ARTICLE pubs.acs.org/Biomac
Synthesis of Multiresponsive and Dynamic Chitosan-Based Hydrogels for Controlled Release of Bioactive Molecules Yaling Zhang,† Lei Tao,*,† Shuxi, Li,† and Yen Wei*,†,‡ †
Department of Chemistry and ‡Key Lab of Organic Optoelectronic & Molecular Engineering of Ministry of Education, Tsinghua University, Beijing, 100084, P. R. China
bS Supporting Information ABSTRACT: An inexpensive, facile, and environmentally benign method has been developed for the preparation of multiresponsive, dynamic, and self-healing chitosan-based hydrogels. A dibenzaldehyde-terminated telechelic poly(ethylene glycol) (PEG) was synthesized and was allowed to form Schiff base linkages between the aldehyde groups and the amino groups in chitosan. Upon mixing the telechelic PEG with chitosan at 20 °C, hydrogels with solid content of 48% by mass were generated rapidly in 2000 u/mg), pyridoxyl hydrochloride (PL-HCl, Sangon Biotech > 99%), rhodamine B (Sigma, ∼95%), Micrococcus lysodeikticus (Ml cell, Sigma), and lysozyme (from chicken egg white, Sigma, 88015 u/ mg) were used as purchased. Tetrahydrofuran (THF) was stored over sodium under a nitrogen atmosphere and distilled prior to use. 2.2. Measurement and Statistical Data Analysis. 1H NMR spectra were recorded on a JEOL 300 MHz spectrometer. Multiplicities were reported as singlet (s), broad singlet (bs), doublet (d), triplet (t), and multiplet (m). FT-IR analyses were carried out using a Perkin-Elmer Spectrum 100 FT-IR spectrometer. UVvis absorption spectra were recorded on a Perkin-Elmer LAMBDA 35 UV/vis system. Rheology analyses were performed on a TA-AR2000ex rheometer with parallel plate geometry (40 mm in diameter) at 37 °C. The statistical data analyses were performed using SPSS 15.0.
3. METHODS 3.1. Synthesis of Difunctionalized PEG (DF-PEG). PEG2000 (3.26 g, 1.63 mmol), 4-formylbenzoic acid (0.98 g, 6.52 mmol), and 2895
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Figure 1. (a) 1H NMR spectrum of DF-PEG in CDCl3 and (b) FT-IR spectra of DF-PEG and PEG2000.
Figure 2. (a) Storage modulus G0 and loss modulus G00 analyses during gelation process (37 °C; frequency: 1.0 Hz; strain: 5.0%). (b) Storage modulus G0 values of hydrogels with different solid % and CHO/NH2 (37 °C; strain: 5.0%). DMAP (0.050 g) were dissolved in 100 mL of dry THF, followed by the addition of DCC (1.68 g, 8.15 mmol) under a nitrogen atmosphere. The system was stirred at 20 °C for 18 h; then, the white solid was filtered. The polymer was obtained as a white solid after repeated dissolution in THF and precipitation in diethyl ether for three times. Upon drying, 3.00 g of dialdehyde-functionalized PEG (DF-PEG) was obtained in 79.8% yield. The synthesis and characterization of monofunctionalized PEG (MF-PEG) were described in the Supporting Information (Figure S1). 1 H NMR (300 MHz, CDCl3, δ): 10.10 (s, 2H, CHO), 8.22 (d, J = 8.3 Hz, 4H, CHCCHO), 7.96 (d, J = 8.3 Hz, 4H, CHCHCCHO), 4.524.49 (m, 4H, COOCH2), 3.863.83 (m, 4H, COOCH2CH2), 3.693.58 (m, 172176H, OCH2CH2O). IR (KBr): ν (cm1) = 3490, 2882, 1976, 1717, 1466, 1345, 1280, 1104, 961, 842. 3.2. Hydrogel Preparation. A 3% (w/w) chitosan solution was prepared by dissolving certain amounts of chitosan in 2.1% (w/w) acetic acid aqueous solution. A 20% (w/w) DF-PEG solution was obtained by dissolving 1.0 g of the polymer in 4.0 g of distilled and deionized water. As a typical hydrogel preparation, DF-PEG solution (0.25 mL) was added to chitosan solution (0.70 g) at 20 °C. The gelation occurred within ∼3040 s of vortex. (See the video in the Supporting Information.) The PEG and MF-PEG were used as controls to mix with chitosan solution under the same conditions, and no gel formation was observed (Supporting Information, Figure S2). The gels for all other analyses were prepared using the same procedures. All % concentrations of solutions were presented based on mass (w/w). The hydrogels containing rhodamine B or lysozyme were incubated at 20 °C for 6 h prior to the controlled release experiments. 3.3. Rheology Test. A series of hydrogels were prepared with different DF-PEG/chitosan ratios to test their mechanical properties. As
Figure 3. Appearance of self-healing hydrogels versus time (5% gelatin gel as control). a typical operation, chitosan solution (0.70 g, 3% in acetic acid aqueous solution) was spread on a parallel plate (diameter: 40 mm). Then, DFPEG aqueous solution (0.27 g, 20%) was evenly added dropwise onto the chitosan solution surface. The storage moduli G0 and loss moduli G00 were measured as a function of time (Figure 2a). For the modulus values versus frequency analyses, the samples were prepared using the same method and incubated at 37 °C for 40 min, followed by the data collection (Figure 2b).
3.4. Self-Healing Experiment 1) Two hydrogel discs were prepared using the above-mentioned method, with a trace amount of rhodamine B added in one disk to 2896
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Figure 4. Rheology analyses of the self-healing process. (a) Storage modulus G0 and loss modulus G00 of original (solid %: 3.8, CHO/NH2: 0.055; 37 °C) and self-healed hydrogels. (b) G0 and G00 versus time during the self-healing process (frequency: 1.0 Hz; strain: 1.0%). (c) G0 and G00 on strain sweep. (d) G0 and G00 in continuous step strain measurements.
Figure 5. pH sensitivity of the hydrogels. (a) Origin hydrogel; (b) hydrogel decomposition after adding HCl aqueous solution; (c) regeneration of the hydrogel after adding NaOH aqueous solution; (d) decomposition of regenerated hydrogel after adding HCl aqueous solution; and (e) regenerated hydrogel after six cycles. give color difference. These hydrogels were cut into two pieces, and two different colored semicircles were put together to form a united disk. A hole (diameter: 0.9 cm) was punched in the middle of the united gel, and photographs at different time intervals were taken to record the appearance of the united gel. As a control, a hot gelatin aqueous solution (5%) was cooled to form a hydrogel disk (Figure 3). 2) Rheology analyses were carried out to monitor qualitatively the self-healing process. In brief, a gel was prepared as abovedescribed (solid %: 3.8, CHO/NH2: 0.055) and tested for the storage moduli G0 (∼1150 Pa, Figure 4a). The gel was subsequently cut into 16 pieces on the plate, and the G0 values versus time of the broken gel were recorded (Figure 4b). The G0 versus shear stress was also carried out, and the G0 of gel decreased quickly when the strain (γ) was g100%. Thus, the profile of G0 values to different amplitude were subsequently tested. Amplitude oscillatory forces were changed from γ = 200 to 20% under the
same frequency (1.0 Hz) to test the recovery of mechanical properties of the hydrogel, and the process was repeated twice. 3.5. Multiresponsive Analyses. The responses of the hydrogels (0.45 g, solid %: 6.0; CHO/NH2: 0.36) to water (1.0 mL), vitamin B6 derivative (PL-HCl, 1.0 mL, 50 mg/mL), amino acid (lysine, 1.0 mL, 100 mg/mL), and enzyme (papain, 1.0 mL, 50 mg/mL) solutions were tested and shown in the Supporting Information (Figures S5 and S6). Changing pH was chosen as a typical stimulus and described as follows: The hydrogel was prepared by mixing 3% chitosan solution (0.70 g) and DF-PEG (250 μL, 0.27 g), as mentioned above, and trace rhodamine B was added for better observation. Concentrated HCl aqueous solution (60 μL, 6 M) was added to the hydrogel, and the hydrogel was liquefied in ∼5 min with vortex. Subsequent addition of concentrated NaOH aqueous solution (60 μL, 6 M), neutralized acid, and the hydrogel was regenerated in ∼60 s. This process could be repeated at least 6 times in our experiments, and the hydrogel could still be regenerated (Figure 5). 2897
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Figure 6. Release of rhodamine B from the hydrogel system under different conditions at 20 °C. (a) Photos of release samples at 0 h; (b) photos of release samples at 2.5 h; and (c) plots of cumulative release of rhodamine B (measured at 555 nm) versus time, in which the absorbance of chitosan + rhodamine B solution at the same concentration was defined as 100%. Data represent mean ( SD (n = 3, * and ** indicate p < 0.05 and 0.01, respectively).
Figure 7. (a) Bioactivity of lysozyme incubated with all elements in release experiment. Data represent mean ( SD (n = 3, * and ** indicate p < 0.05 and 0.01, respectively). (b) Bioactivity of released lysozyme from hydrogel system at 37 °C under different conditions (lysozyme/chitosan and lysozyme/ papain were used as controls as denoted with $), and the bioactivity of lysozyme in water is defined as 100%. Data represent mean ( SD (n = 3, * and ** indicate p < 0.05 and p < 0.01, respectively).
3.6. Controllable Releases of Rhodamine B. Four hydrogels were prepared in four vials by mixing chitosan solutions (0.33 g, 2.5% in acetic acid aqueous solution), DF-PEG solutions (100 μL, 0.12 g, 20%), and rhodamine B aqueous solution (5 μL, 1.0 mg/mL) with approximately 3040 s of vortex. The hydrogels were incubated at 20 °C for 6 h; then, four different stimuli, namely, pure water (1.0 mL), lysozyme (50 mg/mL, 1.0 mL), papain (50 mg/mL, 1.0 mL), and pyridoxyl hydrochloride neutral solution (PL-HCl, 50 mg/mL, 1.0 mL) were added to four vials separately. The control solution was prepared by mixing chitosan (0.33 g, 2.5% in acetic acid aqueous solution), pure water (100 μL + 1.0 mL), and rhodamine B aqueous solution (5 μL, 1.0 mg/mL), and the absorbance of the control solution at UV 555 nm was defined as 100%. We took out 100 μL of sample solutions every 30 min for UV analyses at 555 nm and then put them back in the systems. Before taking samples, the mixtures were shaken with ∼510 s of vortex, and photographs were taken (Figure 6). The statistical data analyses were performed using SPSS 15.0. 3.7. Lysozyme Bioactivity Analyses. Acetic acid, chitosan, DFPEG, PL-HCl, and papain were mixed with lysozyme to test their influences on the protein bioactivity. The lysozyme solutions mixed with different compounds were listed in the Supporting Information
(Table S1). The Micrococcus lysodeikticus (Ml) cells were employed as substrate to test the bioactivity of the protein because lysozyme could damage bacterial cell walls by catalyzing hydrolysis of 1,4-β-linkages between N-acetyl-glucosamine and N-acetylmuramic acid, resulting in lyses of the bacteria.28 Lysozyme aqueous solution (Supporting Information, Table S1, sample 6) was used as the control. Lysozyme-chitosan solution (Supporting Information, Table S1, sample 1) was chosen as a typical protein bioactivity analysis, and the operation was carried out as follows: Lysozymechitosan mixture (2 μL) was diluted to 200 μL using a PBS (pH 6.5, 100 mM) solution. Then, the diluted solution (6 μL) was mixed with Ml cell suspension (1.0 mg/mL, 194 μL), and the absorbance at 450 nm was recorded every 10 s for 3 min. The activity was calculated from the equation: A (unit/mL) = K/(0.001VD), where A is defined as relative lysozyme bioactivity, K is the slope of plot, V is the volume (mL) of sample solution, and D is the dilution coefficient. The relative bioactivity of lysozyme in aqueous solution (Supporting Information, Table S1, sample 6) was tested using the same method and defined as 100%. The results of relative lysozyme bioactivity with different compounds were shown in Figure 7a. 3.8. Releases of Lysozyme. Lysozyme was incorporated in the hydrogel system, and Ml cells were employed as substrate to test the 2898
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Biomacromolecules bioactivity of the released protein. Releases of lysozyme as triggered with various stimuli were carried out as follows. Two hydrogels were prepared separately by homogeneously mixing chitosan solution (100 μL, 3% in acetic acid solution), lysozyme aqueous solution (100 μL, 50 mg/mL), and DF-PEG aqueous solution (50 μL, 20%) together. After they were incubated at 20 °C for 6 h, these two hydrogels were separately added with PL-HCl (0.5 mL, 50 mg/mL) and papain (0.5 mL, 50 mg/mL) solutions. The mixtures were kept in a thermostatic shaker (300 r/min, 37 °C). Samples were taken at certain intervals for bioactivity analyses of released proteins using the same method as described above. The statistical analysis of data was performed using SPSS 15.0. The lysozyme/ chitosan and lysozyme/papain solutions were used as controls, respectively, and the bioactivity of lysozyme in aqueous solution was defined as 100% (Figure 7b).
4. RESULTS AND DISCUSSION 4.1. Polymer Synthesis. The dibenzaldehyde-functionalized polymer (DF-PEG) was prepared by esterification of hydroxylterminated PEG with 4-formylbenzoic acid. In the 1H NMR spectrum of the DF-PEG (Figure 1a), the ether methylene protons on polymer backbone gave signals at 3.64 ppm, and new peaks corresponding, respectively, to the aldehyde (10.10 ppm), benzene ring (8.22, 7.96 ppm), and ester methylene (4.51 ppm) groups were clearly observed. The integration ratios among 8.22, 7.96, 4.51, and 3.64 were 4/4/4/181, close to the theoretical value 4/4/4/174, indicating that all polymer chains were terminated with benzaldehyde groups at both ends. In the FT-IR spectrum (Figure 1b), the aldehyde and ester carbonyls could also be clearly identified (∼1715 cm1). 4.2. Hydrogel Preparation. Because of the conjugation, aromatic Schiff bases are more stable than their aliphatic counterparts,26,27 allowing for dynamic equilibriums between the Schiff base and the aldehyde and amine reactants in aqueous solutions.2931 The ester carbonyl on benzene ring might improve the stability of Schiff bases, leading to the surprisingly fast reaction between DF-PEGs and chitosan to form gel networks. When the DF-PEG aqueous solution (0.27 g, 20%) was mixed with chitosan (0.70 g, 3% in acetic acid aqueous solution), a hydrogel (solid: 7.7% by mass; CHO/NH2: 0.46) was created almost at the end of mixing process (20 °C,
