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

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 ß-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

Focused ultrasound mediated drug delivery from temperature-sensitive liposomes: in-vitro characterization and validation

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Focused ultrasound mediated drug delivery from temperature-sensitive liposomes:In-vitro characterization and validation

\u3cp\u3eNanomedicine-based delivery with non-invasive techniques is a promising approach to increase local drug concentration and to reduce systemic side effects. Focused ultrasound (FUS) has become a promising strategy for non-invasive local drug delivery by mild hyperthermia. In this study, traditional temperature-sensitive liposomes (TTSLs) encapsulating doxorubicin (DOX) were evaluated for FUS-mediated drug delivery with an in-vitro FUS setup. In-vitro studies showed quantitative release of the DOX from the lumen of the temperature-sensitive liposomes when heated to 42 °C with FUS using 1 MHz sinusoidal waves at 1.75 MPa for 10 min. No release was observed when heated at 37°C. Moreover, we showed that DOX released from TTSLs by FUS is as efficiently internalized by glioblastoma cells as free DOX at 37°C. In-vitro therapeutic evaluation showed that exposure of a cell monolayer to FUS-activated TTSLs induced a 60% and a 50% decrease in cell viability compared to cell medium and to TTSLs preheated at 37°C, respectively. Using an in-vitro 3D cell culture model, the results showed that after FUS-mediated hyperthermia, preheated liposomes induced a 1.7-fold decrease in U-87 MG spheroid growth in comparison to the preheated liposomes at 37°C. In conclusion, our results show that in-vitro FUS allows the evaluation of TTSLs and does not modify the cellular uptake of the released DOX nor its cytotoxic activity.\u3c/p\u3

Thermal combination therapies for local drug delivery by magnetic resonance-guided high-intensity focused ultrasound

Several thermal-therapy strategies such as thermal ablation, hyperthermia-triggered drug delivery from temperature-sensitive liposomes (TSLs), and combinations of the above were investigated in a rhabdomyosarcoma rat tumor model (n = 113). Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) was used as a noninvasive heating device with precise temperature control for image-guided drug delivery. For the latter, TSLs were prepared, coencapsulating doxorubicin (dox) and [Gd(HPDO3A)(H2O)], and injected in tumor-bearing rats before MR-HIFU treatment. Four treatment groups were defined: hyperthermia, ablation, hyperthermia followed by ablation, or no HIFU. The intratumoral TSL and dox distribution were analyzed by single-photon emission computed tomography (SPECT)/computed tomography (CT), autoradiography, and fluorescence microscopy. Dox biodistribution was quantified and compared with that of nonliposomal dox. Finally, the treatment efficacy of all heating strategies plus additional control groups (saline, free dox, and Caelyx) was assessed by tumor growth measurements. All HIFU heating strategies combined with TSLs resulted in cellular uptake of dox deep into the interstitial space and a significant increase of tumor drug concentrations compared with a treatment with free dox. Ablation after TSL injection showed [Gd(HPDO3A)(H2O)] and dox release along the tumor rim, mirroring the TSL distribution pattern. Hyperthermia either as standalone treatment or before ablation ensured homogeneous TSL, [Gd(HPDO3A)(H2O)], and dox delivery across the tumor. The combination of hyperthermia-triggered drug delivery followed by ablation showed the best therapeutic outcome compared with all other treatment groups due to direct induction of thermal necrosis in the tumor core and efficient drug delivery to the tumor rim

Geometrical analysis for motion monitoring of rigid bodies with optical surface scanning in radiation oncology

BACKGROUND AND PURPOSE: Surface guided radiotherapy can be used to improve patient setup and for accurate intra-fraction motion monitoring in correspondence to the isocenter. For a clinical relevant motion analysis the actual displacement of the entire clinical target volume (CTV) is necessary. Therefore, the aim of this study was to develop a novel assessment method for intra-fraction motion for rigid body structures based on motion data and a geometrical analysis. MATERIALS AND METHODS: A threshold value on the volume coverage (VC(t)) of the CTV by the planning target volume (PTV) was proposed as online motion monitoring method. Moreover, offline analysis was performed by using heat maps and by calculating VCx, the volume coverage for at least x% of treatment time. The method was applied retrospectively to patient treatment data for whole brain radiation treatment without a thermoplastic mask. RESULTS: In 132 out of 142 fractions in total the proportion of the CTV that was inside the PTV for at least 99% of the time (VC99) was more than 95%, for a CTV-to-PTV margin of 5 mm. The source-voxel heat map showed which part of the CTV had a reduced coverage and the target heat map showed the movement of the CTV. CONCLUSION: Instead of using an action threshold on the movements of the isocenter, a threshold on the VC(t) of the CTV by the PTV was proposed. The heat maps and resulting values of VCx can be used to adapt the VC(t) threshold or the CTV-to-PTV margin for subsequent fractions

Temperature-sensitive liposomes for doxorubicin delivery under MRI guidance

Local drug delivery of doxorubicin holds promise to improve the therapeutic efficacy and to reduce toxicity profiles. Here, we investigated the release of doxorubicin and [Gd(HPDO3A)(H2O)] from different temperature-sensitive liposomes for applications in temperature-induced drug delivery under magnetic resonance image guidance. In particular, two temperature-sensitive systems composed of DPPC:MPPC:DPPE-PEG2000 (low temperature-sensitive liposomes, LTSL) and DPPC:HSPC:cholesterol:DPPE-PEG2000 (traditional temperature-sensitive liposomes, TTSL) were investigated. The co-encapsulation of [Gd(HPDO3A)(H2O)], a clinically approved MRI contrast agent, did not influence the encapsulation and release of doxorubicin. The LTSL system showed a higher leakage of doxorubicin at 37 °C, but a faster release of doxorubicin at 42 °C compared to the TTSL system. Furthermore, the rapid release of both doxorubicin and the MRI contrast agent from the liposomes occurred near the melting phase transition temperature, making it possible to image the release of doxorubicin using MRI

Lipid bilayer carrier for drugs or imaging agents

\u3cp\u3eDisclosed are carriers for drugs and/or MR imaging agents having a lipid bilayer shell comprising a phospholipid having two terminal alkyl chains, one being a short chain having a chain length of at most seven carbon atoms, the other being a long chain having a chain length of at least fifteen carbon atoms. The mixed long/short chain phospholipids serve to tune the release properties of the carrier. Preferred phospholipids are phosphatidylcholines.\u3c/p\u3

Lipid bilayer carrier for drugs or imaging agents

\u3cp\u3eDisclosed are carriers for drugs and/or MR imaging agents having a lipid bilayer shell comprising a phospholipid having two terminal alkyl chains, one being a short chain having a chain length of at most seven carbon atoms, the other being a long chain having a chain length of at least fifteen carbon atoms. The mixed long/short chain phospholipids serve to tune the release properties of the carrier. Preferred phospholipids are phosphatidylcholines.\u3c/p\u3

Thermal combination therapies for local drug delivery by magnetic resonance-guided high-intensity focused ultrasound

\u3cp\u3eSeveral thermal-therapy strategies such as thermal ablation, hyperthermia-triggered drug delivery from temperature-sensitive liposomes (TSLs), and combinations of the above were investigated in a rhabdomyosarcoma rat tumor model (n = 113). Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) was used as a noninvasive heating device with precise temperature control for image-guided drug delivery. For the latter, TSLs were prepared, coencapsulating doxorubicin (dox) and [Gd(HPDO3A)(H2O)], and injected in tumor-bearing rats before MR-HIFU treatment. Four treatment groups were defined: hyperthermia, ablation, hyperthermia followed by ablation, or no HIFU. The intratumoral TSL and dox distribution were analyzed by single-photon emission computed tomography (SPECT)/computed tomography (CT), autoradiography, and fluorescence microscopy. Dox biodistribution was quantified and compared with that of nonliposomal dox. Finally, the treatment efficacy of all heating strategies plus additional control groups (saline, free dox, and Caelyx) was assessed by tumor growth measurements. All HIFU heating strategies combined with TSLs resulted in cellular uptake of dox deep into the interstitial space and a significant increase of tumor drug concentrations compared with a treatment with free dox. Ablation after TSL injection showed [Gd(HPDO3A)(H2O)] and dox release along the tumor rim, mirroring the TSL distribution pattern. Hyperthermia either as standalone treatment or before ablation ensured homogeneous TSL, [Gd(HPDO3A)(H2O)], and dox delivery across the tumor. The combination of hyperthermia-triggered drug delivery followed by ablation showed the best therapeutic outcome compared with all other treatment groups due to direct induction of thermal necrosis in the tumor core and efficient drug delivery to the tumor rim.\u3c/p\u3

Temperature-sensitive paramagnetic liposomes for image-guided drug delivery: Mn2+ versus [Gd(HPDO3A)(H2O)]

Temperature-sensitive liposomes (TSLs) loaded with doxorubicin (Dox), and Magnetic Resonance Imaging contrast agents (CAs), either manganese (Mn2 +) or [Gd(HPDO3A)(H2O)], provide the advantage of drug delivery under MR image guidance. Encapsulated MRI CAs have low longitudinal relaxivity (r1) due to limited transmembrane water exchange. Upon triggered release at hyperthermic temperature, the r1 will increase and hence, provides a means to monitor drug distribution in situ. Here, the effects of encapsulated CAs on the phospholipid bilayer and the resulting change in r1 were investigated using MR titration studies and 1H Nuclear Magnetic Relaxation Dispersion (NMRD) profiles. Our results show that Mn2 + interacted with the phospholipid bilayer of TSLs and consequently, reduced doxorubicin retention capability at 37 °C within the interior of the liposomes over time. Despite that, Mn2 +-phospholipid interaction resulted in higher r1 increase, from 5.1 ± 1.3 mM− 1 s− 1 before heating to 32.2 ± 3 mM− 1 s− 1 after heating at 60 MHz and 37 °C as compared to TSL(Gd,Dox) where the longitudinal relaxivities before and after heating were 1.2 ± 0.3 mM− 1 s− 1 and 4.4 ± 0.3 mM− 1 s− 1, respectively. Upon heating, Dox was released from TSL(Mn,Dox) and complexation of Mn2 + to Dox resulted in a similar Mn2 + release profile. From 25 to 38 °C, r1 of [Gd(HPDO3A)(H2O)] gradually increased due to increase transmembrane water exchange, while no Dox release was observed. From 38 °C, the release of [Gd(HPDO3A)(H2O)] and Dox was irreversible and the release profiles coincided. By understanding the non-covalent interactions between the MRI CAs and phospholipid bilayer, the properties of the paramagnetic TSLs can be tailored for MR guided drug delivery