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Today, imaging procedures in medicine are the most important diagnostics to recognize and locate diseases e.g. cancer, atherosclerosis or diseases of the cardiovascular system. Ultrasound diagnostics is an established and efficient procedure in this field. This technique uses sound waves, which are transmitted by an ultrasound head and sent non-invasively through the tissue where they are selectively reflected. The reflected sound waves are detected and used to generate images and diagnose diseases.
Ultrasound contrast agents can significantly improve imaging by providing contrast enhancement. Another benefit of such contrast-enhancing diagnostics is the visualization of target structures. Thus, the size of the contrast agents as well as their biological properties e.g. biocompatibility play a crucial role. Currently, commercially available ultrasound contrast agents are made from lipids, proteins or polymers. Polymers are particularly suitable for the development of novel ultrasound contrast agents. The advantages of polymeric materials are the wide range of known compounds with versatile physical and chemical properties combined with the possibility of manufacturing specific tailor made structures. So it is possible to adapt and modify polymers regarding density, melting point and electrical conductivity. These properties can be influenced by e.g. the choice of the monomer, the composition and interactions of the chain segments or the degree of polymerization. Furthermore, a variety of different polymerization reactions are established. In case of polyesters, polyamides and polyurethanes, the polymerization takes place via controlled chemical multi-step reaction. Therefore, the polymer grows stepwise by adding monomers. The chain growth reaction for olefinic unsaturated compounds, takes place over a continued prolongation. All methods can be used for the formulation of ultrasound-active diagnostic agents.
The primary objective of this work was the preparation and characterization of a novel polymer-based ultrasound-active contrast agent characterized by their nanoscaled size, biocompatibility and good contrast enhancement. Therefore, n-Butyl cyanoacrylate (Histoacryl®) was used monomer for polymer production and Polysorbate 80 (Tween 80) served as the surfactant for controlling the particle size and for stabilization. The first step involved the preparation of the polymer particles in a specific reactor under defined conditions. Subsequently, detailed physicochemical characterization of the particle dispersions was carried out using dynamic light scattering (DLS), dispersion analysis and laser Doppler anemometry. Using atomic force microscopy, the particles could be visualized and their by size and shape could be determined. The prepared polymeric nanoparticles showed hydrodynamic diameters between 8-12nm and zeta potentials of -6mV to -8mV. The 2% Tween 80 formulation has been proven to be the one with the smallest particles and lowest zeta potential. The following dispersion analysis showed a significant sedimentation process, indicating the presence of different particle size populations. This could be also confirmed by the change in transmission over time in turbidity measurements.
Visualization using an atomic force microscope provided a deeper insight into the morphology of the polymeric particles and confirmed their size. The phase image showed significant differences in the viscoelastic material properties between shell and core of the particles. An obvious explanation is given by the model that the rather hard polymer particles are coated with the "soft" surfactant (protective colloid). The diameter of the measured particles was in the range of 20-50nm. In fact, the visualized diameter is slightly increased compared to the DLS measurements, due to can be assumed particle/slide surface interactions. The second and technologically more complex chapter of the dissertation dealt with the preparation of the ultrasound contrast agents. For this purpose, the nanoscale polymer particles were used as the starting point for the production of air-filled ultrasound contrast agents in which they are used as protective colloids comparable to the known "Pickering" systems. The preparation of the nanoscale ultrasound contrast agent (so-called nanobubbles) was carried out by the application of ultrasound via an ultrasonic homogenizer (bar homogenizer). Depending on the ambient conditions, small air bubbles could be encapsulated and stabilized by the polymeric nanoparticles. Afterwards, the produced micro- and nanobubbles were separated using a Hamilton syringe and further determined by DLS. The measured diameters were between 200-800nm. However, all measured formulations showed a low storage stability. This has been observed as a large diameter increase (2800-3500μm) over time. By adding different NaCl concentrations an improved stability could be achieved.
The following detailed visualization of the nanobubbles was carried out by atomic force microscopy, as well as scanning electron microscopy, transmission electron microscopy and phase-contrast light microscopy. Again, atomic force microscopy revealed clear differences between shell and core. Scanning electron microscopy showed toroidal structures, due to implosion of the particles during sample preparation under vacuum. This confirmed the presence of gas inside the nanobubbles. Transmission electron microscopy clearly showed surfactant-coated structures in the range of about 250nm. Light microscopy was used as a final visualization technique. At this, a "glimmer" of the nanobubbles could be detected under the microscope, which indicates a phase, confirming the assumption of a gas-filled core. Biocompatibility testing was performed using the chorio-allantoic membrane model (CAM-Model) and MTT assay. The CAM model serves as an alternative to the animal model and showed no harmful effect on chicken embryos. On EDD 14 (Egg development day), i.e. four days after injection of the test solution, all chicken embryos survived. In the MTT assay, a cell survival rate of 99% could be observed from a particle concentration of 0.032g/l PBCA. The measurement of contrast enhancement was used to evaluate the effectiveness of the ultrasound contrast agent. For this purpose, the formulations were investigated in a custom build model and compared to the commercially used SonoVue®. The 2% Tween 80 formulation achieved the best contrast enhancement (46.56% ± 2.87% compared to SonoVue®). This relatively low value is nevertheless an excellent result considering the size and stability.
Imaging diagnostics is the most important tool detection and evaluating diseases. At the same time, it has become more and more important to reach specific targets. This defines new requirements to our existing contrast agents. This dissertation describes the preparation and characterization of a contrast agent, which copes with these new requirements and shows excellent biocompatibility.