<p>Use of chemotherapeutics in treatment of solid tumors suffers from insufficient and heterogeneous drug delivery, systemic toxicity and lack of knowledge of delivered drug concentration. The overall objectives of this work were: 1) to address these shortcomings through development and characterization of a treatment system capable of real-time spatiotemporal control of drug distribution and 2) to investigate the role of MR-image-able tissue transport parameters in predicting drug distribution following hyperthermia-triggered drug release from nanoparticles. Towards these objectives, a combination of potentially synergetic technologies was used: 1) image-able low temperature-sensitive liposomes (iLTSLs) for drug delivery, 2) quantitative drug delivery and transport parameter imaging with magnetic resonance imaging (MRI), and 3) control over drug release with magnetic resonance-guided high intensity focused ultrasound (MR-HIFU). The overall hypothesis of this work is that the drug distribution in the targeted zone spatially correlates with the image-able transport-related parameters as well as contrast enhancement due to release of contrast agent during treatment.</p><p>We began by developing and characterizing iLTSLs, which were designed using a lipid formulation similar to one that is in clinical trials in the US (ThermoDox®) and a gadolinium-based MR contrast agent that is in widespread clinical use (Prohance®) and least likelihood of toxicity due to nephrogenic systemic fibrosis (NSF). The resulting liposome was found to stably encapsulate both an anthracycline chemotherapeutic, doxorubicin, and the MR contrast agent. Release rates were similar for these two species in physiologic buffer as well as in human plasma. The next step towards control and imaging of release with this drug delivery system (DDS) was development of algorithms that allowed for large-volume mild hyperthermia with MR-HIFU that would be required to move this combination of technologies into the clinic.</p><p>Optimal drug delivery with iLTSL requires a sustained period of heating of the entire target to the range of temperatures that are optimal for liposomal release and maintenance of perfusion (40 - 45 &#61616;C). The MR-HIFU technology was developed and used mainly for rapid thermal ablation or mechanical disruption of tissue in small ellipsoid volumes. Variability and size of common clinical lesions called for modifications that would enable stable conformal heating of large tumor volumes to the sub-ablative temperature range of mild hyperthermia (40 - 45 &#61616;C). Therefore, we set out to develop an algorithm that would allow rapid attainment and maintenance of mild hyperthermia in larger volumes of variable shape that were typically encountered in the clinic. We approached this goal through a series of successive steps that addressed different aspects of mild hyperthermia treatment: 1) controlled heating to mild hyperthermia, 2) conformity of heating and 3) ability to heat large volumes.</p><p>To achieve controlled heating to mild hyperthermia we implemented a simple binary mild hyperthermia feedback mechanism that adequately maintained mild hyperthermia for extended periods of time in small ellipsoidal volumes. We then developed a conformal small-volume mild hyperthermia algorithm that could provide spatial control over heating in an environment with spatially heterogeneous perfusion. This algorithm used electronic steering of the HIFU focus to heat each MR image voxel with different power, depending on temperature measured within that voxel. Finally, to heat large volumes conformally, we developed an algorithm that combined mechanical displacement of the MR-HIFU transducer (to cover large areas) with electronic deflection of the HIFU beam (to heat sub-volumes conformally). This advancement allowed us to quickly attain mild hyperthermia (<8.1 min to steady state) in larger volumes (cross-sectional area = 8.4 cm, ~12 times larger than previous methods).</p><p>Following their characterization, we examined iLTSL pharmacokinetics and combined MR-HIFU large volume mild hyperthermia with iLTSL to deliver doxorubicin to large Vx2 carcinomas in the hindlimb muscle of rabbits. To determine MR image-able correlates to the intratumoral drug distribution, we assessed the spatial pattern of drug distribution with fluorescence microscopy and examined spatial correlations of this pattern to several parameters measured with MRI, including the spatial distributions of temperature, contrast enhancement following injection of iLTSL, dynamic contrast-enhanced MRI (DCE-MRI) parameters, and maps of apparent diffusion coefficient (ADC). Dynamic contrast-enhanced MRI parameters have been used extensively in literature to approximate a mixture of parameters critical to drug delivery, such as perfusion (F), permeability-vascular surface area product (PS) and vascular volume and ADC has been previously correlated with cellular density in tumors. Possible utility of such spatial correlations was examined for future use in treatment planning, intraprocedural feedback control and post-treatment evaluation.</p><p>Highly perfused peripheral regions of Vx2 tumors in rabbit hindlimb displayed high Ktrans and ve, indicative of high perfusion. Maps of ADC obtained with low b-values also showed high ADC in the periphery of these tumors, indicating high perfusion there. ADC maps that were weighted more towards diffusion (using higher b-values) showed that diffusion was largest in the tumor core, indicating destruction of the cellular membranes and greater mobility of water. Microscopic examination of excised tumors was spatially registered to the MRI datasets and showed that most of the tumor core is necrotic, though some highly vascularized and viable tissue was present in strands or segments that traversed the necrotic regions. Those segments also showed bright doxorubicin fluorescence following treatment with MR-HIFU and iLTSL. The two control groups - free drug and iLTSL without mild hyperthermia - showed minimal to no doxorubicin fluorescence in the tumor.</p><p>Susceptibility effects due to use of contrast agent caused large errors (up to 15 °C) in MR thermometry measurements. To address this phenomenon, experiments were designed to arrive at steady state heating (target temperature = 41 °C), and employ an algorithm to learn the spatiotemporal distribution of power that was needed to maintain steady state heating. This heating pattern was then played back several times to verify maintenance of steady state, and if satisfactory, image-able liposomes were injected. Since temperature feedback was replaced by the learned steady-state heating, injection of image-able liposomes likely did not alter the heating performance. Following injection, changes in T1 and magnetic susceptibility were most pronounced in regions that previously showed greatest enhancement during DCE-MRI and displayed larger values of ADC with perfusion-weighted, low b-value scans. Maps of T1 were obtained in real time using a variable flip angle sequence during heating, and were corrected for inhomogeneity of the B1 field and calibrated against a more accurate, T1 mapping technique.</p><p>After treatment with MR-HIFU and iLTSL, the drug was preferentially distributed in the viable tissue, in and around the tumor. Doxorubicin fluorescence was greatest in zones that were heated, though the drug distribution did not display a clear boundary between heated and unheated tissue. While iLTSL provided intraprocedural feedback via enhancement of T1-weighted image intensity, susceptibility-related effects of iLTSL on MR thermometry complicate their prospects of clinical use, where precise temperature feedback is required for control of therapy and MR thermometry techniques that are in widespread use would be affected. Spatial correlations between drug delivery with iLTSL and MR-imageable parameters may serve as a predictive tool to identify areas that will not receive adequate drug. Such a-priori knowledge of correlates to the approximate tumor drug distribution has the potential to inform treatment planning by revealing the extent to which drug dose could be painted with a combination of LTSL and MR-HIFU. These studies point to an adjustment of course in further development of drug dose painting this combination of technologies, towards informing treatment planning, and not only painting the dose, but predicting it. These results also point to the need to develop rational combinations other treatments, such as ablation and radiation, to treat regions that will not receive sufficient drug.</p> / Dissertation

Nous avons optimisé des liposomes thermosensibles, encapsulant un principe actif anticancéreux, le 5-Fluorouracile (5-FU), afin de déclencher sa libération par une hyperthermie locale modérée (39-42°C) induite par des ultrasons focalisés. L'hyperthermie sera appliquée au niveau de la tumeur, afin d'améliorer l’efficacité thérapeutique et de réduire la toxicité systémique. Les liposomes ont été formulés par hydratation du film lipidique en mélangeant la 1,2-dipalmitoyl-sn-glycéro-3-phosphocholine (DPPC) pour sa thermosensibilité à 41,5 ± 0,5°C, le cholestérol (CHOL) pour favoriser la stabilité des liposomes vis-à-vis des composants du sang, et le 1,2-distéaroyl-sn-glycéro-3-phosphoéthanolamine-N-[méthoxy(polyéthylène glycol)-2000] (DSPE-PEG) pour assurer la furtivité de la formulation. Les expériences ont confirmé que les liposomes formulés à base de DPPC/CHOL/DSPE-PEG dans un ratio molaire 90 : 5 : 5 mol% sont thermosensibles. Des liposomes composés du même mélange lipidique dans un rapport 65 : 30 : 5 mol% ont été considérés comme contrôle négatif non thermosensible. L’optimisation de l’encapsulation passive du 5-FU a permis d’obtenir une efficacité d’encapsulation (5-FU encapsulé/5-FU total) de 13%, mais le 5-FU est très faiblement retenu (12%) dans la cavité aqueuse des liposomes du fait du gradient osmotique à la dilution. La rétention du 5-FU a été optimisée (93%) par la technique d’encapsulation active basée sur la complexation intraliposomale du 5-FU avec le complexe cuivre-polyéthylèneimine préalablement encapsulé dans les liposomes. Cette technique a également permis d’améliorer l’efficacité d’encapsulation d’un facteur trois environ (37%), avec un taux de charge (ratio final 5-FU/lipides, mole/mole) de 50% environ. Nous avons alors obtenu des liposomes thermosensibles d'un diamètre hydrodynamique de 65 nm et de charge de surface de -10 mV. Les liposomes non thermosensibles, ont été caractérisés par un diamètre hydrodynamique de 105 nm et une charge de surface de -4,9 mV. La libération du 5-FU déclenchée par une hyperthermie induite par des ultrasons focalisés a été mesurée in vitro. En réponse à une hyperthermie de 42°C, les liposomes thermosensibles libèrent 68% de leur contenu, au bout de 10 min, alors que les liposomes non thermosensibles en libèrent moins de 20%. En outre, la cytotoxicité des liposomes encapsulant le complexe 5-FU-cuivre-polyéthylèneimine a été évaluée vis-à-vis de la lignée cellulaire HT-29 du carcinome colorectal humain. Les résultats ont révélé que les lipides à une concentration de 800 µM ne sont pas cytotoxiques (80% de viabilité). De plus, la complexation du 5-FU n’influence pas sa cytotoxicité ce qui prouve que la toxicité provient du 5-FU et non des excipients. En revanche, l’encapsulation du complexe 5-FU-cuivre-polyéthylèneimine dans les liposomes induit une diminution de la concentration inhibitrice médiane de 115 (solution du complexe) à 49 µM environ, corrélée à leur internalisation cellulaire. La pharmacocinétique chez des souris porteuses d’un modèle de tumeur colorectale HT-29 xénogreffée a montré que les liposomes permettent de prolonger d’un facteur 1,4 la demi-vie plasmatique de distribution du 5-FU. De plus, les aires sous la courbe des concentrations plasmatiques sur 24 h sont 1,9 et 2,9 fois plus élevées lorsque le 5-FU est administré sous forme de liposomes thermosensibles et non thermosensibles, respectivement, par rapport à la solution de 5-FU. Enfin, les liposomes non thermosensibles augmentent significativement d'un facteur 2 l'accumulation du 5-FU dans la tumeur par rapport à la solution de 5-FU. En conclusion, les liposomes thermosensibles développés présentent un fort intérêt pour une application thérapeutique en combinaison avec des ultrasons focalisés. / We optimized thermosensitive liposomes encapsulating an anticancer drug, 5-Fluorouracil (5-FU), in order to trigger the release upon focused ultrasound-mediated mild hyperthermia at the tumor. This approach would improve drug efficacy and would lower side effects. Liposomes were prepared by the lipid hydration method by mixing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) for its temperature sensitivity at 41.5 ± 0.5°C, cholesterol (CHOL) to promote liposome stability towards blood components, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) to confer stealthiness to the formulation. The experiments confirmed that the liposomes formulated with DPPC/CHOL/DSPE-PEG in a molar ratio 90:5:5 mol% are thermosensitive, while liposomes composed of the same lipid mixture in a ratio 65:30:5 mol% were considered non thermosensitive negative control. The optimization of passive encapsulation of 5-FU yielded an encapsulation efficacy (encapsulated 5-FU/total 5-FU) of 13%. 5-FU was, however, very weakly retained (12%) in the aqueous core of liposomes following dilution due to the generation of an osmotic gradient. The retention of 5-FU has been optimized (93%) by the active encapsulation technique based on the intraliposomal complexation of 5FU with copper-polyethylenimine complex encapsulated beforehand into liposomes. This technique also improved 5-FU encapsulation efficacy by 3-fold (37%), yielding a loading efficiency (final drug/lipid ratio, mol/mol) of approximately 50%. The resulting thermosensitive liposomes and non thermosensitive liposomes have a hydrodynamic diameter and a surface charge around 65 nm and -10 mV, and 105 nm and -4.9 mV, respectively. Heat-triggered drug delivery was evaluated using focused ultrasound, and showed a release of 68% of the encapsulated 5-FU from thermosensitive liposomes, within 10 min, whereas release remained below 20% for the non thermosensitive formulation. Furthermore, the cytotoxicity of 5-FU-copper-polyethylenimine complex-loaded liposomes towards HT-29 human colorectal carcinoma cell line was evaluated. Results revealed that lipids at a concentration of 800 µM are not cytotoxic (80% viability). Moreover, 5-FU complexation has no impact on its cytotoxic activity, disclosing that liposomes toxicity arose from 5-FU and not from the excipients. Nevertheless, 5-FU-copper-polyethylenimine complex-loaded liposomes exhibited a lower half maximal inhibitory concentration of 49 µM compared to 115 µM for complex solution. This enhancement of cytotoxicity was attributed to the cellular internalization of liposomes. Pharmacokinetics in mice bearing HT-29 xenograft tumor showed that liposomes can extend the plasma distribution half-life of 5-FU by a factor 1.4. Furthermore, areas under the concentration-time curve over 24 h were higher by 1.9- and 2.9-fold when the drug was encapsulated into thermosensitive and non thermosensitive liposomes, respectively, compared to free 5-Fluorouracil. Finally, non thermosensitive liposomes significantly increased 5-FU accumulation in tumor by 2-fold, compared to 5-FU solution. In conclusion, these 5-FU-loaded thermosensitive liposomes represent valuable carriers to investigate the therapeutic efficacy following focused ultrasound-mediated heat application.