Synthesis of chitosan and chitosan-catechol hydrogels 

Hydrogels were prepared in three main steps. First chitosan was purified. Then it was modified (or not) by grafting catechol groups. Third, a CH (or CH-Cat) solution was mixed with a gelling agent solution in order to create solutions gelifying around body temperature. These steps are described in greater details below.

Purification of chitosan 

In order to remove the impurities, the commercial powder was first purified as follows (Assaad et al., 2015). Six (6) grams of CH was dissolved in 600 ml of 0.1 M hydrochloric acid (HCl)

solution and stirred overnight at 40° C. The next day, the solution was filtered under vacuum to remove the insoluble particles. The solubilized CH precipitated with stirring by incorporating 0.5 M sodium hydroxide (NaOH) until the pH reached between 8 and 9. Then the mixture was heated to 95 ° C with stirring and 6 ml of sodium dodecyl sulfate (SDS) 10% (w / v) was added. The mixture was kept at 95 °C for 5 min, after which it was cooled to room temperature. The pH was then adjusted to 10 by adding 0.5 M NaOH. The mixture was filtered under vacuum and the precipitated CH was recovered and then washed five times in a beaker containing 600 ml of Milli-Q water, previously heated to 40 ° C, in order to eliminate traces of SDS. Finally, the CH was frozen overnight, freeze-dried for three days and then ground, sieved and stored .

Synthesis of chitosan-catechol (CH-Cat) 

Modifying chitosan by catechol was achieved by grafting hydrocaffeic acid to the carbonated chain of CH .

As detailed below, the protocol of synthesis of CH-Cat was modified from the work previously done by Professor Cerruti’s team at McGill University (Xu et al., 2015) .

The steps of this protocol (which will be named old protocol) were as follows:
A. Dissolve 0.6 gr chitosan in 60 ml deionized (DI) water and HCl (pH = 2.5)
B. Add HCA and EDC previously solvated in a water: ethanol 1: 1 mixture in stoichiometric proportions (1: 0.5: 1.17 of glucosamine: HCA: EDC respectively). Adjust the pH between 5 and 5.5 using 1M NaOH.
C. Let the reaction take place for 12 hours under stirring.
D. Dialyses the solution by using a dialysis membrane tube (MWCO 5,000, Spectrum Laboratories, USA) for three days against a solution of HCl pH 5.
E. Lyophilize the purified product and store it at -20 ° C.

By following the old protocol, the CH-Cat solution obtained after the twelve hours of reaction exhibited a red color, becoming more intense and darkened during the dialysis stages. After freeze-drying, the powder obtained formed a porous network, was pink in color.

Once dissolved in DI water for gel preparation, CH-Cat formed a brown and dense solution which was not permeable to light. Moreover, particles remained suspended in the liquid phase .

Gelling agents 

For this project, different gelling agents were used,  They were prepared using phosphate buffer (PB), BGP and SHC. The PB, at a pH of 8, was prepared by dissolving SPM and SPD salts at molar ratio of 0.073 in Milli-Q water. The SHC and BGP solutions were prepared by dissolving their salts in Milli-Q water. To prepare SHC-PB and SHC-BGP solutions, the SHC salt was solubilized in PB and BGP solutions respectively.

Preparation of chitosan (CH) and chitosan-catechol (CH-cat) hydrogels 

To prepare the CH (CH-Cat) physical hydrogels, the gelling agent solution was mixed with a CH or CH-Cat solution prepared as following:

CH hydrogels: The purified CH powder was solubilised in HCl (0.1 M for K-CH and 0.12 M for the H-CH) at 3.33% (w / v) with intensive stirring for about 3 h. The resulting solution was sterilized by autoclaving (20 min, 121°C) and then stored at 4 ° C .

CH-Cat hydrogel: CH-Cat powder was solubilised in DI water at 3.33% (w / v) with intensive stirring for about 3 h. CH-Cat solution was used freshly to make hydrogel. To study the influence of the pH on gel properties, CH-Cat was also prepared in aqueous solution containing various HCl concentrations (from 0 to 0.09 M).

The CH (CH-Cat) hydrogels were prepared at room temperature by mixing one of the gelling agents with the CH (CH-Cat) solution, by using two syringes and a luer-lock connector , at a volume ratio of 0.4: 0.6 respectively.

All hydrogels contain 2% w/v of CH (Assaad et al., 2015) or CH-Cat. The hydrogels names express their composition. In addition, the gelling agent names express their final concentration in hydrogels (SHC0075-PB004 gelling agent solution means in fact the initial concentrations are 0.19M SHC and 0.1M PB). For example, CH/ SHC0075-PB004 represents a hydrogel containing 2% (w/v) CH, 0.075 M SHC and 0.04 M PB .