Entwicklung und Charakterisierung von nanoskaligen, liposomalen Ultraschallkontrastmitteln zum Plaque-Targeting sowie Aufbau eines in vitro-Plaque-Modells

In this work, new, lipid-based formulations for use as nanoscaled ultrasound contrast agents and for targeting of atherosclerotic plaques were developed and characterized. The aim was a very good contrast enhancement in the use of diagnostic, non-invasive ultrasound at frequencies between 1 and 3 MHz. A method to determine elastic properties of nanoscale systems using AFM was developed and tested with PLGA nanoparticles. And finally an in vitro model has been established and the successful plaque targeting was shown. The basics of ultrasound and the use of ultrasound in diagnosis and therapy, and particularly the use of ultrasound contrast-enhancing agents have been shown in the introduction (Chapter 1). A brief overview of the vascular disease atherosclerosis, the visualization of the disease and in vivo characterization of the lesions were given. The methods used for the preparation of the ultrasound contrast agents and PLGA nanoparticles (film method, salting-out method), the physico-chemical and morphological characterization (PCS, LDA, cryo-TEM, 31P NMR, AFM), measurement of the ultrasound contrast-enhancing properties (flow model) and the results of fibrin targeting (in vitro plaque model) were explained in chapter 2. Chapter 3 dealt with the results of physico-chemical and morphological characterization of the liposomal formulations as well as of the polymer nanoparticles. It has been shown with PCS measurements and also with AFM and cryo-TEM that two size fractions occurred in the preparation of the liposomal ultrasound contrast agents. And with using the 31P-NMR it could be shown that micelles and liposomes were present together. The preparation of other lipid mixtures revealed similar results by PCS measurements. The successful coupling of antibodies to the liposomal formulations and the antigen-antibody interaction could be detected with the AFM. The successful control of the size of the PLGA nanoparticles with different stirring speeds during preparation with the Ultra-Turrax ®, was shown by PCS. Chapter 4 presented the results of the ultrasonic contrast measurements. With the use of a flow model, the contrast intensities could be determined and evaluated as mean gray values using the ImageJ software. All contrast agents were compared to SonoVue ®, a commercially available ultrasound contrast agent with bubbles sizes between 2 and 8 microns. Chapter 5 dealt with the results of the improvement of the contrast intensity by variation of the preparation method, the lipid composition, as well as freeze-drying. The various mixtures were again examined in the flow model. It turned out that the method of preparation had strong influence on the contrast. By the use of the ultrasonic tip, instead of the ultrasonic bath for preparation, significantly better results were achieved. The composition of the liposomal formulations was crucial for the contrast intensity. Lyophilization had also a large influence on the contrast enhancement and almost always led to an improvement in contrast. Even if the preparation of liposomal formulations was done with the ultrasonic bath and the formulations had a weaker ultrasonic contrast, this was after freeze-drying greatly improved. In chapter 6 elastic properties of polymer nanoparticles were measured with the atomic force microscope. Since the elastic properties of the liposomal ultrasound contrast agents could have an effect on the intensity of ultrasound contrast, this model system for the measurement of elasticity was developed. The polymer nanoparticles were prepared with different sizes, and measured. The experiments also revealed degradation phenomena of PLGA nanoparticles. Chapter 7 introduced a self-made in vitro plaque model to study the specific targeting properties. The successful targeting of artificial fibrin plaques, consisting of fibrin powder, and of human plaques, cut into small pieces, was shown. The liposomal formulations with coupled antibodies adhered even under flow on the artificial and human plaques and the plaques could be seen as bright spots in the ultrasound image.