Simultaneous Intravital Optical and Acoustic Monitoring of Ultrasound-Triggered Nanobubble Generation and Extravasation.

Ultrasound-activated nanobubbles are being widely investigated as contrast agents and therapeutic vehicles. Nanobubbles hold potential for accessing the tumor extravascular compartment, though this relies on clinically debated passive accumulation for which evidence to date is indirect. We recently reported ultrasound-triggered conversion of high payload porphyrin-encapsulated microbubbles to nanobubbles, with actively enhanced permeability for local delivery. This platform holds implications for optical/ultrasound-based imaging and therapeutics. While promising, it remains to be established how nanobubbles are generated and whether they extravasate intact. Here, insights into the conversion process are reported, complemented by novel simultaneous intravital and acoustic monitoring in tumor-affected functional circulation. The first direct acoustic evidence of extravascular intact nanobubbles are presented. These insights collectively advance this delivery platform and multimodal micro- and nanobubbles, extending their utility for imaging and therapeutics within and beyond the vasculature.

Microbubble-assisted MRI-guided focused ultrasound for hyperthermia at reduced power levels.

PURPOSE: Ultrasound contrast agent microbubbles were combined with magnetic resonance imaging (MRI)-guided focused ultrasound (MRgFUS) as a means to achieve mild hyperthermia at reduced power levels. METHODS: MRgFUS hyperthermia (42°C for 20 min) was evaluated in rabbit thigh muscle or Vx2 tumors using infusions of microbubbles (Definity, 20 µL/kg) or saline (sham) administered over 5 min. The impact of treatments on drug uptake was assessed with liposomal doxorubicin (Caelyx, 2.5 mg/kg). Applied power levels before and after the injection of microbubbles or saline were compared, and drug uptake was evaluated with fluorometry of tissues harvested 24 hr post-treatment. RESULTS: MRgFUS hyperthermia in muscle and tumors resulted in accurate temperature control (mean =42.0°C, root mean square error (RMSE) = 0.3°C). The power dropped significantly following the injection of microbubbles in muscle and tumors compared to exposures without microbubbles (-21.9% ± 12.5% vs -5.9% ± 7.8%, p = .009 in muscle; -33.8% ± 9.9% vs -3.0% ± 7.2%, p < .001 in tumors). Cavitation monitoring indicated emission of subharmonic, ultraharmonic, and elevated levels of fourth to sixth harmonic frequencies following microbubble injection. The drug delivery was elevated significantly in muscle with the use of microbubble-assisted relative to conventional heating (0.5 ± 0.5 ng/mg vs 0.20 ± 0.04 ng/mg, p = .05), whereas in tumors similar levels were found (11 ± 3 ng/mg vs 16 ± 4 ng/mg, p = .13). CONCLUSIONS: The finding that microbubbles reduce the applied power requirements for hyperthermia has considerable clinical implications. The elevated levels of drug found in muscle but not tumor tissue suggest a complex interplay between the heating effects of microbubbles with those of enhanced permeabilization and possible vascular damage.

Translational dynamics of individual microbubbles with millisecond scale ultrasound pulses.

It is established that radiation forces can be used to transport ultrasound contrast agents, particularly for molecular imaging applications. However, the ability to model and control this process in the context of therapeutic ultrasound is limited by a paucity of data on the translational dynamics of encapsulated microbubbles under the influence of longer pulses. In this work, the translation of individual microbubbles, isolated with optical tweezers, was experimentally investigated over a range of diameters (1.8-8.8 µm, n = 187) and pressures (25, 50, 100, 150, and 200 kPa) with millisecond pulses. Data were compared with theoretical predictions of the translational dynamics, assessing the role of shell and history force effects. A pronounced feature of the displacement curves was an effective threshold size, below which there was only minimal translation. At higher pressures (≥150 kPa) a noticeable structure emerged where multiple local maxima occurred as a function of bubble size. The ability to accurately capture these salient features depended on the encapsulation model employed. In low Reynolds number conditions (i.e., low pressures, or high pressures, off-resonance) the inclusion of history force more accurately fit the data. After pulse cessation, bubbles exhibited substantial displacements consistent with the influence of history effects.

The microscale evolution of the erosion front of blood clots exposed to ultrasound stimulated microbubbles.

Serial two-photon microscopy of blood clots with fluorescently tagged fibrin networks was conducted during microbubble-mediated sonothrombolysis to examine the microscale evolution of the resulting erosion front. The development of a complex zonal erosion pattern was observed, comprised of a cell depleted layer of fibrin network overlying intact clot which then underwent progressive recession. The fibrin zone architecture was dependent on exposure conditions with 0.1 MPa causing no erosion, 0.39 MPa resulting in homogenous structure, and combination 0.39/0.96 MPa pulses forming large-scale tunnels. High speed imaging and Coulter counter data indicated the fibrin zone formation process involves the ejection of intact erythrocytes.

An overview of the influence of therapeutic ultrasound exposures on the vasculature: high intensity ultrasound and microbubble-mediated bioeffects.

It is well established that the interaction of ultrasound with soft tissues can induce a wide range of bioeffects. One of the most important and complex of these interactions in the context of therapeutic ultrasound is with the vasculature. Potential vascular effects range from enhancing microvascular permeability to inducing vascular damage and vessel occlusion. While aspects of these effects are broadly understood, the development of improved approaches to exploit these effects and gain a more detailed mechanistic understanding is ongoing and largely anchored in preclinical research. Here a general overview of this established yet rapidly evolving topic is provided, with a particular emphasis on effects arising from high-intensity focused ultrasound and microbubble-mediated exposures.

The influence of compliant boundary proximity on the fundamental and subharmonic emissions from individual microbubbles.

The proximity of a solid-liquid boundary has been theoretically predicted to affect nonlinear microbubble emissions, but to date there has been no experimental validation of this effect. In this study, individual microbubbles (n = 15) were insonicated at f = 11 MHz as a function of offset distance from a compliant (agarose) planar boundary by employing an optical trapping apparatus. It was found that fundamental scattering increases while subharmonic scattering decreases as the microbubble approaches the boundary. Although a microbubble-boundary model can predict the qualitative trends observed for a subset of encapsulation properties, further modeling efforts are required to completely model compliant boundary-microbubble interactions.

In vivo high-speed microscopy of microbubbles in the chorioallantoic membrane model.

Rationale: The acoustic stimulation of microbubbles within microvessels can elicit a spectrum of therapeutically relevant bioeffects from permeabilization to perfusion shutdown. These bioeffects ultimately arise from complex interactions between microbubbles and microvascular walls, though such interactions are poorly understood particularly at high pressure, due to a paucity of direct in vivo observations. The continued development of focused ultrasound methods hinges in large part on establishing links between microbubble-microvessel interactions, cavitation signals, and bioeffects. Methods: Here, a system was developed to enable simultaneous high-speed intravital imaging and cavitation monitoring of microbubbles in vivo in a chorioallantoic membrane model. Exposures were conducted using the clinical agent DefinityTM under conditions previously associated with microvascular damage (1 MHz, 0.5-3.5 MPa, 5 ms pulse length). Results: Ultrasound-activated microbubbles could be observed and were found to induce localized wall deformations that were more pronounced in smaller microvessels and increased with pressure. A central finding was that microbubbles could extravasate from microvessels (from 34% of vessels at 1 MPa to 79% at 3 MPa) during insonation (94% within 0.5 ms) and that this occurred more frequently and in progressively larger microvessels (up to 180 µm) as pressure was increased. Following microbubble extravasation, transient or sustained red blood cell leakage ensued at the extravasation site in 96% of cases for pressures ≥1 MPa. Conclusions: The results here represent the first high-speed in vivo investigation of high-pressure focused ultrasound-induced microbubble-microvessel interactions. This data provides direct evidence that the process of activated microbubble extravasation can occur in vivo and that it is linked to producing microvessel wall perforations of sufficient size to permit red blood cell leakage. The association of red blood cell leakage with microbubble extravasation provides mechanistic insight into the process of microvessel rupture, which has been widely observed in histology.

Inducing cavitation within hollow cylindrical radially polarized transducers for intravascular applications.

Thrombotic occlusions of large blood vessels are increasingly treated with catheter based mechanical approaches, one of the most prominent being to employ aspiration to extract clots through a hollow catheter lumen. A central technical challenge for aspiration catheters is to achieve sufficient suction force to overcome the resistance of clot material entering into the distal tip. In this study, we examine the feasibility of inducing cavitation within hollow cylindrical transducers with a view to ultimately using them to degrade the mechanical integrity of thrombus within the tip of an aspiration catheter. Hollow cylindrical radially polarized PZT transducers with 3.3/2.5 mm outer/inner diameters were assessed. Finite element simulations and hydrophone experiments were used to investigate the pressure field distribution as a function of element length and resonant mode (thickness, length). Operating in thickness mode (∼5 MHz) was found to be associated with the highest internal pressures, estimated to exceed 23 MPa. Cavitation was demonstrated to be achievable within the transducer under degassed water (10 %) conditions using hydrophone detection and high-frequency ultrasound imaging (40 MHz). Cavitation clouds occupied a substantial portion of the transducer lumen, in a manner that was dependent on the pulsing scheme employed (10 and 100 µs pulse lengths; 1.1, 11, and 110 ms pulse intervals). Collectively the results support the feasibility of achieving cavitation within a transducer compatible with mounting in the tip of an aspiration format catheter.

Synchronous Intravital Imaging and Cavitation Monitoring of Antivascular Focused Ultrasound in Tumor Microvasculature Using Monodisperse Low Boiling Point Nanodroplets.

Focused ultrasound-stimulated microbubbles can induce blood flow shutdown and ischemic necrosis at higher pressures in an approach termed antivascular ultrasound. Combined with conventional therapies of chemotherapy, immunotherapy, and radiation therapy, this approach has demonstrated tumor growth inhibition and profound synergistic antitumor effects. However, the lower cavitation threshold of microbubbles can potentially yield off-target damage that the polydispersity of clinical agent may further exacerbate. Here we investigate the use of a monodisperse nanodroplet formulation for achieving antivascular effects in tumors. We first develop stable low boiling point monodisperse lipid nanodroplets and examine them as an alternative agent to mediate antivascular ultrasound. With synchronous intravital imaging and ultrasound monitoring of focused ultrasound-stimulated nanodroplets in tumor microvasculature, we show that nanodroplets can trigger blood flow shutdown and do so with a sharper pressure threshold and with fewer additional events than conventionally used microbubbles. We further leverage the smaller size and prolonged pharmacokinetic profile of nanodroplets to allow for potential passive accumulation in tumor tissue prior to antivascular ultrasound, which may be a means by which to promote selective tumor targeting. We find that vascular shutdown is accompanied by inertial cavitation and complex-order sub- and ultraharmonic acoustic signatures, presenting an opportunity for effective feedback control of antivascular ultrasound.

Antitumor effects of combining metronomic chemotherapy with the antivascular action of ultrasound stimulated microbubbles.

Considerable effort is being directed toward investigating the use of ultrasound (US) stimulated microbubbles (MB) to promote the uptake of anticancer agents in tumors. In this study we propose and investigate a new method for combining therapeutic ultrasound with anticancer agents, which is to induce antivascular effects and combine these with an antiangiogenic treatment strategy, in this case metronomic chemotherapy. This is effectively a vascular targeting rather than a drug delivery approach. Experiments were conducted on MDA-MB-231 breast cancer tumors implanted in athymic mice. Metronomic cyclophosphamide (MCTX) was employed as an antiangiogenic therapy and was administered through the drinking water. Ultrasound stimulated microbubble treatments (USMB) were conducted at 1 MHz employing short bursts (0.00024 duty cycle) at 1.6 MPa in combination with the commercial microbubble agent Definity. USMB treatments were performed on a weekly basis for 4 weeks and MCTX was administered for 10 weeks. The USMB induced an acute reduction of blood flow as confirmed with US contrast imaging and DiOC7 perfusion staining. Longitudinal experiments demonstrated that significant growth inhibition occurred in MCTX-only and USMB-only treatment groups relative to control tumors. The combined USMB and MCTX treatment group showed significant growth inhibition and survival prolongation relative to the USMB-only (p < 0.01) and MCTX-only treatment groups (p < 0.01). These results indicate the feasibility of a new approach to combining therapeutic ultrasound with an anticancer agent.

Nonlinear resonance behavior and linear shell estimates for Definity™ and MicroMarker™ assessed with acoustic microbubble spectroscopy.

There is a growing interest in microbubble based ultrasound contrast imaging applications in the 5-15 MHz range. In this study, individual microbubbles were insonified at low pressures (≤ 25 kPa) using an "acoustic spectroscopy" approach which entailed transmitting a sequence of tone bursts with center frequencies ranging from 4 to 13.5 MHz. The fundamental (transmit) frequency radial excursion amplitude was calculated from the scattered signals to produce a resonance curve for each bubble. For diameters between 2.5 to 4 µm, 69% of Target-Ready MicroMarker™ (Bracco, Geneva; Visualsonics, Canada) exhibited asymmetric resonance, characterized by a skewing of the resonance curve and indicative of nonlinear behavior. For Definity™ (Lantheus Medical Imaging, N. Billerica, MA), these responses were observed for 8% of diameters between 1.7 to 3.1 µm. For the subset of bubbles exhibiting linear, symmetric resonance curves, resonant frequencies, shell elasticity, and viscosity values were estimated. Between 10 to 12 MHz, for example, Target-Ready MicroMarker between 2.7 to 3.3 µm in diameter was resonant, where Definity was resonant between 1.7 to 2.6 µm. From 4 to 13.5 MHz, Target-Ready MicroMarker is characterized by a stiffer shell (3 < χ(0) < 5) N/m than Definity (0.5 < χ(0) < 2.5) N/m, and distinct strain-softening and shear-thinning rheological behavior. For Definity, no clear strain or shear-rate dependence of the shell properties is evident.

An optical and acoustic investigation of microbubble cavitation in small channels under therapeutic ultrasound conditions.

Therapeutic focused ultrasound in combination with encapsulated microbubbles is being widely investigated for its ability to elicit bioeffects in the microvasculature, such as transient permeabilization for drug delivery or at higher pressures to achieve 'antivascular' effects. While it is well established that the behaviors of microbubbles are altered when they are situated within sufficiently small vessels, there is a paucity of data examining how the bubble population dynamics and emissions change as a function of channel (vessel) diameter over a size range relevant to therapeutic ultrasound, particularly at pressures relevant to antivascular ultrasound. Here we use acoustic emissions detection and high-speed microscopy (10 kframes/s) to examine the behavior of a polydisperse clinically employed agent (Definity®) in wall-less channels as their diameters are scaled from 1200 to 15 µm. Pressures are varied from 0.1 to 3 MPa using either a 5 ms pulse or a sequence of 0.1 ms pulses spaced at 1 ms, both of which have been previously employed in an in vivo context. With increasing pressure, the 1200 µm channel - on the order of small arteries and veins - exhibited inertial cavitation, 1/2 subharmonics and 3/2 ultraharmonics, consistent with numerous previous reports. The 200 and 100 µm channels - in the size range of larger microvessels less affected by therapeutic focused ultrasound - exhibited a distinctly different behavior, having muted development of 1/2 subharmonics and 3/2 ultraharmonics and reduced persistence. These were associated with radiation forces displacing bubbles to the distal wall and inducing clusters that then rapidly dissipated along with emissions. As the diameter transitioned to 50 and then 15 µm - a size regime that is most relevant to therapeutic focused ultrasound - there was a higher threshold for the onset of inertial cavitation as well as subharmonics and ultraharmonics, which importantly had more complex orders that are not normally reported. Clusters also occurred in these channels (e.g. at 3 MPa, the mean lateral and axial sizes were 23 and 72 µm in the 15 µm channel; 50 and 90 µm in the 50 µm channel), however in this case they occupied the entire lumens and displaced the wall boundaries. Damage to the 15 µm channel was observed for both pulse types, but at a lower pressure for the long pulse. Experiments conducted with a 'nanobubble' (<0.45 µm) subpopulation of Definity followed broadly similar features to 'native' Definity, albeit at a higher pressure threshold for inertial cavitation. These results provide new insights into the behavior of microbubbles in small vessels at higher pressures and have implications for therapeutic focused ultrasound cavitation monitoring and control.

Intravital imaging and cavitation monitoring of antivascular ultrasound in tumor microvasculature.

Rationale: Focused ultrasound-stimulated microbubbles have been shown to be capable of inducing blood flow shutdown and necrosis in a range of tissue types in an approach termed antivascular ultrasound or nonthermal ablation. In oncology, this approach has demonstrated tumor growth inhibition, and profound synergistic antitumor effects when combined with traditional platforms of chemo-, radiation- and immune-therapies. However, the exposure schemes employed have been broad and underlying mechanisms remain unclear with fundamental questions about exposures, vessel types and sizes involved, and the nature of bubble behaviors and their acoustic emissions resulting in vascular damage - impeding the establishment of standard protocols. Methods: Here, ultrasound transmitters and receivers are integrated into a murine dorsal window chamber tumor model for intravital microscopy studies capable of real-time visual and acoustic monitoring during antivascular ultrasound. Vessel type (normal and tumor-affected), caliber, and viability are assessed under higher pressure conditions (1, 2, and 3 MPa), and cavitation signatures are linked to the biological effects. Results: Vascular events occurred preferentially in tumor-affected vessels with greater incidence in smaller vessels and with more severity as a function of increasing pressure. Vascular blood flow shutdown was found to be due to a combination of focal disruption events and network-related flow changes. Acoustic emissions displayed elevated broadband noise and distinct sub- and ultra-harmonics and their associated third-order peaks with increasing pressure. Conclusions: The observed vascular events taken collectively with identified cavitation signatures provide an improved mechanistic understanding of antivascular ultrasound at the microscale, with implications for establishing a specific treatment protocol and control platform.

Spatially segmented SVD clutter filtering in cardiac blood flow imaging with diverging waves.

Ultrafast ultrasound imaging enables the visualization of rapidly changing blood flow dynamics in the chambers of the heart. Singular value decomposition (SVD) filters outperform conventional high pass clutter rejection filters for ultrafast blood flow imaging of small and shallow fields of view (e.g., functional imaging of brain activity). However, implementing SVD filters can be challenging in cardiac imaging due to the complex spatially and temporally varying tissue characteristics. To address this challenge, we describe a method that involves excluding the proximal portion of the image (near the chest wall) and divides the reduced field of view into overlapped segments, within which tissue signals are expected to be spatially and temporally coherent. SVD filtering with automatic selection of cut-off singular vector orders to remove tissue and noise signals is implemented for each segment. Auto-thresholding is based on the coherence of spatial singular vectors, delineating tissue, blood, and noise subspaces within a spatial similarity matrix calculated for each segment. Filtered blood flow signals from the segments are reconstructed and then combined and Doppler processing is used to form a set of blood flow images. Preliminary experimental results suggest that the spatially segmented approach improves the separation of the tissue and blood subsets in the spatial similarity matrix so that automatic thresholding is significantly improved, and tissue clutter can then be rejected more effectively in cardiac ultrafast imaging, compared to using the full field of view. In the case studied, spatially segmented SVD improved the rate of correct automatic selection of thresholds from 78% to 98.7% for the investigated cases and improved the post-filter power of blood signals by an average of more than 10 dB during a cardiac cycle.

Porphyrin shell microbubbles with intrinsic ultrasound and photoacoustic properties.

Porphyrin-phospholipid conjugates were used to create photonic microbubbles (MBs) having a porphyrin shell ("porshe"), and their acoustic and photoacoustic properties were investigated. The inclusion of porphyrin-lipid in the MB shell increased the yield, improved the serum stability, and generated a narrow volumetric size distribution with a peak size of 2.7 ± 0.2 µm. Using an acoustic model, we calculated the porshe stiffness to be 3-5 times greater than that of commercial lipid MBs. Porshe MBs were found to be intrinsically suitable for both ultrasound and photoacoustic imaging with a resonance frequency of 9-10 MHz. The distinctive properties of porshe MBs make them potentially advantageous for a broad range of biomedical imaging and therapeutic applications.

High frequency ultrasound nonlinear scattering from porphyrin nanobubbles.

Emerging contrast imaging studies have highlighted the potential of nanobubbles for both intravascular and extravascular applications. Reports to date on nanobubbles have generally utilized low frequencies (<12 MHz), high concentrations (>109 mL-1), and B-mode or contrast-mode on preclinical and clinical systems. However, none of these studies directly examined nanobubble acoustic signatures systematically to implement nonlinear imaging schemes in a methodical manner based on nanobubble behaviour. Here, nanobubble nonlinear behaviour is investigated at high frequencies (12.5, 25, 30 MHz) and low concentration (106 mL-1) in a channel phantom, with different pulse types in single- and multi-pulse sequences to examine behaviour under conditions relevant to high frequency imaging. Porphyrin nanobubbles are demonstrated to initiate nonlinear scattering at high frequencies in a pressure-threshold dependent manner, as previously observed at low frequencies. This threshold behaviour was then utilized to demonstrate enhanced nanobubble imaging with pulse inversion, amplitude modulation, and a combination of the two, progressing towards the improved sensitivity and expanded utility of these ultrasound contrast agents.

High-Frequency Array-Based Nanobubble Nonlinear Imaging in a Phantom and In Vivo.

There has been growing interest in nanobubbles (NBs) for vascular and extravascular ultrasound contrast imaging and therapeutic applications. Studies to date have generally utilized low frequencies (<12 MHz), high concentrations (>109 mL-1), and uncalibrated B-mode or contrast-mode on commercial systems without reporting investigations on NB signatures upon which the imaging protocols should be based. We recently demonstrated that low concentrations (106 mL-1) of porphyrin-lipid-encapsulated NBs scatter nonlinearly at low (2.5, 8 MHz) and high (12.5, 25, 30 MHz) frequencies in a pressure threshold-dependent manner that is advantageous for amplitude modulation (AM) imaging. Here, we implement pressure-calibrated AM at high frequency on a commercial preclinical array system to enhance sensitivity to nonlinear scattering of three phospholipid-based NB formulations. With this approach, improvements in contrast to tissue ratio relative to B-mode between 12.4 and 22.8 dB are demonstrated in a tissue-mimicking phantom, and between 6.7 and 14.8 dB in vivo.

Self-demodulation of high-frequency ultrasound.

High-frequency (>10 MHz) ultrasound is used in, e.g., small animal imaging or intravascular applications. Currently available ultrasound contrast agents (UCAs) have a suboptimal response for high frequencies. This study therefore investigates the nonlinear propagation effects in a high-frequency ultrasound field (25 MHz) and its use for standard UCA and diagnostic frequencies (1-3 MHz). Nonlinear mixing of two high-frequency carrier waves produces a low-frequency wave, known as the self-demodulation or parametric array effect. Hydrophone experiments showed that the self-demodulated field of a focused 25 MHz transducer (850 kPa source pressure) has an amplitude of 45 kPa at 1.5 MHz in water. Such pressure level is sufficient for UCA excitation. Experimental values are confirmed by numerical simulations using the Khokhlov-Zabolotskaya-Kuznetsov equation on a spatially convergent grid.

Concurrent visual and acoustic tracking of passive and active delivery of nanobubbles to tumors.

Background: There has been growing interest in nanobubbles for their potential to extend bubble-mediated ultrasound approaches beyond that of their larger microbubble counterparts. In particular, the smaller scale of nanobubbles may enable them to access the tumor extravascular compartment for imaging and therapy in closer proximity to cancer cells. Compelling preliminary demonstrations of the imaging and therapeutic abilities of nanobubbles have thus emerged, with emphasis on their ability to extravasate. However, studies to date rely on indirect histologic evidence that cannot confirm whether the structures remain intact beyond the vasculature - leaving their extravascular potential largely untapped. Methods: Nanobubble acoustic scattering was assessed using a recently reported ultra-stable formulation at low concentration (106 mL-1) and frequency (1 MHz), over a range of pressures (100-1500 kPa) in a channel phantom. The pressure-dependent response was utilized as a basis for in vivo experiments where ultrasound transmitters and receivers were integrated into a window chamber for simultaneous intravital multiphoton microscopy and acoustic monitoring in tumor-affected microcirculation. Microscopy and acoustic data were utilized to assess passive and active delivery of nanobubbles and determine whether they remained intact beyond the vasculature. Results: Nanobubbles exhibit pressure-dependent nonlinear acoustic scattering. Nanobubbles are also found to have prolonged acoustic vascular pharmacokinetics, and passively extravasate intact into tumors. Ultrasound stimulation of nanobubbles is shown to actively enhance the delivery of both intact nanobubbles and shell material, increasing their spatial bioavailability deeper into the extravascular space. A range of acute vascular effects were also observed. Conclusion: This study presents the first direct evidence that nanobubbles passively and actively extravasate intact in tumor tissue, and is the first to directly capture acute vascular events from ultrasound-stimulation of nanobubbles. The insights gained here demonstrate an important step towards unlocking the potential of nanobubbles and extending ultrasound-based applications.

Novel fractionated ultrashort thermal exposures with MRI-guided focused ultrasound for treating tumors with thermosensitive drugs.

Thermosensitive liposomes represent an important paradigm in oncology, where hyperthermia-mediated release coupled with thermal bioeffects enhance the effectiveness of chemotherapy. Their widespread clinical adoption hinges upon performing controlled targeted hyperthermia, and a leading candidate to achieve this is temperature-based magnetic resonance imaging (MRI)-guided focused ultrasound (MRgFUS). However, the current approach to hyperthermia involves exposures lasting tens of minutes to hours, which is not possible to achieve in many circumstances because of blood vessel cooling and respiratory motion. Here, we investigate a novel approach to overcome these limitations: to use fractionated ultrashort (~30 s) thermal exposures (~41° to 45°C) to release doxorubicin from a thermosensitive liposome. This is first demonstrated in a dorsal chamber tumor model using two-photon microscopy. Thermal exposures were then conducted with a rabbit tumor model using a custom MRgFUS system incorporating temperature feedback control. Drug release was confirmed, and longitudinal experiments demonstrated profoundly enhanced tumor growth inhibition and survival.