid) hydrogels and their controlled release behaviours. Radiat. Phys. Chem. 55, 113 118. Ilavsky, M., 1993. Effect of phase transition on swelling and mechanicalbehaviour of synthetic hydrogels. In: Dusek, K. (Ed.), Responsive Gels: Volume Transition I, Vol. 109. Springer, Berlin, pp. 173 206. Kaetsu, I., 1996. Biomedicalmaterials, devices and drug delivery systems by radiation technique. Radiat. Phys. Chem. 47, 419 424. Karada&g, E., Saraydın, D., G.uven, O., 1996. Interaction of some cationic dyes with acrylamide/itaconic acid hydrogels. J. Appl. Polym. Sci. 61, 2367 2372. Kayaman, N., Kazan, D., Erarslan, A., Okay, O., Baysal, B.M., 1998. Structure and protein separation efficiency of poly(n-isopropylacrylamide) gels: effect of synthesis conditions. J. Appl. Polym. Sci. 64, 805 814. Korsmeyer, R.W., Peppas, N.A., 1983. Macromolecular and modelling aspects of swelling controlled system in controlled release delivery systems. In: Roseman, T.J., Mansdorf, S.Z. (Eds.), Controlled Release Delivery Systems, Marcel Dekker, New York, pp. 77 90. Kuckling, D., Adler, H.P., Arbdt, K.F., Ling, L., Habicher, W.D., 2000. Temperature and ph dependent solubility of novelpoly(n-isopropylacrylamide) copolymers. Macromol. Chem. Phys. 201, 273 280. Lee, W., Shieh, C., 1999a. ph-thermoreversible hydrogels. 1. Synthesis and swelling behaviors of the (N-isopropylacrylamide-co-acrylamide-co-2-hydroxyethylmethacrylate) copolymeric hydrogels. J. Appl. Polym. Sci. 71, 221 231.

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