Preparation of N-Succinic Chitosan (NSC)

Aqueous Acetic acid solution was put in the three-necked flask . Chitosan was added in small doses through the flask neck under heavy mechanical stirring. Therefore a lab stirrer was needed, not just a stir bar, because the solution becomes very viscous. The solution was stirred until becoming a homogenous semi-fluid. Now 20 mL of a saturated succinic anhydride solution in acetone were prepared and added drop-wise to the reaction flask under heavy mechanical stirring for 30 minutes. The reaction mixture was left overnight at room temperature. The following day, it was centrifuged to separate the precipitate from the left, non reacted Chitosan.

Preparation of thin-layer Chitosan/N-Succinic Chitosan

Spin coating is a suitable technique to prepare thin layers out of viscose fluids. In principle, a spin coater device works as following: Firstly, a surface is clamped onto a centrifuge. Through fast rotation of the surface, a fluid is then distributed evenly to create the coating. We used a microscope slide as surface and coated it with our Chitosan/N-Succinic Chitosan mixture from the previous step. To determine the best fluid properties we performed four test with different amounts of chitosan and acetic acid concentrations. Every coating step with the spin-coater was performed at 400 rpm for 20 seconds and then 2000 rpm for 60 seconds.

Succinyl saturation of Chitin

To ensure a sufficient amount of N-Succinic Chitosan in our product we performed a second succinylation of the thin-layer. For this step, we tested two different procedures.
1. Succinic anhydride in Acetone
2.5 g Succinic anhydride was solvated in 100 ml aceton in a glas petri dish and we put our microscope slide with the thin layer of thin-layer Chitosan/N-Succinic Chitosan in. The petri dish was sealed with parafilm and we shook it for 72 hours at 37 °C. The evaporated aceton was replaced every 12 hours
2. Succinic anhydride in DMSO
We put 50 ml DMSO and 2.5g succinic anhydride in a closable Glass and and shook it for 2 hours at 60 °C. After the succinic anhydride was completely dissolved we place your microscope sildes in the glas and continue shooking t 60 °C for 24 hours.

Grafting of Ala-Ala-Phe-7-Amido-4-methylcoumarin (Ala-Ala-Phe-AMC) to N-Succinic Chitosan

The linkage of the Ala-Ala-Phe-AMC was performed in two steps. First 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was dissolved in methanol and 1-Hydroxy-2,5-pyrrolidindion (NHS) was added. A separate solution of Ala-Ala-Phe-AMP in methanol was prepared. The immobilized NSC film was immersed in the EDC/NHS solution for 60 minutes while shaking. The film was rinsed with methanol. Afterwards, the NSC film was immersed in the Ala-Ala-Phe-AMP solution for 60 minutes. The film was rinsed again with methanol and immersed in methanol for 60 minutes, the methanol was replaced every 15 minutes. The film was rinsed for the last time with methanol an dried.

Characterization of Ala-Ala-Phe-AMP-NSC

Fluorescence Emission

To check if the coupling was sucessful we used UV fluorescence spectroscopy with a exication wavelength of 325 nm. A flourescence emission at 390 nm would mean a sucessful coupling.

Cleavage of the 7 AMC with bonvine chymotrypsin and the Fluorescence Emission

For the cleavage we used the bovine chymotrypsin. We put the enzyme in the buffer and dipped our produced hydrogel in the solution. We shook it at room temperature for 20 minutes and check the fluorescence with UV light immediately after the 20 minutes. After the cleavage the absorption should shift from 390 nm to 450 nm because the concentration of uncleaved product decreases while the concentration of free 7-AMC increases. This fluorescence emission is visible with the naked eye.