Team:TU Darmstadt/project/chemistry
ChiTUcare
Chemical modification of Chitosan for the use in protease-sensing wound coatings.
Chitosan can be modified at the amino group with succinyl anhydride and a variable peptide with a fluorogenic substrate to form a reliable structure to detect proteases. In this study, we reproduce the findings of the paper “Enzyme-Sensing Chitosan Hydrogels” by Mir Morteza Sadat Ebrahimi and Holger Schönherr from the university of Siegen [1] and use the fluorogenic substrate alanyl-alanyl-phenylalanine-7-amido-4-methylcoumarin (Ala-Ala-Phe-AMC) to detect α-chymotrypsin. This protease is secreted by Staphylococcus aureus or Pseudomonas aeruginosa that are examples of pathogenic bacteria that can infect wounds.
Introduction
Badly healing wounds are still a big issue in clinical medicine all over the world. Especially inflamed wounds often exhibit impaired healing properties and are prone to infections of opportunistic pathogenic bacteria. On the one hand, wounds have to be screened for infections extensively, on the other hand, they have to be kept wet and in an oxygen-free atmosphere for optimal healing conditions. Thus there is an obvious contradiction between the best healing conditions and the commonly used infection swab test,where the wound coating has to be removed. Furthermore, current swab tests need a few days to evaluate the presence of pathogenic bacteria, but it is important to get this information as soon as possible and to start the suitable treatment. Ebrahimi and Schönherr developed a quick and non invasive detection method for wound infections without the necessity to remove the wound coating. The principle of the test is the modification of an amino group of chitosan with succinic anhydride to gain a carboxyl group that can be linked to the amino group of our alanyl-alanyl-phenylalanine peptide linker. This peptide linker is fused to our actual detectable unit, the methylcoumarin.
This linker was chosen due to the fact that chymotrysin cleaves peptides n-terminal of aromatic amino acids, so here it would cleave n-terminalof Phenylalanin.
In our studies, we present an uncomplicated method to repeat the work of Ebrahimi and Schönherr in a way that works for iGEMers and FabLabers. For that, we combined a few instructions to guarantee a working product without the need of expensive instrumental analysis.
Methods
Material:
Chitosan (high molecular weight, 310-375 kDa, >75% deacetylated), succinic anhydride, ala-ala-phe-7-amido-4-methylcoumarin,
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-Hydroxy-2,5-pyrrolidindion were purchased from Sigma-Aldrich.
Acetic acid and Acetone were given from AK Fessner (TU Darmstadt).
We used 250 mL-three-necked flask with a dropping funnel, a spin coater and laboratory shakers.
Laboratory and Lab equipment were provided by AK Fessner and AK Kolmar (TU Darmstadt) and the centrifuge was provided by AK Hausch. Spin-coating was performed in the Laboratory of Prof. Koeppl (TU Darmstadt).
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.
Results
To determine the best combination of amounts and concentrations for the preparation of the NCS film we made four different films.
| Nr. | Chitosan | Acetic acid | Results |
|---|---|---|---|
| 1 | 2g | 80mL, 1% wt | Stable, even film |
| 2 | 2g | 80mL, 0,5% wt | Much too dry immediately after coating |
| 3 | 2g | 100mL, 2% wtt | Stable, even film |
| 4 | 0.7g | 80mL, 1% wtt | Much too fluid, no stable film |
Films Nr.1 and Nr.3 were used for further treatment.
Acknowledgements:
We would like to thank Prof. Schönher from the University of Siegen for helping us with the problems in our early project. We also thank Prof. Kolmar and Prof. Fessner for the opportunity to use their Laboratories. Additionally, we thank Marie-Luise Reif for supporting us in the chemical Lab. We express our special gratitude to Dr. Avrutina who helped us with many different issues and always had an ear for our problems and missing chemicals and enzymes. In addition to that, we want to thank Prof Koeppl for using his Spin Coater and Francois-Xavier Lehr and Tim Prangemeier for showing us how to use it.
References
| [1] | Enzyme-Sensing Chitosan Hydrogels
Mir Morteza Sadat Ebrahimi and Holger Schönherr*
Physical Chemistry I, Department of Chemistry and Biology, University of Siegen, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany
DOI: 10.1021/la501482u |
