pH- and Temperature-Responsive Hydrogels (PDF)

J Polym Res (2012) 19:9944 DOI 10.1007/s10965-012-9944-z ORIGINAL PAPER pH- and temperature-responsive behaviors of hydrogels resulting from the photopolymerization of allylated chitosan and N-isopropylacrylamide, and their drug release profiles San-Ping Zhao & Feng Zhou & Li-Yan Li Received: 19...

J Polym Res (2012) 19:9944 DOI 10.1007/s10965-012-9944-z ORIGINAL PAPER pH- and temperature-responsive behaviors of hydrogels resulting from the photopolymerization of allylated chitosan and N-isopropylacrylamide, and their drug release profiles San-Ping Zhao & Feng Zhou & Li-Yan Li Received: 19 February 2012 / Accepted: 18 July 2012 / Published online: 3 August 2012 # Springer Science+Business Media B.V. 2012 Abstract Allyl glycidyl ether (AGE)-functionalized chito- Introduction san (CS-AGE), a macromolecular crosslinker, was synthe- sized and then copolymerized with N-isopropylacrylamide During the past few decades, temperature- and pH-sensitive (NIPAAm) monomer under UV irradiation to produce polymeric hydrogels have received special interest due to hydrogels. The allylated chitosan and the resulting hydro- their tunable swelling behaviors, permeability, and mechan- gels were characterized by 1 H NMR and FT IR, respective- ical strength in response to temperature and/or pH modula- ly. The interior morphologies of the hydrogels were tions [1, 2]. These hydrogels have been extensively investigated by scanning electron microscopy (SEM) after investigated for a variety of biomedical applications, such freeze drying them in the equilibrium state in buffer solution as controlled drug delivery, tissue engineering, and biosepa- at pH 2.0. Their swelling kinetics were found to be sensitive ration [3–5]. Numerous synthetic polymers, such as PEO- to both temperature and pH, so it was possible to modulate PPO-PEO Pluronic triblock copolymers, PLGA-PEG- the swelling by adjusting the pH or the temperature of the PLGA triblock copolymers, and copolymers derived from medium containing the hydrogel and the proportion of the poly(methacrylic acid) (PMAA) or poly(acrylic acid) CS derivative with respect to the NIPAAm monomer. Rhe- (PAA), have been shown to exhibit temperature- or pH- ological measurements were utilized to investigate the me- sensitive phase-transition behaviors in aqueous solution chanical properties of the hydrogels. The in vitro release [6–9]. In particular, natural polymers such as gelatin, cellu- profiles of the model drugs methyl orange (MO) and bovine lose derivatives, carrageenans, alginate, and hyaluronic acid serum albumin (BSA) from the hydrogels were also exam- have received a great deal of attention from scientists in ined. The results revealed that the drug release rate could be relation to the fabrication of temperature- or pH-responsive tuned by adjusting the pH of the medium and the hydrogel hydrogels, due to their biocompatibility, biodegradability, composition. and the fact that they can be used for the controlled release of bioactive molecules—a very desirable property [10–13]. Keywords Chitosan. pH sensitivity. Temperature Chitosan, a cationic amino-polysaccharide derived by sensitivity. Rheological properties. Drug delivery the partial deacetylation of the naturally occurring bio- polymer chitin, has been widely used for drug carriers and in other biomedical fields in various chemical or physical gel forms because of its appealing intrinsic properties, such as biodegradability, biocompatibility, S.-P. Zhao (*) : F. Zhou : L.-Y. Li bioadhesivity, polyfunctionality, hydrophilicity, and me- Key Laboratory of Green Processing and Functional Textiles of chanical properties [14, 15]. Chitosan-based hydrogels New Textile Materials of Ministry of Education, display pH-dependent swelling behaviors due to the Wuhan Textile University, Wuhan 430073, Peoples Republic of China ionization of amino groups in the chitosan chain in e-mail: [email protected] response to external pH variations, and these hydrogels Page 2 of 9 J Polym Res (2012) 19:9944 can also be used as drug delivery devices due to their Experimental ionic binding affinity (meaning that they have affinities toward molecules such as DNA). In addition, the Materials abundant reactive functional groups of chitosan, espe- cially its –NH2 groups, allow specific modifications to Chitosan (M w 04.5 × 105; degree of deacetylation DD 0 be made to chitosan without too many difficulties, 90 %) was purchased from Ruji Biotech Development Co., yielding numerous useful materials for different domains Ltd (Shanghai, China). N-isopropylacrylamide (NIPAAm) of application [17, 18]. (97 %, Aldrich, St. Louis, MO, USA) was purified by Poly(N-isopropylacrylamide) (PNIPAAm) hydrogel is recrystallization from a 65:35 (v/v) mixture of hexane and one of most thoroughly investigated thermosensitive benzene and dried under vacuum for two days. Allyl gly- polymers. It displays phase separation around the lower cidyl ether (AGE) (99 %, Aldrich), 1-vinyl-2-pyrrolidone critical solution temperature (LCST), and exhibits a (NVP) (97 %, Fluka, St. Louis, MO, USA) and the photo- sudden decrease in volume upon heating. The transi- initiator, 2,2-dimethoxy-2-phenylacetophenone (DMPA) tion is controlled by hydrophilic/hydrophobic interac- (99 %, Acros, Fair Lawn, NJ, USA), were used as received. tions in the NIPAAm unit, associated with the Other chemicals used were of analytical grade and were hydrophilic (amide groups) and hydrophobic (isopropyl used without further treatment. groups) regions of the PNIPAAm, which change with temperature. This thermogelation property of PNI- Instrumental analysis and measurements PAAm hydrogel has been widely used in many devi- ces; for example, in on-off switches for controlled drug The 1H NMR spectrum of the chitosan derivative CS- release, for the dewatering of protein solutions, and in AGE was recorded on a Varian (Palo Alto, CA, USA) gene carriers [20, 21]. Inovia 500 NB spectrometer at 500 MHz using DCl/ In recent years, different methods of fabricating chito- D2O as the mixed solvent. FT-IR spectra of CS-AGE san/PNIPAAm hydrogels have been developed that utilize and the dry gel samples were obtained using KBr pel- a combination of the advantages of synthetic and natural lets on a Nicolet (Madison, WI, USA) 670IR spectrom- polymers. Cho et al. prepared thermosensitive chitosan eter. The interior morphology of the hydrogels was hydrogels by grafting chitosan with PNIPAAm, and these observed by scanning electron microscopy (SEM). The were used in tissue engineering. Kim et al. fabricated dry gel samples were immersed into a PBS solution, pH chemically crosslinked chitosan-g-PNIPAAm copolymer 2.0, at 25 °C to reach swelling equilibrium, and then hydrogels by utilizing glutaraldehyde as a crosslinker frozen at −40 °С in a low temperature freezer and. Alvarez-Lorenzo et al. prepared interpenetrated freeze-dried for 24 h. After being covered with gold polymer network (IPN) hydrogels of PNIPAAm and chi- on an aluminum holder, the freeze-dried sample was tosan based on bis(acrylamide) crosslinked PNIPAAm and examined using a scanning electron microscopy (SEM- glutaraldehyde postcrosslinked chitosan. We synthe- JSM 5510-LV, JEOL, Tokyo, Japan) with a sub- sized semi-IPN chitosan/PNIPAAm hydrogels via the in accelerating voltage of 20 kV. The gravimetric method situ photopolymerization of NIPAAm monomer, using was applied to determine the equilibrium swelling ratios PEG-co-PCL macromer as a crosslinker in the presence (ESRs) of the hydrogels. Buffer solutions with different of chitosan. Verestiuc et al. produced copolymerized pH ranges (2.0–4.0, 4.0–6.0, and 6.0–8.0) were prepared hydrogels of acrylic-acid-modified chitosan and NIPAAm using hydrochloric acid/potassium hydrogen phthalate, monomer. sodium hydroxide/potassium hydrogen phthalate, and In this work, chitosan functionalized with photopoly- sodium hydroxide/sodium dihydrogen phosphate, respec- merizable allyl groups was synthesized and then copo- tively. The ionic strength of the buffer solution was kept lymerized with NIPAAm monomer to fabricate chitosan/ constant at a value of 0.1 mol/L by adding potassium PNIPAAm hydrogels under UV irradiation. The interior chloride when required. The equilibrium swelling stud- morphologies of the hydrogels were observed by scan- ies were performed in buffer solutions with two differ- ning electron microscopy (SEM). The swelling behav- ent pH values (2.0 and 7.4) and at different oirs of the hydrogels in response to pH and temperature temperatures (from 13 °С to 53 °С), and in buffer were investigated. The mechanical properties of the solutions with various pH values (from 2.0 to 8.0) at resulting hydrogels were studied by performing rheolog- 25 °С. The hydrogels were immersed in the buffer ical measurements. The in vitro release kinetics of the solutions and allowed to reach equilibrium swelling at model drugs MO and BSA from the hydrogels were each predetermined temperature or predetermined pH, also evaluated to determine their potential applicability and the weights of the equilibrated swollen hydrogels as drug carriers. were recorded after the removal of excess surface water J Polym Res (2012) 19:9944 Page 3 of 9 with a moistened filter paper. All experiments were into MO solution at pH 2.0 (50 mg/L) for two days at performed in triplicate for each of the samples, and room temperature to achieve adsorption equilibrium. the average value of three measurements was taken. The initial loading of the drug was calculated based The equilibrium swelling ratio (ESR) was defined as on the change in the concentration of the MO solution follows: before adsorption and after absorption. BSA-loaded hydrogel was prepared using a similar method to that ESRð%Þ ¼ ½ðWe  Wd Þ=Wd   100 ð1Þ employed to prepare the hydrogel, but 0.5 wt% BSA (relative to the total weight of the hydrogel formulation) where We and Wd represent the weights of the equili- was added, and the solution was mixed homogeneously brated swollen hydrogels and dried gels, respectively. before photopolymerization. The drug-loaded hydrogels The oscillatory swelling behaviors of the hydrogels were placed in a tube containing 12 mL of fresh buffer were observed in buffer solutions with pH values be- solution with horizontal shaking at 37 °С (pH 2.0 and tween 2.0 and 7.4 at 20 °С, and in a buffer solution of pH 7.4). At predetermined time intervals, 2 mL of the pH 2.0 maintained at alternate temperatures of 20 °С solution were taken out and placed in another 2 mL of and 45 °С, respectively. The hydrogel samples were fresh buffer solution. The concentrations of MO and immersed in buffer solutions at pH 2.0 and 20 °С for BSA released were analyzed using a UV spectropho- at least 12 h to reach equilibrium swelling. The swollen tometer (UV-2550, Shimadzu, Kyoto, Japan). The max- samples were transferred to buffer solution at pH 7.4 imum absorbance wavelengths of MO and BSA were and 20 °С or buffer solutions at pH 2.0 and 45 °С, 508 and 280 nm, respectively. For each sample, the before the weights of the samples were measured gravi- experiments were performed in triplicate, and the aver- metrically and the swelling ratio was calculated accord- age value of three measurements was taken. ing to Eq. 1. After 3 h, the hydrogel samples were placed in buffer solution at pH 2.0 and 20 °С, and Synthesis of allyl glycidyl ether modified chitosan the swelling ratio was determined. After 3 h, the hydro- (CS-AGE) gels were transferred to buffer solution at pH 7.4 and 20 °С or at pH 2.0 and 45 °С. This process was Allylated chitosan CS-AGE was synthesized using a similar repeated four times. The experiments were performed method to that reported by Flores et al. Briefly, 2.5 g of in triplicate, and the final results were calculated as chitosan were dissolved in acetic acid solution adjusted to the averages of these triplicate results. The rheological pH 3.8 by KOH, and 9.3 g of AGE were added to the behaviors of the copolymerized hydrogels were investi- solution. The mixture was then reacted under mechanical gated at different temperatures using a strain-controlled stirring for 8 h at 60 °C in an Ar atmosphere. The solution AR2000ex rheometer (TA Instruments, New Castle, DE, was poured into an excess amount of acetonitrile to obtain a USA) with a stainless-steel parallel plate geometry. Stor- pale yellow precipitate, and the precipitate was washed age moduli were measured as a function of the frequen- several times with acetone and dried under vacuum at room cy at a strain of 0.1 %. Test samples were made to temperature to obtain the chitosan derivative CS-AGE. match the diameter of the parallel plate (40 mm). Meas- urements were performed as dynamic frequency sweep Preparation of CS-AGE/PNIPAAm copolymerized tests at 25 °C and 40 °C, respectively. A solvent trap hydrogels was used to prevent water evaporating from the hydro- gels. Methyl orange (MO) and BSA were used as model To synthesize the copolymerized hydrogels, NIPAAm drugs to investigate the release profiles of the hydrogels monomer and the chitosan derivative were dissolved in when used as drug carriers. MO-loaded hydrogel sam- different ratios into a 2 wt% acetic aqueous solution at room ples were obtained by immersing the dry gel samples temperature (see Table 1). A photoinitiator solution of Table 1 Feed compositions used to generate the copolymer- Sample code Feed composition Gel content (%) ized hydrogels investigated in this study CS-AGE (g) NIPAAm (g) 2 wt% acetic acid DMAP (mg) aqueous solution (g) CN-30 0.735 0.315 4.9 11 91.2 CN-50 0.525 0.525 4.9 11 90.1 CN-70 0.315 0.735 4.9 11 88.5 Page 4 of 9 J Polym Res (2012) 19:9944 DMPA in NVP (150 mg/mL) was then added to the mixture opening reaction between the –NH2 groups of chitosan (1 wt% DMPA with respect to the total amount of the and the epoxy group of AGE. Figure 2 presents the 1H chitosan derivative and NIPAAm monomer), the resulting NMR spectrum of the CS-AGE. The signals at 4.9– solution was homogeneously mixed, and it was added to a 5.8 ppm belong to the protons of –CH0CH2 in AGE. The Teflon (PTFE) mold with a diameter of 5 cm. Following signal at around 4.0 ppm can be attributed to H-1 of chito- this, the mixture was exposed to 365 nm LWUV radiation san. The signal at 3.95 ppm belongs to the protons of –CH2– produced by an 8 W lamp (ZF-7A type, Shanghai Jinhui O–CH2–CH0CH2 in AGE. The signals at 3.2–3.9 ppm re- Scientific Instrumental Co., Ltd., Shanghai, China) for sult from the protons H-3, 4, 5, and 6 of chitosan. The signal 15 min to ensure sufficient copolymerization of the at 3.0 ppm can be assigned to the protons of –CH2CH NIPAAm monomer with the allylated chitosan derivative. (OH)CH2– in AGE. The signal at 2.9 ppm can be attributed The distance between the reaction mixture and the light to the proton H-2 of chitosan. The degree of substitution was source was maintained at 3 cm. calculated from the signal intensity ratio of the –CH0CH2 The hydrogel was carefully punched into disks (10 mm in protons in AGE and protons H-3, 4, 5, 6 of chitosan, and diameter and 3 mm in thickness). The hydrogel samples was found to be about 12.3 %. These results show that we were immersed in distilled water at 20 °C for three days to successfully synthesized the photopolymerizable chitosan remove residual unreacted monomer and free linear PNI- derivative used as a crosslinking agent. PAAm chains. The distilled water was refreshed every day. The aqueous solutions of CS-AGE and NIPAAm under- Finally, the samples were dried at room temperature for went rapid polymerization to form a macromolecular cross- two days, and then dried at 45 °C under reduced pressure linked network under UV irradiation using DMAP as a for another two days. photoinitiator. This polymer network consisted of covalently crosslinked PNIPAAm and chitosan chains. Figure 3 shows the FT-IR spectra of the chitosan deriv- Results and discussion ative CS-AGE and the copolymerized hydrogel samples. All of the samples exhibited a wide spectral band at around Synthesis of the chitosan derivative and the copolymerized 3432 cm−1, which could be assigned to stretching vibrations hydrogels of –NH and –OH in the chitosan chains and –NH in the PNIPAAm chains. The peaks at 2975, 2927, and 2872 cm−1 The structures of CS, AGE, and NIPAAm, and the synthetic were attributed to C–N, methyl, and ethylene in the polymer routes to CS-AGE and the CS-AGE/PNIPAAm copolymer- backbone. Two characteristic bands, amide I and amide II, ized hydrogels are shown in Fig. 1. The photopolymerizable from the amide groups of the chitosan derivative and the chitosan derivative CS-AGE was synthesized via a ring- copolymerized hydrogels were observed at around 1655 and 1551 cm−1, respectively. For the copolymerized hydro- NH2 CH2OH gels, the intensity of these characteristic peaks clearly in- HO O O O O O + O creased with increasing feed ratio of the NIPAAm CH2OH NHCOCH3 AGE comonomer to the chitosan derivative. Chitosan HAc/KOH pH 3.8 pH dependence of the swelling ratio OH CH CH2 O CH2 CH2 O CH=CH2 Chitosan (CS) is a weak base, and is insoluble in water and CH2=CH C NH CH2OH organic solvents. However, it is soluble in dilute aqueous O NH HO O + O CH O O OH c H3C CH3 CH2 OH NHCOCH3 b a f CH d CH2 CH2 e CH2 O CH=CH2 NIPAAm CS-AGE O NH CH2OH Photoinitiator HO 3 5 O UV 2 1O O O 4 NHCOCH3 CH2OH 6 CS chain PNIPAAm moieties Copolymerized hydrogel Fig. 1 Synthetic routes to CS-AGE and the pH- and temperature- responsive copolymerized hydrogels Fig. 2 1H NMR spectrum of CS-AGE in DCl/D2O J Polym Res (2012) 19:9944 Page 5 of 9 CS-AGE the hydrogel network. In basic solution, the –NH2 groups remain in the form –NH2, which may induce the formation of inter- and intramolecular hydrogen bonding, such that CN-70 hydrogen bonds become dominant in the polymer network. This enhancement of the interactions between polymer Transmittance (%) chains causes the observed decrease in the ESR of the CN-50 hydrogels. Figure 4 also implies a dependence of the ESR on the feed ratio of CS-AGE to NIPAAm in the resulting CN-30 hydrogels. The ESR of the copolymerized hydrogels in- creased with increasing content of the chitosan derivative at lower pH values, which may be attributed to the presence of more positively charged –NH2 groups, leading to an expansion in the hydrogel network. 4000 3500 3000 2500 2000 1500 1000 500 -1 Figure 5 depicts the pH-dependent reversible swelling– Wavelength (cm ) deswelling behaviors of the copolymerized hydrogels in Fig. 3 FT-IR spectra of CS-AGE and the hydrogel samples buffer solutions with pH values between 2.0 and 7.4 at 25 °C. The experimental data indicate that the resulting hydrogels show good swell/shrink reversibility upon alter- acidic solution, which can convert the glucosamine units ing the pH of the medium. This may be ascribed to changes into the soluble form R–NH3+. Such pH-dependent solubil- in the proportion of protonated –NH2 groups (i.e., –NH3+ ity could affect the swelling properties of the hydrogels groups) upon varying the local pH. As seen in Fig. 5, the pH containing the chitosan derivative. The effect of pH on the sensitivity of the copolymerized hydrogels decreased as the swelling ratios of the copolymerized hydrogels was there- content of NIPAAm monomer in the hydrogel increased. fore determined in external buffer solutions in the pH range from 2.0 to 8.0 at a fixed ionic strength of 0.1 mol/L at 25 ° SEM micrographs of the hydrogels C. As shown in Fig. 4, the ESR of all of the copolymerized hydrogels gradually decreased with increasing pH of the The swollen hydrogels were frozen at the low temperature buffer solution. This phenomenon may be attributed to the of −40 °C and freeze-dried at –50 °C in order to preserve ionization behavior of the free amino groups in the chitosan their open, microporous structure. SEM photos of the inter- derivative in response to external pH changes. Since the pKa nal morphology of the CS-AGE/PNIPAAm copolymerized of the amino group of a glucosamine residue is about 6.5 hydrogels equilibrated in buffer solution at pH 2.0 and 25 ° , most of the –NH2 groups of chitosan chains are pos- C are presented in Fig. 6. The microphotos clearly display a itively charged in acidic media, and electrostatic repulsion porous structure, and the inner structure was dependent on between the –NH3+ groups of chitosan chains causes the the composition ratio of the hydrogel. At pH 2.0, sample polymer network to expand. This attracts more water into CN-30 (with a high CS-AGE content) exhibited a large pore 1150 1000 CN-70 1100 CN-50 900 CN-70 1050 CN-30 CN-50 800 CN-30 1000 pH 7.4 700 950 900 600 ESR (%) 850 SR (%) 500 800 400 750 700 300 650 pH 2.0 200 600 100 550 500 0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 100 200 300 400 500 600 700 pH Time (min) Fig. 4 pH dependence of the equilibrium swelling ratios of the hydro- Fig. 5 The dynamic pH-dependent swelling/deswelling behaviors of gels at 25 °C the copolymerized hydrogels at 25 °C Page 6 of 9 J Polym Res (2012) 19:9944 1400 CN-30 (pH2.0) CN-70 (pH7.4) 1300 CN-50 (pH2.0) CN-50 (pH7.4) 1200 CN-70 (pH2.0) CN-30 (pH7.4) 1100 1000 ESR (%) 900 800 700 600 500 400 300 10 20 30 40 50 o Temp ( C) Fig. 7 ESR as a function of temperature for different hydrogels in buffer solutions at pH 2.0 and pH 7.4 of the ESR in buffer solutions at different pH values (2.0 and 7.4). The ESR of every hydrogel displayed a tendency to decrease when the local temperature was increased, and the temperature sensitivity of the hydrogels increased with increasing feed ratio of the NIPAAm monomer to the CS- AGE derivative. However, the hydrogels showed different transition behaviors in buffer solutions at pH 7.4 and pH 2.0, which was due to the introduction of a pH-sensitive CS moiety as the crosslinker. At pH 2.0, as seen in Fig. 7, the ESR of the hydrogels showed a relatively slow transition region, and the phase-transition temperature shifted to a higher temperature than that of pure PNIPAAm, which dis- played a low critical solution temperature (LCST) of around 32 °C. This can be attributed to the ionization of –NH2 Fig. 6 SEM micrographs of hydrogel samples equilibrated in buffer groups in the CS component; the electrostatic repulsion solution at pH 2.0 and 25 °C: a CN-30, b CN-70 among the –NH3+ groups may offset the aggregation caused by the thermosensitive PNIPAAm component, increasing size, which can be attributed to the expansion of the network the phase transition temperature. At pH 7.4, the –NH2 induced by the strong electrostatic repulsion among the – groups of the CS component remain unionized, and the NH3+ groups in the chitosan chain, which was in turn caused formation of hydrogen bonds results in a decrease in the by the relatively high CS-AGE content and the low pH. ESR of the hydrogels. As the amount of CS-AGE (crosslinker) is increased, the hydrogen-bond interactions Temperature dependence of the swelling ratio and the crosslinking density increases, leading to a much lower ESR for the hydrogels, even at lower temperatures. As PNIPAAm is one of the most extensively investigated ther- seen in Fig. 7, the differences in the ESR at pH 2.0 were mosensitive materials because it uniquely exhibits thermor- more obvious than those present at pH 7.4. This may be eversible gelation. Chemically crosslinked hydrogels explained by the fact that the PNIPAAm chains in the containing PNIPAAm were also seen to exhibit phase- hydrogels were influenced by the chitosan chains due to transitional behavior in response to changes in external the covalently crosslinked structure of the hydrogel. temperature. However, when the pH-dependent chitosan At pH 2.0, chitosan exhibited a highly swollen state and the derivative CS-AGE is covalently crosslinked to the PNI- PNIPAAm chains were relatively free to move around. As a PAAm polymer to form a copolymerized hydrogel network, result, the differences in the ESR were larger than those at the temperature sensitivity of the resulting hydrogel should pH 7.4. also be affected by the pH of the medium containing the Figure 8 shows the stepwise swelling behaviors of copo- hydrogel. Figure 7 exhibits the temperature dependence lymerized hydrogels in buffer solutions at pH 2.0 and J Polym Res (2012) 19:9944 Page 7 of 9 1300 CN-70 displayed solid-like behavior, with G’ showing barely any 1200 CN-50 frequency dependence. However, changing the temperature CN-30 had different effects on the rheological behavior of the 1100 0 45 C 0 hydrogels. At 20 °C, as we can see, hydrogel CN-30 had a 1000 20 C higher G’ than hydrogel CN-70. At the lower temperature, the hydrogels presented higher swelling ratios due to ioni- SR (%) 900 zation of the amino groups on the chitosan moieties and 800 strong hydrogen-bond interactions between the amide 700 groups and water molecules derived from the PNIPAAm chains. Increasing the CS derivative content increased the 600 crosslinking density in the hydrogel network, resulting in an 500 increase in G’. However, at 40 °C, the PNIPAAm chains 0 100 200 300 400 500 600 700 appeared shrunken because of the strengthened hydrophobic Time (min) interactions between the hydrophobic groups in the PNI- PAAm chains, which led to an increase in the intermolecular Fig. 8 The dynamic temperature-dependent swelling/deswelling crosslinking in the hydrogel network. Therefore, the hydro- behaviors of the copolymerized hydrogels in buffer solution at pH 2.0 gels had higher G’ values at 40°C, and the G’ value in- creased as the content of PNIPAAm increased. On the other temperatures alternating between 20 and 45 °C. The swell- hand, at higher frequencies (>2), all of the hydrogels ing/deswelling process of every hydrogel was found to be showed an increase in G’, and a higher increase in response rapid and repeatable, in accordance with the temperature to increased rigidity of the network strands. The mag- alterations. However, as the feed ratio of the NIPAAm nitude of the viscoelastic response elicited by a polymeric monomer to the chitosan derivative was increased, the network is governed primarily by the length of the flexible swelling ratios of the hydrogens during the swelling and polymer chains and the nature of the imposed mechanical deswelling process also increased. motion. At higher frequencies, hydrogels with relative- ly high crosslink density fail to rearrange themselves during Rheological properties of the hydrogels the timescale of the imposed motion and therefore stiffen up, thus presenting more “solid-like” behavior, characterized by Frequency sweep measurements are widely applied to ob- an obvious increase in G’. tain information about the stability of the three-dimensional crosslinked networks in hydrogels. Figure 9 shows the In vitro drug release profiles of the hydrogels dependence of the storage modulus (G’) on the frequency for the hydrogels in their equilibrated states in buffer solu- Hydrogels have been extensively investigated as drug tion at pH 2.0 and at 20 and 40 °C. All of the hydrogels delivery carriers due to their excellent physicochemical properties. Drug release from hydrogel matrices is af- fected by many factors, such as their swelling proper- 7000 ties, network porosity, and the erosion of the hydrogel 6500 0 CN-30 (40 C) matrix, as well as drug–polymer interactions. Herein, 0 6000 CN-70 (40 C) MO and BSA were chosen as a small-molecule model 0 CN-30 (20 C) 5500 0 drug and a water-soluble macromolecular model drug, CN-70 (20 C) 5000 respectively, to evaluate the in vitro release profiles of Modulus (MPa) 4500 the hydrogels. 4000 MO is an acid dye bearing a sulfonate group that can bind 3500 with chitosan through an ionic interaction. Its molecu- 3000 lar structure is illustrated in Fig. 10. The binding of MO to 2500 chitosan is dependent on the pH of the local medium. 2000 1500 1000 0.1 1 10 100 -1 Frequency (s ) Fig. 9 Rheological behaviors of the hydrogel samples in buffer solu- Fig. 10 The molecular structure of the model drug methyl orange tion at pH 2.0 and different temperatures (MO) Page 8 of 9 J Polym Res (2012) 19:9944 100 80 90 CN-30 CN-50 80 pH 7.4 CN-70 60 Cumulative release (%) Cumulative release (%) 70 60 50 40 40 30 20 20 10 pH 2.0 0 0 0 10 20 30 40 50 0 10 20 30 40 50 Time (h) Time (h) Fig. 11 Cumulative release profiles of MO from the hydrogel sample CN-30 in buffer solutions at pH 2.0 and pH 7.4 Fig. 12 In vitro BSA release profiles for various hydrogel samples in buffer solution at pH 7.4 and 37 °C Figure 11 shows the release profiles of MO from hydrogel solution at pH 7.4 and 37 °C. This may be due to the sample CN-30 in different buffer solutions (pH 2.0 and pH swelling behavior of the copolymerized hydrogels. A 7.4) at 37 °C. The release of MO was highly dependent on much denser network structure may form as the content the pH of the medium, and the percentage cumulative re- of NIPAAm monomer is increased, which retards the lease from the matrix was much lower in pH 2.0 buffer diffusion of BSA from the hydrogel into the external solution than in pH 7.4 buffer solution. This can be medium. The loading of BSA in the hydrogel could be explained by the fact that the amino groups of the chitosan regulated by adjusting the formulation of the copoly- chains are positively charged at pH 2.0, which induces the merized hydrogel. formation of strong ionic interactions between the –NH3+ groups of chitosan and the –SO3− groups of MO molecules. On the other hand, the amino groups of chitosan remain Conclusions unionized in buffer solution at pH 7.4, which greatly decreases the interaction between chitosan and MO mole- pH- and temperature-sensitive hydrogels were prepared cules, so the MO molecules readily diffuse from the hydro- via the photopolymerization of an allylated chitosan gel matrix, even though the swell ratio of the hydrogel is derivative (the crosslinker) and NIPAAm monomer. low at pH 7.4. As the feed ratio of the chitosan derivative to NIPAAm Figure 12 depicts the cumulative BSA release pro- monomer was increased, the pH sensitivity of the files for the hydrogels. The initial release rate of pro- hydrogels also increased. The interior network struc- tein from the hydrogels was rapid, but it decreased tures of the hydrogels became more porous in acidic after several hours. This initial burst release may be media. The temperature sensitivity of the swelling ra- attributed to the rapid diffusion of BSA that was load- tios of the hydrogels could be adjusted by varying the ed close to the surface of the hydrogel due to the pH of the medium containing the hydrogel and the deswelling of the drug-loaded hydrogel in buffer solu- formulation of the hydrogel itself. The mechanical tion at pH 7.4 and 37 °C. Later on, protein was properties of the hydrogels were therefore found to be released more slowly from the hydrogels. This was dependent on pH and temperature, as well as the com- mostly because the hydrogel forms a tight polymer position of the hydrogel. In vitro release results network in buffer solution at pH 7.4 and 37 °C, effec- showed that the pH of the medium containing the tively retarding the diffusion of BSA from the hydro- hydrogel had a strong effect on the release rate of gels. The release of BSA from the hydrogels is MO from the hydrogel matrix due to the interactions considered to be mostly controlled by diffusion, similar between the MO molecules and the chitosan in the to the release characteristics seen in our previous study hydrogel network, and the release of BSA from the. As can be seen from Fig. 10, the BSA release hydrogels could be controlled by adjusting the compo- profile was found to decrease as the feed ratio of sition of the copolymerized hydrogel in buffer solution NIPAAm to chitosan derivative was increased in buffer at pH 7.4 and 37 °C. J Polym Res (2012) 19:9944 Page 9 of 9 Acknowledgements The authors are grateful for the financial sup- 17. Yu LMY, Kazazian K, Shoichet MS (2007) J Biomed Mater Res port provided by the Nature Science Foundation of Hubei Province 82A:243–255 (2010CDB04903), the Foundation of Hubei Educational Committee 18. Mourya VK, Inamdar NN (2008) React Funct Polym 68:1013– (Q20101603), and SRF for ROCS, SEM. 1051 19. 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