Entwicklung und Charakterisierung nanoskaliger Lipidformulierungen als Ultraschallkontrastmittel zur sonothrombolytischen Therapie

In this work development, characterisation and first in vitro experiments with a new, nanoscaled ultrasound contrast agent (NUSCA) are presented. The aim of this work was improving the therapy of thromboembolic diseases with the use of a NUSCA. Chapter 1 gives an introduction into the topic of this thesis and chapter 2 summarises the methods used. In chapter 3 the influence of different preparation methods und lipid mixtures on the physicochemical properties of the NUSCA’s are shown. Effects of extrusion, lyophilisation, DPPG addition and changes of PEG40S concentration in combination with additional sonication with an US-tip on size, Zetapotential and structure of the NUSCA were examined. Liposomal structure was visible for DPPC/CH formulations while PEG40S containing formulations form a mixture of liposomes and micelles. Extrusion led to a reduction of hydrodynamic diameter of all the formulations while lyophilisation, as a possibility to increase storage stability, increases particle size. Addition of the negative charged lipid DPPG to prevent particle aggregation didn’t change particle size and structure but the Zetapotential decreases to less than -20 mV. Additional sonication with an ultrasound-tip decreases the size of the formulation. An influence of PEG40S concentration on the structure as well as the hydrodynamic diameter could be shown. The echogenicity of the NUSCA was tested in a custom build flow-model and the results are summarised in chapter 4. Variations of the preparation method had mostly negative effect on the echogenicity. After extrusion no contrast enhancement was measurable for DPPC/CH liposomes and the echogenicity of PEG40S containing formulations was weakened. Lyophilisation, normally known to increase echogenicity, also reduces the contrast enhancement of our formulation, beside an addition of PEG4000 to the DPPC/CH liposomes. Depending on the concentration of DPPG the echogenicity increases, but never exceed echogenicity of formulation without DPPG. Additional sonication increased the contrast enhancement dependent on the PEG40S concentration. The ratio of liposomes to micelles seems to influence the echogenicity, however a difference between DPPC and DSPC formulations was found. The miscibility of the compound was therefore investigated in chapter 5. Langmuir monolayer studies revealed miscibility in fluid-expanded phase for DPPC and PEG40S while DSPC and PEG40S were not miscible. Epifluorescence measurements after adding a fluorescent labelled lipid ensure these results. Based on these results the regime of PEG-chains was derived as a further explanation for PEG40S concentration dependent echogenicity of the formulations. For the DPPC/PEG40S formulations the PEG chains exist longer in the supercoiled mushroom regime due to the equal distribution of the PEG40S on the surface. Increase of the PEG40S concentration leads to interaction of the PEG chains and thus a change of the conformation to brush regime. For the immiscible DSPC and PEG40S a formation of PEG40S domains was visible. A strong interaction of the PEG chains in this domains cause stronger PEG chain interaction at lower concentration and thus an early change of the regime. PEG chains in the brush regime extend further into the surrounding medium leading to stronger interaction with ultrasound (US) and thus higher echogenicity. Chapter 6 summarises the results of an in vitro sonothrombolysis study using the best NUSCA. Blood clots of human whole blood were exposed to diagnostic US and different combination of thrombolytic drugs and NUSCA. The clot mass loss was higher than in comparable in vitro studies. Furthermore an effect of the NUSCA on the fibrin network could be proven. NUSCA and US lead to a clearly visible increase in pore size of the fibrin network. After treatment with US, NUSCA and thrombolytic agent no fibrin could be detected at the thrombus surface. These results make our NUSCA a promising new approach for sonothrombolytic therapy.