Team:Groningen/Template/MODULE/project/bandage

Most people would think it is a bad thing to have a bacteria inside a bandage. And in many ways this makes a lot of sense, since most will damage wounds more then heal them. But this one won't! One of the biggest problems in hospitals right now, are drug resistant bacteria. However, despite the hesitation of a large group of people we think we can change a lot in bandages by having a inducible secreting system. In order to achieve a safe and easy solution a lot had to be discussed. This is our idea: Bacteria inside a hydrogel.

Lactococcus lactis would be the perfect candidate as a chassis for our engineered construct mainly because the unlikeliness of infecting humans and its ability to produce lactic acid. Of course there is a lot of expertise of L. lactis in our building which enables us to get acces to a lot of information. Before we started it was already proven that L. lactis cells can survive the harsh conditions of freeze drying¹. What also counted is that this bacteria is already used in production process in the dairy industry². These caracteristics make ourproduct more approachable to the public.

Although it is a relatively save bacteria, it is still a risk to have bacteria this close to the wound. That is why we designed the bandage, a containment device to store and activate bacteria at the right time. This bandage consists of 3 layers. A top membrane that diffuses gases like oxygen and carbon dioxide, a middle layer (a hydrogel) containing freeze dried bacteria and a bottom membrane that makes sure our bacteria stay inside the bandage. This last membrane also allows diffusion of several molecules/peptides.

Although the needs for this bandage were pretty clear from the start, finding the specific materials was harder than expected. For our top membrane a polymer from the iGEM 2012 team could be used. This is called TPX or polymethylpentene. TPX is available as a thin, transparent sheet, while it still stays strong. For our middle membrane tests with polyacrylamide gels are carried out. Although acrylamide is toxic when unpolymerized, it will no longer be toxic when polymerized and properly washed. Polyacrylamide was chosen because it is a cheap and easy to handle material. For our final product we could use a different hydrogel named PMBVF/PVA. This material is harder to handle and more expansive, but it has been tested before with bacteia inside³. Our last part is the bottom membrane. A Cellulose Nitrate membrane with a pore size of 0.2µm could be used. This is small enough to keep the bacteria inside and large enough for the molecules to diffuse freely through the membrane.

More information about the bandage design and the experiments are stated below.

We want to add a color system which shows whether there is an infection of S. aureus or P. aeruginosa in the wound. The detection will activate as soon as the first bacteria is in sight. For the testing the detection constructs can be coupled to a GFP(or a lumniscent) signal.

Our bandage should be transparent. In this way you can look through the bandage and judge whether it should be changed or it can stay on for another day, or whether you need to see a doctor. Seeing through the bandage without having to remove it is one of the criteria for the burn wound center as well. Of course the bandage should be as thin as possible. We are aiming at a maximum thickness of one centimeter and a minimum thickness of 3 millimeter.

We have to make sure our bacteria stays inside the gel. Although it is unlikely that L. lactis infects a human, it is possible when the immune system is compromised. We can make sure the bacteria can not leave the bandage by adjusting the pore-size of this gel. Another extra safety point is the usage of a bottom membrane. This membrane will have a pore-size of 0.2um to make sure our molecules can diffuse, but the bacteria are stuck inside. Since we are aiming for hospital usage the bandage can be autoclaved or burned after use, and thrown away in the regular waste.

The bandage should not stick, burns are painful wounds and we don't want to make it worse. Secondly it is important that the bandage can be activated easily. This should be within 30 minutes minimize the risk of getting infected. Also one of the requirements is the thickness the bandage, and mainly the thickness of the hydrogel. The thinner the bandage is the quicker the diffusion, and thus the quicker the treatment. Also the flexibillity of the bandage is important, the bandage should be waerable.

So far we have done a lot of experiments with acrylamide gels. We mainly wanted to proof that bacteria can survive extreme conditions and encapsulation in a gel. We realize it wouldn't be a perfect material for an end product, but for us it is an easy and quite cheap way of testing. There will be a lot of testing, this started with what percentage of acrylamide gave the best flexibility results for us, we started with using 10-15-20-25-30 % acrylamide. Here 30% and 25% were to sturdy and thick, the 20% seems to give the best results for us. It’s the right combination between sturdy and flexible. After this we've tried freeze drying the cells itself and checking whether these survive after they are rehydrated. Until now we’re not that certain about our results. After encapsulation in the gel our cells did show a lot of GFP. Unfortunately we haven’t seen a single cell divide inside the gel which of course is the best proof of a living cell. Another thing was checking if the cells can't leave the gel, this was checked by putting cells in the gel. These parts of gel were put in a GM17 medium overnight, and if there was growth inside the media the next morning the bacteria could come out. We've tried this with ruptured gel and healthy, we clearly saw that cells could come out more easy when it was ruptured.