Introduction

Badly healing wounds are still a huge issue in clinical medicine all over the world. Especially inflamed wounds often exhibit reduced healing properties and are prone to infections of opportunistic pathogenic bacteria. On one hand, wounds have to be extensively screened for infections, 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, during which the wound coating has to be removed. Furthermore, current swab tests need a few days to be evaluated for the presence of pathogenic bacteria. It is important to get this information as soon as possible to be able to start 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 in chitosan with succinic anhydride. A carboxyl group that can be linked to the amino group of our alanyl‑alanyl‑phenylalanine peptide linker is gained. This peptide linker is fused to our actual detectable unit, the 4‑methylcoumarin.

The linker was chosen due to chymotrypsin's ability to cleave peptides behind their aromatic amino acids. In our case, it would cleave behind phenylalanine.

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 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 purchased from Roth.
We used a 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), while 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)

Preparation of saturated N‑Succinic Chitosan‑Hydrogel

To increase the saturation of the succinic‑chitosan binding we followed quite the same procedure like in the protocol of the preparation of N‑succinic chitosan except the point that the step where succinic anhydride in acetone was added was executed twice. Also the centrifugation was left out, because and replaced by just washing the hydrogel with a tiny amount of acetone to get rid of the non‑reacted succinic anhydride.

Preparation of thin-layer Chitosan/N-Succinic Chitosan

Spincoating is a suitable technique to prepare thin layers out of viscous fluids. In general, 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 evenly distributed to create the coating. We used microscope slides as a surface and coated it with our chitosan/N‑succinic chitosan mixture from the previous step. To determine the best fluidic properties, we performed four tests 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.

Succinic Saturation of the Chitosan-Film

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 were dissolved in 100 mL aceton in a glas petri dish. Our microscope slide with the thin‑layer chitosan/N‑succinic chitosan was placed inside. The petri dish was sealed with parafilm and shaken 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 2.5 g succinic anhydride in a closable glass bottle and and shook it for 2 hours at 60 °C. After the succinic anhydride was completely dissolved, we placed our microscope sildes inside the glass and continued shaking at 60 °C for 24 hours.

Grafting of Ala‑Ala‑Phe‑7‑Amido‑4‑methylcoumarin (Ala‑Ala‑Phe‑AMC) to the N‑Succinic chitosan‑film

The linkage of 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‑AMC in methanol was prepared. While shaking, the immobilized NSC film was immersed in EDC/NHS solution for 60 minutes. The film was rinsed with methanol. Afterwards, the NSC film was immersed in Ala‑Ala‑Phe‑AMP solution for 60 minutes. The film was rinsed again and immersed in methanol for 60 minutes. Every 15 minutes, the methanol was replaced. The film was rinsed one last time with methanol and afterwards dried.

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

Due to the fact that the saturated N‑succinic chitosan‑hydrogel is almost completely soluble in water, it is possible to carry out the coupling in a liquid phase. 100 mL of hydrogel were solved in the same amount of water. Subsequently, EDC (~ 1 mol: 1 mol COOH) and NHS (~ 1 mol: 1 mol COOH) were dissolved in methanol, as well as Ala‑Ala‑Phe‑7AMC in a different solution. The EDC/NHS solution was added to the reaction flask. After 60 minutes, while shaking, the Ala‑Ala‑Phe‑7AMC solution was added. After another hour under shaking, the hydrogel was filtered fast and dried at room temperature.

Spin Coat a thin layer of Ala-Ala-Phe-7-Amido-4-methylcoumarin-N-Succinic-Chitosanhydrogel

The hydrogel can either be dried at room temperature as described in the last entry, but it can also be spincoated on to another hydrogel to save material. For this purpose the hydrogels prepared by our hydrogel team were used. The compounds of the hydrogel were a 1:1 mixture of Agar and Chitosan in acetic acid.

Verification of the Ala-Ala-Phe-7AMC and the successful coupling to the Film and the Hydrogel

Fluorescence Emission via Fluorometer

To verify that the protease can actually cleaved the 7-AMC from the peptide, we checked the fluorescence emission of the solved peptide in methanol before and after the treatment with the protease. The Ala‑Ala‑Phe‑7AMC was solved in methanol and protease was added. The film was dipped in a protease solution and checked after 15 min. The hydrogel was moistened with protease solution and checked after 5 minutes. The 7‑AMC has an excitation wavelength of 325 nm, its emission wavelength lies at 450 nm and therefore in the visible spectrum. The used protease was bovine chymotrypsin.

Fluorescence Emission detectable by eye

For the cleavage we used bovine chymotrypsin. The dissolved peptide was put in a cuvette and protease was added. The fluorescence became visible with an ordinary UV lamp (366 nm) because of the emission wavelength of 450 nm (blue light) of the 7‑AMC. The film was dipped in a protease solution and also observed using UV light (366 nm). Last but not least, we moistened the hydrogel with protease solution and observed using UV light.

Results

The different Gels

To determine the best combination of amount and concentration for the preparation of NCS films and hydrogels, we produced five different gels with differing characteristics.

The first gel with the concentration of wt 1 % acetic acid showed a semi stable consistency and was really sticky on glassy surfaces. Due to these characteristics, it was easy to spincoat a flat even film on a microscope slide.

The second gel with the concentration of wt 0.5 %, showed a more liquid consistency than the first one but was still stable enough to form a hydrogel. When it came to the spincoating the film, it appeared that the thin layer was too dry and could not form a hydrogel. Moreover it seemed that the N‑succunic chitosan crystalized on the microscope silde, meaning that we could not modify it as a hydrogel.

The third gel with the concentration of wt 2 %, showed a much more stable and solid consistency than the first and the second gels. Nevertheless, we were able to spincoat it on a microscope slide, although the layer was not as even as the first gel.

The fourth gel with the concentration of 1 % but only 0.7 g of chitosan, did not create a gel. It was just liquid containing chitosan-gel fragments. It was not possible to spincoat a film with this solution.

The fifth gel with the concentration wt 1 %, where the succinic anhydride coupling was done twice, formed a stable film that could be dissolved in approximately double the amount of water. If it is dried, it forms a hydrogel again. When moistened with water, it swells. This procedure is repeatable. With the water you may be able to control the consistency. If the gel is spin coated, the best ratio of water and gel is 1:1. The film is even and very stable. It is even possible to form a thick gel layer when dried at room temperature overnight.

Films №1, №3 and №5 were used for further treatment.

The Fluorescence Emission

Measurement via Fluorometer

The Ala-Ala-Phe-7AMC/methanol solution has a autofluorescense peak at 390 nm before the protease was added.This wavelength is not visble for the human eye. After the cleavage with proteases, a shift of the wavelength peak to 450 nm was observed. Light with the wavelength of 450 nm is normal visble blue light. All the measurements were repeated with different concentrations of Ala‑Ala‑Phe‑7AMC and protease. It could be observed that with decreasing concentration of the Ala‑Ala‑Phe‑7AMC, the intensity of the fluorescence also went down. And with decreasing concentration of the protease, the effect of the digestion got lower until it was almost undetectable in 25 minutes. We decided that a time beyond this limit is not any more practical for a fast detection of the proteases.

Measurement via naked Eye

The Ala-Ala-Phe-7AMC/methanol solution in the cuvettes with and without protease was placed on a UV lamp table (366 nm). We came to the result that it is possible to observe fluorescence caused by proteases in concentrations common for inflamed wounds (up to 500 µg) [2]. A leading role of the visible feedback at such a low concentration has the concentration of the peptide in the gel (Figure. 5). At a concentration of 2 mmol it is still easy for the naked eye to see the fluorescence at protease concentrations of 500 µg.

Considering the outcome of the measurement with the dissolved peptide, the films and hydrogels confirmed the results. The detection is completed between 5 to 10 minutes and it is easy to spot the cleavage of the fluorescence dye under an UV lamp. In addition, it is enough to coat a thin layer of this hydrogel on a surface or another hydrogel to still have this strong effect.

It is possible to form a cheap, reliable enzyme detection system for proteases out of chitosan hydrogels, which is able to detect the proteases within 5 minutes.

Conclusion

The purpose of this study was to provide a principle technique for the production of an enzyme-sensing hydrogel out of biosynthetically produced chitosan, which is reliable and capable even for low budget laboratories. Especially the cheap production of the chitosan layers with the fluorophor peptides, capable of detecting pathogenic bacteria in wounds, was a huge success.
In the first experiments, we struggled to form a hydrogel that showed the wanted characteristics. That the hydrogel was not dissolvable in water was a problem at first. Subsequently, we decided to spin coat the produced hydrogels and modify the layers of the gels. This worked fine and the results were encouraging us to keep on going. Therefore, we decided to form a full, thick hydrogel, which we modified to be a complete detection system for proteases.
For this purpose, we get back to the succinic modification of the whole hydrogel not just the layer. After some trials, we were capable to saturate a hydrogel with the succinic anhydride. As the hydrogel is partly soluble in water, the coupling efficency of the peptide improved. After the desiccation, the gel showed perfect characteristics to be used as an easy way to detect whether proteases were present. If we apply these results to our problem of highly sensitive infected wounds, the hydrogel represents an easy and cheap solution for fast detection. Compared to the standard methods of infection detection, our smart protease sensing hydrogel is non-invasive and fast in detecting infections, without any expertise.

Outlook

In the future, this enzyme-sensing hydrogel could be optimized by adding individualized peptides to detect many different proteases. This system could be used to detect specific pathogenic bacteria in wounds based on the proteases excreted. There are plenty of applications that also can be solved by layers that are packed with peptides or maybe even whole enzymes. After further research, we should even be capable to link drugs or antimicrobial peptides to the hydrogels and use it as a treatment for several illnesses. Another point to be improved upon is to add chitosan with reduced molecular weight. We made the hydrogel from long polymers of chitosan, but if we were able to add the chitosan we produced in E. coli, it can have plenty of great effects on wound healing. Short oligomers, like the one the chitin deacetylases produces, increases the antimicrobial activity on gram-negative bacteria and reduces the overall activity of gram-positive bacteria. [3]. Another nice effect is the scar-free wound healing also connected to chitosan oligomers.
We encourage every iGEM team to improve upon this project to determine the way to a get a multifunctional wound healing tool.

Acknowledgements

We would like to thank Prof. Dr. Schönherr from the University of Siegen for helping us with the problems in our early project. We also thank Prof. Dr. Kolmar and Prof. Dr. Fessner for the opportunity to use their laboratories. Additionally, we thank Marie-Luise Reif for supporting us inside the chemical lab. We express our special gratitude to Dr. Avrutina who helped us with many different issues and always had an open ear for our problems. She also provided us with chemicals and enzymes. In addition to that, we want to thank Prof Koeppl for using his Spin Coater, as well as Francois-Xavier Lehr and Tim Prangemeier for showing us how to use it.

Group Picture

References

iGEM Technische Universität Darmstadt

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