This information is available free of charge via the internet at http://pubs.acs.org/. Supporting Information Synthesis of Multi-responsive and Dynamic Chitosanbased Hydrogels for Controlled Release of Bioactive Molecules Yaling Zhang 1, Lei Tao 1*, Shuxi Li 1, Yen Wei 1,2* 1 Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China 2 Key Lab of rganic ptoelectronic & Molecular Engineering of Ministry of Education, Tsinghua University, Beijing 100084, P. R. China. Tel: +86-10-62792604 leitao@mail.tsinghua.edu.cn; weiyen@tsinghua.edu.cn. 1
1. Synthesis of mono functionalized PEG (MF-PEG). 43 H + H H DCC/DMAP 43 Methyl ether PEG (mpeg 1900, 2.00 g, 1.05 mmol), 4-formylbenzoic acid (0.66 g, 4.40 mmol) and DMAP (0.05 g) were dissolved in 50 ml of dry THF, DCC (1.10 g, 5.33 mmol) was added under nitrogen atmosphere. The system was stirred at 20 C for 20 h. After removing the white solid by filtration, the polymer was obtained as a white solid by precipitation from THF to diethyl ether for three times (1.80 g, 84.5 %). 1 H NMR (300 MHz, CDCl 3, δ/ppm): 10.10 (s, 1H, CH), 8.22 (d, J = 8.3 Hz, 2H, CHCCH), 7.96 (d, J = 8.3 Hz, 2H, CHCHCCH), 4.53-4.49 (m, 2H, CCH 2 ), 3.88-3.84 (m, 2H, CCH 2 CH 2 ), 3.71-3.56 (m, 168-169H, CH 2 CH 2 ), 3.39 (s, 3H, CH 3 ). IR (KBr): ν (cm -1 ) = 3493, 2883, 1976, 1717, 1467, 1345, 1279, 1104, 962, 843. The 1 H NMR and FT-IR spectra of the MF-PEG were exhibited as below: Figure S1. a) 1 H NMR spectrum of MF-PEG in CDCl 3 ; b) FT-IR spectra of MF-PEG and PEG 2000. 2. Hydrogel preparation. A 3 % Chitosan solution was obtained by dissolving certain amounts of chitosan into 2.1 % acetic acid aqueous solution. A 20 % DF-PEG solution was obtained by dissolving polymer (1.0 g) in deionized water (4.0 g). A typical hydrogel preparation was described as follow: 0.25 ml (0.27 g) of 2
DF-PEG solution was added into 0.70 g of chitosan solution, the gelation occurred with ~ 30-40 seconds of vortex. The PEG and MF-PEG were used as controls to mix with chitosan solution under same conditions, and no gel formed (Fig. S2). D 2 (~ 20 % v/v) was added in the mixture of MF-PEG/ chitosan solution, and 1 H NMR analysis was performed (Fig. S3). Figure S2. a) Mixtures of chitosan with PEG, MF-PEG and DF-PEG; b) upside down of the mixed solutions. Figure S3. 1 H NMR of monofunctionalized PEG with chitosan (CH/NH 2 : 1.0, ~ 20 % v/v D 2 ). 3
The gels for all other analyses were prepared using same methods. The hydrogels containing rhodamine B or lysozyme were incubated at 20 o C for 6 hours prior to the control-release experiments. 3. Rheological analyses of hydrogels. A series of hydrogels were prepared with different ratios of DF-PEG/chitosan to test their mechanical properties. A typical operation was described as below: Chitosan solution (0.70 g, 3 % in acetic acid aqueous solution) was spread on a round plate (diameter: 40 mm). Then DF-PEG aqueous solution (0.27 g, 20 %) was added dropwise evenly onto the chitosan solution surface. Then the data of storage modulus G and loss modulus G versus time were collected (Fig. S4). For the modulus values versus frequency analyses, the samples were prepared with same method and incubated at 37 C for 40 mins, then the data were collected. Figure S4. The Storage modulus G and loss modulus G analyses during gelation process (37 C; frequency: 1.0 Hz; strain: 5.0 %). a) solid %: 7.7, CH/NH 2 : 0.46; b) solid %: 3.4, CH/NH 2 : 0.027. 4. Multi-responsive analyses. 4.1 H 2 The hydrogel was prepared by mixing 3% chitosan solution (0.33 g) and DF-PEG (100 µl, 0.12 g) as mentioned above (soild %: 6.0; CH/NH 2 : 0.36), trace rhodamine B was added for better observation. Pure water (1.0 ml) was added to the hydrogel and the mixture was kept for 72 h, then a homogeneous swollen hydrogel formed (Fig. S5). 4
Figure S5. Hydrogel incubated in water. a) t = 0 h; b) t = 72 h. 4.2 Vitamine B 6 derivatives. Pyridoxyl hydrochloride (PL-HCl) neutral aqueous solution (1.0 ml, 50 mg/ml) was added to the hydrogel prepared using same method as above (0.45 g, solid %: 6.0; CH/NH 2 : 0.36). The mixture was kept at ambient temperature with gentle shaking and the hydrogel was decomposed after approximate 1.5 h (Fig. S6). 4.3 Amino acids. Lysine solution was neutralized using acetic acid and adjusted to 100 mg/ml with pure water, the amino acid solution (1.0 ml, 100 mg/ml) was added to the hydrogel prepared using same method as above (0.45 g, solid %: 6.0; CH/NH 2 : 0.36). The mixture was kept at ambient temperature with gentle shaking and the hydrogel was decomposed after approximate 3 h (Fig. S6). 4.4 Enzymes. Papain aqueous solution (1.0 ml, 50 mg/ml) was added to the hydrogel prepared using same method as above (0.45 g, solid %: 6.0; CH/NH 2 : 0.36). The mixture was kept at ambient temperature with gentle shaking and the hydrogel was decomposed after approximate 2 h (Fig. S6). Lysozyme was also used to incubate with the hydrogel and it was found no obvious decomposition of the hydrogel, indicating that digestion of chitosan by lysozyme under this condition was negligible. It is notable that this process was different with others for its irreversibility. 5
Figure S6. The different responses of hydrogels under different conditions, trace rhodamine B were added in hydrogels for better observation. a) t = 0 h; b) t = 1.5 h; c) t = 3h. 5. Lysozyme bioactivity with all ingredients in the hydrogel. Acetic acid, chitosan, PEG dimer, PL-HCl and papain were mixed with lysozyme respectively to test their effects on the protein bioactivities. The lysozyme solutions with different compounds were listed in Table S1. The Micrococcus lysodeikticus (Ml) cells were employed as substitutes to test the bioactivity of the released protein, and lysozyme aqueous solution (sample 6 in Table S1) was used as the control. Table S1. Lysozyme solutions mixed with different compounds. Lysozyme a Chitosan b DF-PEG c Acetic acid d PL-HCL e Papain f Water 1 100 100 50 250 2 100 50 100 250 3 100 100 50 250 4 100 100 50 250 5 100 100 50 250 6 100 150 250 a. Lysozyme was prepared in an aqueous solution (50 mg/ml). b. Chitosan was prepared in a 2.1 % acetic acid (w/w) aqueous solution (3.0 % w/w). c. DF-PEGs were prepared in an aqueous solution (20 % w/w). d. Acetic acid was prepared in an aqueous solution (2.1 % w/w). e. PL-HCl was prepared in an aqueous solurion (50 mg/ml). f. Papain was prepared in an aqueous solution (50 mg/ml). Total Lysozyme-chitosan solution (sample 1 in Table S1) was chosen as a typical protein bioactivity analysis, and the operation was carried out as below: 6
Lysozyme-chitosan mixture (2 µl) was diluted in 198 µl PBS (ph 6.5, 100 mm) buffer solution. Then 6 µl of the diluted solution was mixed with Ml cell suspension (1.0 mg/ml, 194 µl), and the absorbance at 450 nm were recorded every 10 seconds for 3 minutes. 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 graph, V is the volume (ml) of sample solution and D is the dilution coefficient. The relative bioactivity of lysozyme in aqueous solution (sample 6) was tested using same method and defined as 100 %. 7