Development of a release system for antimicrobial substances based on an autologous plasma clot

Status:

completed 03/2014

Aims:

It was the purpose of this study to develop drug-loaded autologous plasma clots for the deposition and the release of antimicrobial substances for bone fracture therapy. These clots should be used for prevention as well as for treatment of infected bone defects.

Activities/Methods:

Platelet-free plasma clots containing antibiotics (5 to 300 µg/ml vancomycin, gentamicin, linezolid), 5 to 80 µg/ml silver ions (Ag-acetate) or PVP-silver-nanoparticles (spherical, Ø <75 nm) were produced. The bioactivity of antimicrobial agents released from plasma matrices was tested against Staphylococcus aureus using bacterial adhesion assay, the BacLight-cytotoxicity test and bacterial susceptibility tests (modified microdilution assay and modified agar diffusion assay). Tissue compatibility was analyzed by live/dead assay (calcein AM/propidium iodide) using mesenchymal stem cells (MSC). The release kinetics for each antimicrobial agent was carried out by reversed phase high-performance liquid chromatography (RP-HPLC) and atomic absorption spectroscopy (AAS).

Results:

All antibiotics showed a concentration-dependent maximal release from the plasma clots within 30 min. The duration of antimicrobial activity ≥ (MIC) was max. 6 days for gentamicin, max. 4 days for vancomycin and only max. 3 hours for linezolid. A correlated antimicrobial activity within the clot matrix and the inhibition of bacterial adhesion to clot surfaces were determined. Contrary to these antibiotics, release of silver acetate or silver-nanoparticles from plasma clots was inhibited. An antimicrobial effect in the respective eluates could not be achieved for PVP-Ag-nanoparticles and for silver acetate only above tissue toxic concentrations (200-500 µg/ml).

In contrast to linezolid, the release of gentamicin and vancomycin from plasma clots resulted in an antimicrobial activity lasting several days. Due to the lack of antimicrobial effect in the eluate and the plasma matrix, loading with tissue-compatible concentrations of silver ions (silver acetate) or silver-nanoparticles is not suitable.

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