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Jochen Reinbold,1 Teresa Hierlemann,1 Helena Hinkel,1 Ingrid Müller,2 Martin E Maier,3 Tobias Weindl,4 Christian Schlensak,1 Hans Peter Wendel,1 Stefanie Krajewski1 1Department of Thoracic, Cardiac and Vascular Surgery, University Hospital Tuebingen, Tuebingen, 2Department of Pharmaceutical Engineering, Albstadt-Sigmaringen University, Sigmaringen, 3Institute of Organic Chemistry, University Tuebingen, Tuebingen, 4Aimecs GmbH, Pfarrkirchen, Germany Abstract: Biodegradable polymers, especially poly(lactide-co-glycolide) (PLGA), have good biocompatibility and toxicological properties. In combination with active ingredients, a specialized drug delivery system can be generated. The aim of the present study was to develop a drug delivery system consisting of PLGA microspheres loaded with the natural active ingredient totarol, which has several antimicrobial mechanisms. Totarol, isolated from the Podocarpus totara tree, was purified using column chromatography, and the eluate was checked for purity using thin layer chromatography. The spherically shaped microspheres with mean diameters of 147.21±3.45µm and 131.14±3.69µm (totarol-loaded and -unloaded microspheres, respectively) were created using the single emulsion evaporation method. Furthermore, the encapsulation efficiency, in a range of 84.72%±6.68% to 92.36%±0.99%, was measured via UV/vis spectroscopy. In a 90-day in vitro drug release study, the release of totarol was investigated by UV/vis spectroscopy as well, showing a release of 53.76%. The toxicity on cells was determined using BJ fibroblasts or Human Embryonic Kidney cells and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, which showed no influence on the cell growth. The minimal inhibitory concentration was ascertained. A totarol concentration between 64µg/mL and 128µg/mL was necessary to inhibit the bacterial growth over a period of 24hours. Biofilm formation on the surface of totarol-loaded microspheres was determined using transmission electron microscopy. No biofilm formation could be detected, even if the totarol concentration was below the minimal inhibitory concentration. The hemocompatibility investigations on various markers with fresh heparinized blood (1.5IU/mL) showed that totarol and totarol-loaded microspheres have no influence on different blood parameters. The PLGA microspheres characterized by slow release of totarol and great entrapment efficiency represent a novel drug delivery system, which may be highly beneficial for the long-term therapy of bacterial infections. Keywords: totarol, drug delivery system, Staphylococcus aureus, antibacterial, cytotoxicity

Background During cardiac surgery with heart-lung-machine support, abdominal swabs are routinely used to adsorb blood from the operation field. In part, abdominal swabs exhibit procoagulant activity, which is usually considered harmless. However, coagulation induction and abnormal clot formation on the surface of abdominal swabs in the operation field may, if the blood is retransfused into the extracorporeal circuit, lead to severe thromboembolic complications. The aim of the present study was to elucidate the origin of the unexpected blood clotting upon contact with hypercoagulant swabs. Methods The coagulant properties of three abdominal swabs were characterized using a simple clotting test and human whole blood, which was anticoagulated with different heparin concentrations. Eluates prepared from the abdominal swabs and the color stabilizer polydiallyamine (PDAA) were incubated with blood and blood clotting was investigated. Furthermore, the effects of the abdominal swabs on blood clotting time and on heparin were investigated. Results Our data show that the three abdominal swabs as well as the respective eluates exhibit distinctive coagulant properties. The abdominal swab with the highest hypercoagulant effect significantly reduced blood clotting time and also led to a reduction in free heparin. PDAA does not induce activation of the coagulation system. Conclusion The data indicate that the hypercoagulant swab reduces the clotting time and the concentration of free heparin. Abdominal swabs used during complex cardiac surgery with heart-lung-machine support should definitely be tested for their coagulant properties using appropriate tests before clinical applications, as it cannot be specified what leads to their hypercoagulant property.