MR-HIFU mediated local drug delivery using temperature-sensitive liposomes

Abstract

In this thesis, temperature-sensitive liposomes co-encapsulating the chemotherapeutic drug doxorubicin and the MRI contrast agent [Gd(HPDO3A)(H2O)] were investigated for the use of Magnetic Resonance-guided High Intensity Focused Ultrasound (MR-HIFU)-mediated local drug delivery. In Chapter 2, the preparation and in vitro characterization of different liposomal formulations is discussed. Two temperature-sensitive systems (LTSL and TTSL) were investigated and non-temperature sensitive liposomes were used as a control. The co-release of doxorubicin together with [Gd(HPDO3A)(H2O)], a paramagnetic MRI contrast agent, from the aqueous lumen of liposomes was studied in great detail. The composition of the lipid bilayer determined the leakage of doxorubicin at body temperature as well as the release kinetic at elevated temperatures. The LTSL showed a higher leakage of doxorubicin at 37 °C, but a faster release of doxorubicin at 42 °C compared to the TTSL system. The biodistribution of free doxorubicin and [Gd(HPDO3A)(H2O)] is well known, however encapsulation into liposomes alters the biodistribution of these compounds radically. Altered drug and contrast agent distribution, coupled with tissue-dependent differences in metabolism of these compounds, could play an important role in therapeutic effects and toxicity. Therefore, it is important to study the biodistribution of all the injected compounds. In Chapter 3, two different methods for quantification of doxorubicin in blood and tissue samples were setup and validated. One is based on the quantification of doxorubicin fluorescence with High Performance Liquid Chromatography (HPLC) after chemical extraction. The other method requires the use of 14C-labeled doxorubicin, which is a s-emitter that can be quantified with Liquid Scintillation Counting. Subsequently, the blood kinetics and biodistribution of 111In-labeled temperature-sensitive liposomes and their encapsulated compounds, doxorubicin and the MRI contrast agent [Gd(HPDO3A)(H2O)], was investigated in Chapter 4. The influence of HIFU-mediated local hyperthermia of the tumor on the biodistribution was studied using SPECT/CT imaging. The highest uptake of 111In-labeled TSLs was observed in the spleen and liver and was similar for the control and HIFU-treated rats. Although a large intratumoral variation was found, HIFU-mediated hyperthermia of the tumor resulted in a 4.4-fold higher uptake of the radiolabeled TSL in the tumor (t = 48h) compared to control experiments without HIFU, while the doxorubicin concentration was increased by a factor 7.9. This increased accumulation of doxorubicin-filled liposomes at longer time points may have an important contribution to the therapeutic outcome of HIFU-mediated drug delivery. In Chapter 5, an in vivo proof-of-concept study for image-guided local drug delivery was performed. The local temperature-triggered release of [Gd(HPDO3A)(H2O)] was monitored with interleaved T1 mapping of the tumor tissue and correlated with the co-release of doxorubicin. A good correlation between the ?R1, the uptake of doxorubicin and the gadolinium concentration in the tumor was found, implying that the in vivo release of doxorubicin from TSLs can be probed in situ with the longitudinal relaxation time of the co-released MRI contrast agents. Furthermore, an increase with a factor of 11 of doxorubicin concentrations in the tumor at 90 min after TSL injection was observed due to HIFU treatment. In Chapter 6, the intratumoral distribution of the TSLs and their encapsulated compounds was investigated, after HIFU-mediated hyperthermia induced local drug release. The presence of radiolabeled liposomal carriers and the intratumoral distribution of doxorubicin were imaged ex vivo with autoradiography and fluorescence microscopy, respectively. In hyperthermia treated tumors, liposomes were distributed more homogeneously across the tumor than in the control tumors. At 48h after injection, the liposomal accumulation in the tumor was enhanced in the hyperthermia group in comparison with the controls. In control tumors, doxorubicin uptake was observed in endothelial cells only, while in the HIFU-treated tumors the delivered drug was spread over a much larger area and was also taken up by tumor cells at a larger distance from blood vessels. Finally, the therapeutic effect of the HIFU-mediated hyperthermia treatment with administration of TSLs was studied and compared with saline, free doxorubicin and clinically available non-temperature sensitive liposomal doxorubicin (Caelyx®) (Chapter 7). TSL+HIFU showed a 2 to 4-fold increase in the time to reach two times the initial tumor size in comparison with the other groups. Furthermore, a correlation was found between the ?R1 and the relative tumor size after 7 days, showing that the MR measurements can be used as a prediction for the therapeutic effect.