Response of fibroin matrices to enzymes of infected wounds

Infection is more than ever a global problem of traumatic, post-surgical or chronic wounds. Patients with infected wounds often suffer from pain, impairment or social isolation. The quality of life can be reduced dramatically and in worst-case lead to hospitalization and eventually sepsis and death. The early treatment of an incipient wound infection is crucial for the healing process. But so far medical doctors still have to rely on conventional methods for the diagnosis of wound infection which are based on the evaluation of classical signs like rubor (redness), calor (heat), tumor (swelling) and dolor (pain). Other methods like the cultivation of bacteria are even more time consuming. Consequently the main problem with the methods used today is that an infection can only be reliably diagnosed in a late stage. A diagnostic device for the early diagnosis of wound infection would therefore be a great benefit for clinicians and patients. Recently, the enzymes lysozyme, neutrophil serine proteases like elastase or cathepsin G and myeloperoxidase produced by neutrophil granulocytes were successfully tested for their use as infection markers as they show enhanced activity in infected wounds. By detecting the activity of these enzymes using colored marker molecules released from a matrix upon enzyme reaction, the development of wound infections could be monitored and controlled. The biomaterial silk fibroin would be an ideal candidate working as a matrix for the described sensor. This material has outstanding mechanical properties and the protein structure is ideal to serve as a substrate for elastase or proteases present in infected wounds. Hence an indicator molecule entrapped in a silk fibroin matrix could be released upon enzyme degradation and give a visible signal indicating the presence of elastase. The specific aim of this project is to investigate silk fibroin based matrices with potential in sensors for early stage wound infections. Different variations of silk fibroin scaffolds based on particles or capsules will be studied. The particles will be analyzed in detail regarding formulation, stability, loading levels and release kinetics of signal molecules in combination with enzymatic degradation by elastase. Additionally, a screen of other enzymes in wound fluids will be performed to either exclude a non-specific signal release by non-infected wounds or to broaden the utility of the sensor as other infection specific enzymes that could also have the ability to release signal molecules. Together with the host, Prof. David Kaplan, who has a broad expertise on bio-inspired and high technology materials based on silk fibroin for medical applications and the applicants expertise on protein nanoparticles this collaboration is the ideal basis to achieve the major goals of this proposal.

The natural protein silk fibroin was investigated for its applicability in sensors for the detection of wound infections. Infection is more than ever a global problem of traumatic, post-surgical or chronic wounds. Patients with infected wounds often suffer from pain, impairment or social isolation. The quality of life can be reduced dramatically and in worst-case lead to hospitalization and eventually sepsis and death. The early treatment of an incipient wound infection is crucial for the healing process. But so far medical doctors still have to rely on conventional methods for the diagnosis of wound infection which are based on the evaluation of classical signs like rubor (redness), calor (heat), tumor (swelling) and dolor (pain). Other methods like the cultivation of bacteria are even more time consuming. Consequently the main problem with the methods used today is that an infection can only be reliably diagnosed in a late stage. A diagnostic device for the early diagnosis of wound infection would therefore be a great benefit for clinicians and patients. Recently, the enzymes lysozyme, neutrophil serine proteases like elastase or cathepsin G and myeloperoxidase produced by neutrophil granulocytes were successfully tested for their use as infection markers as they show enhanced activity in infected wounds. By detecting the activity of these enzymes using colored marker molecules released from a matrix upon enzyme reaction, the development of wound infections could be monitored and controlled. In this project matrices from silk fibroin a protein naturally produced by the silk worm Bombyx morii were investigated regarding their potential as sensor material. In an early stage silk fibroin films were prepared and functionalized, to study the effects of various dye molecules on the protein and to perform degradation studies with different elastases. It was proven that silk fibroin indeed is accepted as a substrate by human neutrophil elastase. Hence, in a next stage new methods for the production of functionalized silk particles were established to provide a suitable material for later industrial applications like printing the substrates on sensor surfaces. A new natural substrate for infection marker enzymes was discovered and optimized for its use in sensors for the fast and early detection of wound infections.