Effect of ultrasound dosimetry on drug release from liposomes: Implications for development of novel acoustically sensitive liposomes
The aim was to design acoustic liposomes and develop ultrasound equipment. The liposomes containing encapsulated cytostatic drug are supposed to burst under ultrasound releasing their contents in a tumour. This supplementary technology will enhance the efficacy of chemotherapy by selective drug delivery and reducing drug dose and adverse effects.A series of studies were conducted related to the implementation and optimisation of ultrasound system for drug release experiments and concurrent release quantification methodology. 1) Ultrasound set-up "chamber system" has been established, which consists of the first chamber were an ultrasound transducer is placed, the second sample chamber where liposomes are injected, and the third chamber to dissipate ultrasound wave so that only direct wave emitted by the transducer affects the sample. Water is circulated between the first and the third chambers providing ultrasound transmission medium. The chambers are separated by a thin ultrasound transparent membrane. Reflections can be minimized by placing acoustic absorber (porous foam polyurethane) in the third chamber or by positioning the system at a certain tilting angle. The chamber system was designed in collaboration with CancerCure AS and manufactured at the workshop of the Radiumhospital. Acoustic power outputs and waveforms in the sample chamber were tested by oscilloscope. 2) Different compositions of liposomes were synthesized by CancerCure AS. Non-toxic fluorescent drug calcein was encapsulated in the liposomes as a model drug to study release profiles under various ultrasound and treatment conditions. Release profiles were evaluated using multivariate analysis, which allowed selecting most promising formulations. The selected liposomes showed higher drug release (50-100%) than Caelyx (around 20%) after 6 minutes exposure to ultrasound. For most formulations drug release increases at lower lipid concentration (higher dilution). For some formulations, release is higher at 37 degrees compared to that at 22 degrees centigrade. 3) Mechanical cavitation is the main cause for ultrasound mediated drug release from liposomes. Cavitation induces high temperature bursts up to 5000 K in a microenvironment, however resulting in an increase by only around 5 degrees in a bulk solution after 6 minutes of ultrasound exposure. Addition of a thermal inhibitor sulphur hexafluoride (SonoVue) into the liposomes prevented ultrasound mediated drug release by almost 50%. Sonochemical oxidation of radical scavengers under ultrasound was measured; however antioxidants did not significantly prevent drug release. 4) Release profiles of the same liposome formulation using different ultrasound generation setups were evaluated. For the proprietary Sonics generator and transducer, pressure measured in the second chamber was around 400 kPa peak-to-peak for 20 kHz and 230 kPa peak-to-peak for 40 kHz, which is above cavitation threshold (around 150 kPa peak-to-peak) and effectively induces drug release from the liposomes. However, the proprietary generator emits ultrasound at a fixed frequency. Performance of a non-proprietary broadband generator was tested. However, acoustic pressures were considerably lower resulting in practically no cavitation and low drug release profiles. The reason for this is a mismatch between electric output impedance of the generator and input impedance of the Sonics transducers. Further development of such a more sophisticated system would allow testing behaviour of liposomes in a wide range of acoustic powers (below and above cavitation threshold). In conclusion, drug release studies can now be routinely performed using chamber systems for two frequencies 20 kHz and 40 kHz. Developed liposomal formulations show 50-100% drug release after ultrasound exposure in vitro.