Hyperthermia-induced doxorubicin delivery from thermosensitive liposomes via MR-HIFU in a pig model.

PURPOSE: Encapsulation of cytotoxic drugs for a localized release is an effective way to increase the therapeutic window of such agents. In this article we present the localized release of doxorubicin (DOX) from phosphatidyldiglycerol (DPPG2) based thermosensitive liposomes using MR-HIFU mediated hyperthermia in a swine model. MATERIALS AND METHODS: German landrace pigs of weights between 37.5 and 53.5 kg received a 30-min infusion of DOX containing thermosensitive liposomes (50 mg DOX/m2). The pigs' biceps femoris was treated locally in two separate target areas with mild hyperthermia using magnetic resonance guided high intensity focused ultrasound, starting 10 min and 60 min after initiation of the infusion, respectively. The pharmacokinetics and biodistribution of DOX were determined and an analysis of the treatment parameters' influence was performed. RESULTS: Compared to untreated tissue, we found a 15-fold and a 7-fold increase in DOX concentration in the muscle volumes that had undergone hyperthermia starting 10 min and 60 min after the beginning of the infusion, respectively. The pharmacokinetic analysis showed a prolonged circulation time of DOX and a correlation between the AUC of extra-liposomal DOX in the bloodstream and the amount of DOX accumulated in the target tissue. CONCLUSIONS: We have demonstrated a workflow for MR-HIFU hyperthermia drug delivery that can be adapted to a clinical setting, showing that HIFU-hyperthermia is a suitable method for local drug release of DOX using DPPG2 based thermosensitive liposomes in stationary targets. Using the developed pharmacokinetic model, an optimization of the drug quantity deposited in the target via the timing of infusion and hyperthermia should be possible.

Visualization of thermal washout due to spatiotemporally heterogenous perfusion in the application of a model-based control algorithm for MR-HIFU mediated hyperthermia.

PURPOSE: This article will report results from the in-vivo application of a previously published model-predictive control algorithm for MR-HIFU hyperthermia. The purpose of the investigation was to test the controller's in-vivo performance and behavior in the presence of heterogeneous perfusion. MATERIALS AND METHODS: Hyperthermia at 42°C was induced and maintained for up to 30 min in a circular section of a thermometry slice in the biceps femoris of German landrace pigs (n=5) using a commercial MR-HIFU system and a recently developed MPC algorithm. The heating power allocation was correlated with heat sink maps and contrast-enhanced MRI images. The temporal change in perfusion was estimated based on the power required to maintain hyperthermia. RESULTS: The controller performed well throughout the treatments with an absolute average tracking error of 0.27 ± 0.15 °C and an average difference of 1.25 ± 0.22 °C between T10 and T90. The MPC algorithm allocates additional heating power to sub-volumes with elevated heat sink effects, which are colocalized with blood vessels visible on contrast-enhanced MRI. The perfusion appeared to have increased by at least a factor of ∼1.86 on average. CONCLUSIONS: The MPC controller generates temperature distributions with a narrow spectrum of voxel temperatures inside the target ROI despite the presence of spatiotemporally heterogeneous perfusion due to the rapid thermometry feedback available with MR-HIFU and the flexible allocation of heating power. The visualization of spatiotemporally heterogeneous perfusion presents new research opportunities for the investigation of stimulated perfusion in hypoxic tumor regions.

Heating technology for malignant tumors: a review.

The therapeutic application of heat is very effective in cancer treatment. Both hyperthermia, i.e., heating to 39-45 °C to induce sensitization to radiotherapy and chemotherapy, and thermal ablation, where temperatures beyond 50 °C destroy tumor cells directly are frequently applied in the clinic. Achievement of an effective treatment requires high quality heating equipment, precise thermal dosimetry, and adequate quality assurance. Several types of devices, antennas and heating or power delivery systems have been proposed and developed in recent decades. These vary considerably in technique, heating depth, ability to focus, and in the size of the heating focus. Clinically used heating techniques involve electromagnetic and ultrasonic heating, hyperthermic perfusion and conductive heating. Depending on clinical objectives and available technology, thermal therapies can be subdivided into three broad categories: local, locoregional, or whole body heating. Clinically used local heating techniques include interstitial hyperthermia and ablation, high intensity focused ultrasound (HIFU), scanned focused ultrasound (SFUS), electroporation, nanoparticle heating, intraluminal heating and superficial heating. Locoregional heating techniques include phased array systems, capacitive systems and isolated perfusion. Whole body techniques focus on prevention of heat loss supplemented with energy deposition in the body, e.g., by infrared radiation. This review presents an overview of clinical hyperthermia and ablation devices used for local, locoregional, and whole body therapy. Proven and experimental clinical applications of thermal ablation and hyperthermia are listed. Methods for temperature measurement and the role of treatment planning to control treatments are discussed briefly, as well as future perspectives for heating technology for the treatment of tumors.

Magnetic Resonance-Guided High-Intensity Focused Ultrasound (MR-HIFU): Technical Background and Overview of Current Clinical Applications (Part 1).

BACKGROUND: Extracorporeal high-intensity focused ultrasound (HIFU) is a promising method for the noninvasive thermal ablation of benign and malignant tissue. Current HIFU treatments are performed under ultrasound (US-HIFU) or magnetic resonance (MR-HIFU) image guidance offering integrated therapy planning, real-time control (spatial and temperature guidance) and evaluation. METHODS: This review is based on publications in peer-reviewed journals addressing thermal ablation using HIFU and includes our own clinical results as well. The technical background of HIFU is explained with an emphasis on MR-HIFU applications. A brief overview of the most commonly performed CE-approved clinical applications for MR-HIFU is given. RESULTS: Over the last decade, several HIFU-based applications have received clinical approval in various countries. In particular, MR-HIFU is now approved for the clinical treatment of uterine fibroids, palliation of bone pain, ablation of the prostate and treatment of essential tremor as a first neurological application. CONCLUSION: MR-HIFU is a patient-friendly noninvasive method for thermal ablation which has received clinical approval for several applications. Overall, clinical data demonstrate treatment efficacy, safety and cost efficiency. KEY POINTS: · HIFU is a promising technique for noninvasive thermal ablation of tissue.. · HIFU is typically performed under image guidance using either diagnostic ultrasound (US-HIFU) or MRI (MR-HIFU).. · The preferred image guidance modality depends on the application.. · MR guidance offers improved soft-tissue contrast for treatment planning, near real-time and noninvasive temperature monitoring and post-interventional therapy evaluation.. · MR-HIFU is CE-approved for treatment of uterine fibroids, alleviation of bone pain, prostate tissue ablation and treatment of essential tremor.. CITATION FORMAT: · Siedek F, Yeo S, Heijman E et al. Magnetic Resonance-Guided High-Intensity Focused Ultrasound (MR-HIFU): Technical Background and Overview of Current Clinical Applications (Part 1). Fortschr Röntgenstr 2019; 191: 522 - 530.

Development and evaluation of an isolated limb infusion model for investigation of drug delivery kinetics to solid tumors by thermosensitive liposomes and hyperthermia.

The combined administration of thermosensitive liposomes (TSLs) and hyperthermia (HT) has been increasingly shown to be a powerful tool for the treatment of solid tumors. At present, it is hypothesized that the circulation of TSLs through the vasculature of a heated tumor results in the rapid release of the entrapped drug, followed by its uptake and distribution within the tumor microenvironment. However, simple questions on the transport kinetics of TSLs through the heated tumor and how much drug is retained upon passage of TSLs through the tumor microcirculation have not been investigated in an experimental setting to-date. The present work describes a novel methodology for investigating these parameters by isolated limb infusion (ILI), developed in a rat model of sarcoma. This approach was used to assess the efficacy of Doxorubicin (Dox) delivery by TSL in a heated (42°C) tumor following a single passage of TSL through the tumor vasculature. Analysis of the effluent post-ILI, whole-tumor histological sections, and tissue homogenates revealed that upon a single passage, Dox delivery by TSL at 42°C did not exceed delivery under conventional (i.e. free Dox) or physiological (i.e. TSL at 37°C, or normothermia; NT) conditions. In fact, mathematical modeling demonstrated that at least thirteen passages are required to obtain the intratumoral Dox levels typically achieved using TSL (i.e. ~5%ID/g). Overall, this work investigates TSL-based determinants for achieving efficacious drug delivery using a model of ILI in tumor-bearing rats and the results bear important implications for TSL disposition in vivo.

Interleaved Mapping of Temperature and Longitudinal Relaxation Rate to Monitor Drug Delivery During Magnetic Resonance-Guided High-Intensity Focused Ultrasound-Induced Hyperthermia.

OBJECTIVES: Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) is a method to heat lesions noninvasively to a stable, elevated temperature and a well-suited method to induce local hyperthermia (41°C-43°C) in deep-seated tissues. Magnetic Resonance (MR) imaging provides therapy planning on anatomical images and offers temperature feedback based on near-real-time MR thermometry. Although constant acquisition of MR thermometry data is crucial to ensure prolonged hyperthermia, it limits the freedom to perform measurements of other MR parameters, which are of interest during hyperthermia treatments. In image-guided drug delivery applications, co-encapsulation of paramagnetic MR contrast agents with a drug inside temperature-sensitive liposomes (TSLs) allows to visualize hyperthermia-triggered drug delivery through changes of the longitudinal relaxation rate R1. While the drug accumulates in the heated tumor tissue, R1 changes can be used for an estimate of the tumor drug concentration. The main objective of this study was to demonstrate that interleaved MR sequences are able to monitor temperature with an adequate temporal resolution and could give a reasonable estimate of the achieved tumor drug concentration through R1 changes. To this aim, in vitro validation tests and an in vivo proof-of-concept study were performed. MATERIALS AND METHODS: All experiments were performed on a clinical 3-T MR-HIFU system adapted with a preclinical setup. The validity of the R1 values and the temperature maps stability were evaluated in phantom experiments and in ex vivo porcine muscle tissue. In vivo experiments were performed on rats bearing a 9L glioma tumor on their hind limb. All animals (n = 4 HIFU-treated, n = 4 no HIFU) were injected intravenously with TSLs co-encapsulating doxorubicin and gadoteridol as contrast agent. The TSL injection was followed by either 2 times 15 minutes of MR-HIFU-induced hyperthermia or a sham treatment. R1 maps were acquired before, during, and after sonication, using a single slice Inversion Recovery Look-Locker (IR-LL) sequence (field of view [FOV], 50 × 69 mm; in-plane resolution, 0.52 × 0.71 mm; slice thickness, 3 mm; 23 phases of 130 milliseconds; 1 full R1 map every 2 minutes). The R1 maps acquired during treatment were interleaved with 2 perpendicular proton resonance frequency shift (PRFS) MR thermometry slices (dynamic repetition time, 8.6 seconds; FOV, 250 × 250 mm; 1.4 × 1.4 mm in-plane resolution; 4 mm slice thickness). Tumor doxorubicin concentrations were determined fluorometrically. RESULTS: In vitro results showed a slight but consistent overestimation of the measured R1 values compared with calibrated R1 values, regardless whether the R1 was acquired with noninterleaved IR-LL or interleaved. The average treatment cell temperature had a slightly higher temporal standard deviation for the interleaved PRFS sequence compared with the noninterleaved PRFS sequence (0.186°C vs 0.101°C, respectively). The prolonged time in between temperature maps due to the interleaved IR-LL sequence did not degrade the temperature stability during MR-HIFU treatment (Taverage = 40.9°C ± 0.3°C). Upon heat treatment, some tumors showed an R1 increase in a large part of the tumor while other tumors hardly showed any ΔR1. The tumor doxorubicin concentration showed a linear correlation with the average ΔR1 during both sonications (n = 8, Radj = 0.933), which was higher than for the ΔR1 measured after tumor cooldown (averaged for both sonications, n = 8, Radj = 0.877). CONCLUSIONS: The new approach of interleaving different MR sequences was applied to simultaneously acquire R1 maps and PRFS thermometry scans during a feedback-controlled MR-HIFU-induced hyperthermia treatment. Interleaved acquisition did not compromise speed or accuracy of each scan. The ΔR1 acquired during treatment was used to visualize and quantify hyperthermia-triggered release of gadoteridol from TSLs and better reflected the intratumoral doxorubicin concentrations than the ΔR1 measured after cooldown of the tumor, exemplifying the benefit of interleaving R1 maps with temperature maps during drug delivery. Our study serves as an example for interleaved MR acquisition schemes, which introduce a higher flexibility in speed, sequence optimization, and timing.

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.

Comparing the therapeutic potential of thermosensitive liposomes and hyperthermia in two distinct subtypes of breast cancer.

Local drug delivery of Doxorubicin (Dox) with thermosensitive liposomes (TSL) and hyperthermia (HT) has shown preclinically to achieve high local drug concentrations with good therapeutic efficacy. Currently, this is clinically studied for treatment of chest wall recurrence of breast cancer, however with various outcomes. This study examines the potency of neoadjuvant TSL HT combination therapy in two orthotopic mouse models of human breast cancer, MDA-MB-231 and T-47D, which morphologically correlate to mesenchymal and epithelial phenotypes, respectively. Both cell lines showed improved in vitro chemosensitivity and Dox uptake at HT. Dox-loaded TSL (TSLDox) was stable in vitro in FBS, BALB/c-nu plasma and human plasma, although release of the drug at HT was incomplete for the latter two. Combination treatment with TSLDox and HT in vivo was significantly more effective against MDA-MB-231 tumors, whereas T-47D tumors showed no significant therapeutic response. Ex vivo investigation revealed a higher mean vessel density and poorly differentiated extracellular matrix (ECM) in MDA-MB-231 tumors relative to T-47D tumors. Although in vitro results of the TSLDox and HT treatment were favorable for both cell types, the therapeutic efficacy in vivo was remarkably different. The well-differentiated and slowly-growing T-47D tumors may provide a microenvironment that limits drug delivery to the target cell and therefore renders the therapy ineffective. Mesenchymal and invasive MDA-MB-231 tumors display higher vascularization and less mature ECM, significantly enhancing tumor response to TSLDox and HT treatment. These results yield insight into the efficacy of TSL treatment within different tumor microenvironments, and further advance our understanding of factors that contribute to heterogeneous therapeutic outcomes in clinical trials.

Investigation of Particle Accumulation, Chemosensitivity and Thermosensitivity for Effective Solid Tumor Therapy Using Thermosensitive Liposomes and Hyperthermia.

Doxorubicin (Dox) loaded thermosensitive liposomes (TSLs) have shown promising results for hyperthermia-induced local drug delivery to solid tumors. Typically, the tumor is heated to hyperthermic temperatures (41-42 °C), which induced intravascular drug release from TSLs within the tumor tissue leading to high local drug concentrations (1-step delivery protocol). Next to providing a trigger for drug release, hyperthermia (HT) has been shown to be cytotoxic to tumor tissue, to enhance chemosensitivity and to increase particle extravasation from the vasculature into the tumor interstitial space. The latter can be exploited for a 2-step delivery protocol, where HT is applied prior to i.v. TSL injection to enhance tumor uptake, and after 4 hours waiting time for a second time to induce drug release. In this study, we compare the 1- and 2-step delivery protocols and investigate which factors are of importance for a therapeutic response. In murine B16 melanoma and BFS-1 sarcoma cell lines, HT induced an enhanced Dox uptake in 2D and 3D models, resulting in enhanced chemosensitivity. In vivo, therapeutic efficacy studies were performed for both tumor models, showing a therapeutic response for only the 1-step delivery protocol. SPECT/CT imaging allowed quantification of the liposomal accumulation in both tumor models at physiological temperatures and after a HT treatment. A simple two compartment model was used to derive respective rates for liposomal uptake, washout and retention, showing that the B16 model has a twofold higher liposomal uptake compared to the BFS-1 tumor. HT increases uptake and retention of liposomes in both tumors models by the same factor of 1.66 maintaining the absolute differences between the two models. Histology showed that HT induced apoptosis, blood vessel integrity and interstitial structures are important factors for TSL accumulation in the investigated tumor types. However, modeling data indicated that the intraliposomal Dox fraction did not reach therapeutic relevant concentrations in the tumor tissue in a 2-step delivery protocol due to the leaking of the drug from its liposomal carrier providing an explanation for the observed lack of efficacy.

In vivo temperature controlled ultrasound-mediated intracellular delivery of cell-impermeable compounds.

Many chemotherapeutic drugs are characterized by high systemic toxicity and/or suffer from limited bioavailability. Thermosensitive liposomes (TSLs) encapsulating drugs in their aqueous lumen are promising activatable nanocarriers for ultrasound (US)-mediated drug delivery in response to mild hyperthermia. On the other hand, US is known to locally break biological barriers and as a consequence enable internalization of molecules. In this work, a two-step protocol for intracellular delivery of cell-impermeable molecules comprising of US-induced permeabilization followed by temperature-controlled release of the model drug from thermosensitive liposomes has been developed. TSLs containing TO-PRO-3, a cell-impermeable molecule that displays a significant increase in fluorescence upon binding to nucleic acids thus serving as a 'sensor' for internalization have been prepared and characterized in detail. US-mediated permeabilization followed by temperature-controlled release was applied to tumor bearing mice following i.v. injection of TSLs and microbubbles. The efficacy of this approach was evaluated by in vivo fluorescence imaging followed by histological analysis. A 2.4-fold increase of fluorescence signal was observed and intracellular delivery of TO-PRO-3 was confirmed by a characteristic nuclear staining. These results demonstrate the feasibility of novel drug delivery system to tumors comprising of local cell permeabilization by US followed by in situ release of the payload from thermosensitive liposomes. Possible applications include local and controlled intracellular delivery of molecules with otherwise limited bioavailability.

Tumour hyperthermia and ablation in rats using a clinical MR-HIFU system equipped with a dedicated small animal set-up.

PURPOSE: We report on the design, performance, and specifications of a dedicated set-up for the treatment of rats on a clinical magnetic resonance high intensity focused ultrasound (MR-HIFU) system. MATERIALS AND METHODS: The small animal HIFU-compatible 4-channel MR receiver volume coil and animal support were designed as add-on to a clinical 3T Philips Sonalleve MR-HIFU system. Prolonged hyperthermia (T ≈ 42°C, 15 min) and thermal ablation (T = 65°C) was performed in vivo on subcutaneous rat tumours using 1.44 MHz acoustic frequency. The direct treatment effect was assessed with T(2)-weighted imaging and dynamic contrast enhanced (DCE-) MRI as well as histology. RESULTS: The developed HIFU-compatible coil provided an image quality that was comparable to conventional small animal volume coils (i.e. without acoustic window), and a SNR increase by a factor of 10 as compared to the coil set-up used for clinical MR-HIFU therapy. The use of an animal support minimised far field heating and allowed precise regulation of the animal body core temperature, which varied <1°C during treatment. CONCLUSIONS: The results demonstrated that, by using a designated set-up, both controlled hyperthermia and thermal ablation treatment of malignant tumours in rodents can be performed on a clinical MR-HIFU system. This approach provides all the advantages of clinical MR-HIFU, such as volumetric heating, temperature feedback control and a clinical software interface for use in rodent treatment. The use of a clinical system moreover facilitates a rapid translation of the developed protocols into the clinic.

Block-copolymer-stabilized iodinated emulsions for use as CT contrast agents.

The objective of this study was to develop radiopaque iodinated emulsions for use as CT blood pool contrast agents. Three hydrophobic iodinated oils were synthesized based on the 2,3,5-triiodobenzoate moiety and formulated into emulsions using either phospholipids or amphiphilic polymers, i.e. Pluronic F68 and poly(butadiene)-b-poly(ethylene glycol) (PBD-PEO), as emulsifiers. The size, stability and cell viability was investigated for all stabilized emulsions. Three emulsions stabilized with either lipids or PBD-PEO were subsequently tested in vivo as a CT blood pool contrast agent in mice. While the lipid-stabilized emulsions turned out unstable in vivo, polymer-stabilized emulsions performed well in vivo. In blood, a contrast enhancement of 220 Hounsfield Units (HU) was measured directly after intravenous administration of 520 mg I/kg. The blood circulation half-life of a PBD-PEO stabilized emulsion was approximately 3 h and no noticeable in vivo toxicity was observed. These results show the potential of above emulsions for use as blood pool agents in contrast enhanced CT imaging.