Enhanced capillary delivery with nanobubble-mediated blood-brain barrier opening and advanced high resolution vascular segmentation.

Overcoming the blood-brain barrier (BBB) is essential to enhance brain therapy. Here, we utilized nanobubbles with focused ultrasound for targeted and improved BBB opening in mice. A microscopy technique method assessed BBB opening at a single blood vessel resolution employing a dual-dye labeling technique using green fluorescent molecules to label blood vessels and Evans blue brain-impermeable dye for quantifying BBB extravasation. A deep learning architecture enabled blood vessels segmentation, delivering comparable accuracy to manual segmentation with a significant time reduction. Segmentation outcomes were applied to the Evans blue channel to quantify extravasation of each blood vessel. Results were compared to microbubble-mediated BBB opening, where reduced extravasation was observed in capillaries with a diameter of 2-6 µm. In comparison, nanobubbles yield an improved opening in these capillaries, and equivalent efficacy to that of microbubbles in larger vessels. These results indicate the potential of nanobubbles to serve as enhanced agents for BBB opening, amplifying bioeffects in capillaries while preserving comparable opening in larger vessels.

Nonlinear Frequency Mixing Ultrasound Imaging of Nanoscale Contrast Agents.

OBJECTIVE: Nanoscale ultrasound contrast agents show promise as alternatives for diagnostics and therapies due to their enhanced stability and ability to traverse blood vessels. Nonetheless, their reduced size limits echogenicity. This study introduces an enhanced nanobubble frequency mixing ultrasound imaging method, by capitalizing on their nonlinear acoustic response to dual-frequency excitation. METHODS: A single broadband transducer (L12-3v) controlled by a programmable ultrasound system was used to transmit a dual-frequency single-cycle wavefront. The frequency mixing effect enabled simultaneous transducer capture of nanobubble-generated sum and difference frequencies in real time without the need for additional hardware or post-processing, by substituting the single-frequency wavefront in a standard contrast harmonic pulse inversion imaging protocol, with the dual-frequency wavefront. RESULTS: Optimization experiments were conducted in tissue mimicking phantoms. Among the dual-frequency combinations that were tested, the highest contrast was obtained using 4&8 MHz. The nanobubble contrast improved with increased mechanical index, and achieved a maximal contrast improvement of 8.4 ± 0.5 dB compared to 4 MHz pulse inversion imaging. In imaging of a breast cancer tumor mouse model, after a systemic nanobubble injection, the contrast was improved by 3.4 ± 1.7, 4.8 ± 1.8, and 6.3 ± 1.6 dB for mechanical indices of 0.04, 0.08, and 0.1, respectively. CONCLUSION: Nonlinear frequency mixing significantly improved the nanobubble contrast, which facilitated their imaging in-vivo. SIGNIFICANCE: This study offers a new avenue to enhance ultrasound imaging utilizing nanobubbles, potentially leading to advancements in other diagnostic applications.

Protocol to assess extravasation of fluorescent molecules in mice after ultrasound-mediated blood-brain barrier opening.

Blood-brain barrier disruption (BBBD) using focused ultrasound (FUS) and microbubbles (MBs) is an effective tool for therapeutic delivery to the brain. Here, we present an optimized protocol for quantifying fluorescent molecules extravasation in mice. We describe steps for ultrasound treatment, injection of MBs and fluorescent dyes, brain harvesting, microscopy imaging, and image postprocessing algorithm. Our protocol has proven to successfully conduct a diameter-dependent analysis that measures vascular leakage following FUS-mediated BBBD at a single blood vessel resolution. For complete details on the use and execution of this protocol, please refer to Katz et al.1.

Versatile Ultrasound-Compatible Microfluidic Platform for In Vitro Microvasculature Flow Research and Imaging Optimization.

Ultrasound localization microscopy (ULM) enables the creation of super-resolved images and velocity maps by localizing and tracking microbubble contrast agents through a vascular network over thousands of frames of ultrafast plane wave images. However, a significant challenge lies in developing ultrasound-compatible microvasculature phantoms to investigate microbubble flow and distribution in controlled environments. In this study, we introduce a new class of gelatin-based microfluidic-inspired phantoms uniquely tailored for ULM studies. These devices allow for the creation of complex and reproducible microvascular networks featuring channel diameters as small as 100 µm. Our experiments focused on microbubble behavior under ULM conditions within bifurcating and converging vessel phantoms. We evaluated the impact of bifurcation angles (25, 45, and 55°) and flow rates (0.01, 0.02, and 0.03 mL/min) on the acquisition time of branching channels. Additionally, we explored the saturation time effect of narrow channels branching off larger ones. Significantly longer acquisition times were observed for the narrow vessels, with an average increase of 72% when a 100 µm channel branched off from a 300 µm channel and an average increase of 90% for a 200 µm channel branching off from a 500 µm channel. The robustness of our fabrication method is demonstrated through the creation of two trifurcating microfluidic phantoms, including one that converges back into a single channel, a configuration that cannot be achieved through traditional methods. This new class of ULM phantoms serves as a versatile platform for noninvasively studying complex flow patterns using ultrasound imaging, unlocking new possibilities for in vitro microvasculature research and imaging optimization.

Nanobubble-mediated cancer cell sonoporation using low-frequency ultrasound.

Ultrasound insonation of microbubbles can form transient pores in cell membranes that enable the delivery of non-permeable extracellular molecules to the cells. Reducing the size of microbubble contrast agents to the nanometer range could facilitate cancer sonoporation. This size reduction can enhance the extravasation of nanobubbles into tumors after an intravenous injection, thus providing a noninvasive sonoporation platform. However, drug delivery efficacy depends on the oscillations of the bubbles, the ultrasound parameters and the size of the target compared to the membrane pores. The formation of large pores is advantageous for the delivery of large molecules, however the small size of the nanobubbles limit the bioeffects when operating near the nanobubble resonance frequency at the MHz range. Here, we show that by coupling nanobubbles with 250 kHz low frequency ultrasound, high amplitude oscillations can be achieved, which facilitate low energy sonoporation of cancer cells. This is beneficial both for increasing the uptake of a specific molecule and to improve large molecule delivery. The method was optimized for the delivery of four fluorescent molecules ranging in size from 1.2 to 70 kDa to breast cancer cells, while comparing the results to targeted microbubbles. Depending on the fluorescent molecule size, the optimal ultrasound peak negative pressure was found to range between 300 and 500 kPa. Increasing the pressure to 800 kPa reduced the fraction of fluorescent cells for all molecules sizes. The optimal uptake for the smaller molecule size of 4 kDa resulted in a fraction of 19.9 ± 1.8% of fluorescent cells, whereas delivery of 20 kDa and 70 kDa molecules yielded 14 ± 0.8% and 4.1 ± 1.1%, respectively. These values were similar to targeted microbubble-mediated sonoporation, suggesting that nanobubbles can serve as noninvasive sonoporation agents with a similar potency, and at a reduced bubble size. The nanobubbles effectively reduced cell viability and may thus potentially reduce the tumor burden, which is crucial for the success of cancer treatment. This method provides a non-invasive and low-energy tumor sonoporation theranostic platform, which can be combined with other therapies to maximize the therapeutic benefits of cancer treatment or be harnessed in gene therapy applications.

Dense speed-of-sound shift imaging for ultrasonic thermometry.

Objective. Develop a dense algorithm for calculating the speed-of-sound shift between consecutive acoustic acquisitions as a noninvasive means to evaluating temperature change during thermal ablation.Methods. An algorithm for dense speed-of-sound shift imaging (DSI) was developed to simultaneously incorporate information from the entire field of view using a combination of dense optical flow and inverse problem regularization, thus speeding up the calculation and introducing spatial agreement between pixels natively. Thermal ablation monitoring consisted of two main steps: pixel shift tracking using Farneback optical flow, and mathematical modeling of the relationship between the pixel displacement and temperature change as an inverse problem to find the speed-of-sound shift. A calibration constant translates from speed-of-sound shift to temperature change. The method performance was tested inex vivosamples and compared to standard thermal strain imaging (TSI) methods.Main results. Thermal ablation at a frequency of 2 MHz was applied to an agarose phantom that created a speed-of-sound shift measured by an L12-5 imaging transducer. A focal spot was reconstructed by solving the inverse problem. Next, a thermocouple measured the temperature rise during thermal ablation ofex vivochicken breast to calibrate the setup. Temperature changes between 3 °C and 15 °C was measured with high thermometry precision of less than 2 °C error for temperature changes as low as 8 °C. The DSI method outperformed standard TSI in both spatial coherence and runtime in high-intensity focused ultrasound-induced hyperthermia.Significance. Dense ultrasonic speed-of-sound shift imaging can successfully monitor the speed-of-sound shift introduced by thermal ablation. This technique is faster and more robust than current methods, and therefore can be used as a noninvasive, real time and cost-effective thermometry method, with high clinical applicability.

Diameter-dependent assessment of microvascular leakage following ultrasound-mediated blood-brain barrier opening.

Blood brain barrier disruption (BBBD) using focused ultrasound (FUS) and microbubbles (MB) is an effective tool for therapeutic delivery to the brain. BBBD depends to a great extent on MB oscillations. Because the brain vasculature is heterogenic in diameter, reduced MB oscillations in smaller blood vessels, together with a lower number of MBs in capillaries, can lead to variations in BBBD. Therefore, evaluating the impact of microvasculature diameter on BBBD is of great importance. We present a method to characterize molecules extravasation following FUS-mediated BBBD, at a single blood vessel resolution. Evans blue (EB) leakage was used as marker for BBBD, whereas blood vessels localization was done using FITC labeled Dextran. Automated image processing pipeline was developed to quantify the extent of extravasation as function of microvasculature diameter, including a wide range of vascular morphological parameters. Variations in MB vibrational response were observed in blood vessel mimicking fibers with varied diameters. Higher peak negative pressures (PNP) were required to initiate stable cavitation in fibers with smaller diameters. In vivo in the treated brains, EB extravasation increased as a function of blood vessel diameter. The percentage of strong BBBD blood vessels increased from 9.75% for 2-3 µm blood vessels to 91.67% for 9-10 µm. Using this method, it is possible to conduct a diameter-dependent analysis that measures vascular leakage resulting from FUS-mediated BBBD at a single blood vessel resolution.

Ratiometric Fluorescent Detection of Ultrasound-Regulated ATP Release: An Ultrasound-Resistant Cu,N-Doped Carbon Nanosphere.

Focused ultrasound, as a protocol of cancer therapy, might induce extracellular adenosine triphosphate (ATP) release, which could enhance cancer immunotherapy and be monitored as a therapeutic marker. To achieve an ATP-detecting probe resistant to ultrasound irradiation, we constructed a Cu/N-doped carbon nanosphere (CNS), which has two fluorescence (FL) emissions at 438 and 578 nm to detect ultrasound-regulated ATP release. The addition of ATP to Cu/N-doped CNS was conducted to recover the FL intensity at 438 nm, where ATP enhanced the FL intensity probably via intramolecular charge transfer (ICT) primarily and hydrogen-bond-induced emission (HBIE) secondarily. The ratiometric probe was sensitive to detect micro ATP (0.2-0.6 µM) with the limit of detection (LOD) of 0.068 µM. The detection of ultrasound-regulated ATP release by Cu,N-CNS/RhB showed that ATP release was enhanced by the long-pulsed ultrasound irradiation at 1.1 MHz (+37%, p < 0.01) and reduced by the short-pulsed ultrasound irradiation at 5 MHz (-78%, p < 0.001). Moreover, no significant difference in ATP release was detected between the control group and the dual-frequency ultrasound irradiation group (+4%). It is consistent with the results of ATP detection by the ATP-kit. Besides, all-ATP detection was developed to prove that the CNS had ultrasound-resistant properties, which means it could bear the irradiation of focused ultrasound in different patterns and detect all-ATP in real time. In the study, the ultrasound-resistant probe has the advantages of simple preparation, high specificity, low limit of detection, good biocompatibility, and cell imaging ability. It has great potential to act as a multifunctional ultrasound theranostic agent for simultaneous ultrasound therapy, ATP detection, and monitoring.

Deep Learning-Based Ultrasound Beam Shaping for Spatiotemporal Acoustic Holograms Generation.

Acoustical hologram generation can be achieved via controlled beam shaping by engineering the transmitted phases to create a desired pattern. Optically inspired phase retrieval algorithms and standard beam shaping methods assume continuous wave (CW) insonation, which successfully generate acoustic holograms for therapeutic applications that involve long burst transmissions. However, a phase engineering technique designed for single-cycle transmission and capable of achieving spatiotemporal interference of the transmitted pulses is needed for imaging applications. Toward this goal, we developed a multilevel residual deep convolutional network for calculating the inverse process that will yield the phase map for the creation of a multifoci pattern. The ultrasound deep learning (USDL) method was trained on simulated training pairs of multifoci patterns in the focal plane and their corresponding phase maps in the transducer plane, where propagation between the planes was performed via singe cycle transmission. The USDL method outperformed the standard Gerchberg-Saxton (GS) method, when transmitted with single cycle excitation, in parameters including the number of focal spots that were generated successfully and their pressure and uniformity. In addition, the USDL method was shown to be flexible in generating patterns with large focal spacing, uneven spacing, and nonuniform amplitudes. In simulations, the largest improvement was obtained for four foci patterns, where the GS method succeeded in creating 25% of the requested patterns, while the USDL method successfully created 60% of the patterns. These results were confirmed experimentally via hydrophone measurements. Our findings suggest that deep learning-based beam shaping can facilitate the next generation of acoustical holograms for ultrasound imaging applications.

Dual ligand-capped gold nanoclusters for the smart detection of specific reactive oxygen species.

Quantitative detection of different types of reactive oxygen species (ROS) is vital for understanding the crucial roles of them in biological processes. However, few researches achieved the detection of multiple types of ROS with one probe until now. Given this, we designed and prepared fluorescent gold nanoclusters capped by dual ligand bovine serum albumin and lysozyme (BSA-LYS-AuNCs), which could detect 3 specific types of ROS based on its different fluorescent responses to H2O2, •OH and ClO-, respectively. The limit of detection (LOD) of H2O2, •OH, and ClO- was as low as 0.82 µM, 0.45 µM, and 0.62 µM. Moreover, as an important ROS type, ClO- was detected with high sensitivity and low LOD by BSA-LYS-AuNCs. It was also proved that the crosslinking of protein mainly contributed to the unique fluorescent characteristics of the probe exposing to ClO-. Furthermore, the fluorescent probe achieved the smart detection of hROS (including •OH and ClO-) and wROS (the form of H2O2) in the real sample, which could also been applied specifically to the detection of antioxidants, e.g. ascorbic acid. The gold nanoclusters developed have high potential for the smart detection of multiple ROS in the body fluid of organisms.

On-demand regulation and enhancement of the nucleation in acoustic droplet vaporization using dual-frequency focused ultrasound.

Acoustic droplet vaporization (ADV) plays an important role in focused ultrasound theranostics. Better understanding of the relationship between the ultrasound parameters and the ADV nucleation could provide an on-demand regulation and enhancement of ADV for improved treatment outcome. In this work, ADV nucleation was performed in a dual-frequency focused ultrasound configuration that consisted of a continuous low-frequency ultrasound and a short high-frequency pulse. The combination was modelled to investigate the effects of the driving frequency and acoustic power on the nucleation rate, efficiency, onset time, and dimensions of the nucleation region. The results showed that the inclusion of short pulsed high-frequency ultrasound significantly increased the nucleation rate with less energy, reduced the nucleation onset time, and changed the length-width ratio of the nucleation region, indicating the dual-frequency ultrasound mode yields an efficient enhancement of the ADV nucleation, compared to a single-frequency ultrasound mode. Furthermore, the acoustic and temperature fields varied independently with the dual-frequency ultrasound parameters. This facilitated the spatial and temporal control over the ADV nucleation, and opens the door to the possibility to realize on-demand regulation of the ADV occurrence in ultrasound theranostics. In addition, the improved energy efficacy that is obtained with the dual-frequency configuration lowered the requirements on hardware system, increasing its flexibility and could facilitate its implementation in practical applications.

Development of an ultrasound guided focused ultrasound system for 3D volumetric low energy nanodroplet-mediated histotripsy.

Low pressure histotripsy is likely to facilitate current treatments that require extremely high pressures. An ultrasound guided focused ultrasound system was designed to accommodate a rotating imaging transducer within a low frequency therapeutic transducer that operates at a center frequency of 105 kHz. The implementation of this integrated system provides real-time therapeutic and volumetric imaging functions, that are used here for low-cost, low-energy 3D volumetric ultrasound histotripsy using nanodroplets. A two-step approach for low pressure histotripsy is implemented with this dual-array. Vaporization of nanodroplets into gaseous microbubbles was performed via the 1D rotating imaging probe. The therapeutic transducer is then used to detonate the vaporized nanodroplets and trigger potent mechanical effects in the surrounding tissue. Rotating the imaging transducer creates a circular vaporized nanodroplet shape which generates a round lesion upon detonation. This contrasts with the elongated lesion formed when using a standard 1D imaging transducer for nanodroplet activation. Optimization experiments show that maximal nanodroplet activation can be achieved with a 2-cycle excitation pulse at a center frequency of 3.5 MHz, and a peak negative pressure of 3.4 MPa (a mechanical index of 1.84). Vaporized nanodroplet detonation was achieved by applying a low frequency treatment at a center frequency of 105 kHz and mechanical index of 0.9. In ex-vivo samples, the rotated nanodroplet activation method yielded the largest lesion area, with a mean of 4.7 ± 0.5 mm2, and a rounded shape. In comparison, standard fixed transducer nanodroplet activation resulted in an average lesion area of 2.6 ± 0.4 mm2, and an elongated shape. This hybrid system enables to achieve volumetric low energy histotripsy, and thus facilitates the creation of precise, large-volume mechanical lesions in tissues, while reducing the pressure threshold required for standard histotripsy by over an order of magnitude.

Efficacy optimization of low frequency microbubble-mediated sonoporation as a drug delivery platform to cancer cells.

Ultrasound insonation of microbubbles can be used to form pores in cell membranes and facilitate the local trans-membrane transport of drugs and genes. An important factor in efficient delivery is the size of the delivered target compared to the generated membrane pores. Large molecule delivery remains a challenge, and can affect the resulting therapeutic outcomes. To facilitate large molecule delivery, large pores need to be formed. While ultrasound typically uses megahertz frequencies, it was recently shown that when microbubbles are excited at a frequency of 250 kHz (an order of magnitude below the resonance frequency of these agents), their oscillations are significantly enhanced as compared to the megahertz range. Here, to promote the delivery of large molecules, we suggest using this low frequency and inducing large pore formation through the high-amplitude oscillations of microbubbles. We assessed the impact of low frequency microbubble-mediated sonoporation on breast cancer cell uptake by optimizing the delivery of 4 fluorescent molecules ranging from 1.2 to 70 kDa in size. The optimal ultrasound peak negative pressure was found to be 500 kPa. Increasing the pressure did not enhance the fraction of fluorescent cells, and in fact reduced cell viability. For the smaller molecule sizes, 1.2 kDa and 4 kDa, the groups treated with an ultrasound pressure of 500 kPa and MB resulted in a fraction of 58% and 29% of fluorescent cells respectively, whereas delivery of 20 kDa and 70 kDa molecules yielded 10% and 5%, respectively. These findings suggest that low-frequency (e.g., 250 kHz) insonation of microbubbles results in high amplitude oscillation in vitro that increase the uptake of large molecules. Successful ultrasound-mediated molecule delivery requires the careful selection of insonation parameters to maximize the therapeutic effect by increasing cell uptake.

Low frequency nanobubble-enhanced ultrasound mechanotherapy for noninvasive cancer surgery.

Scaling down the size of microbubble contrast agents to the nanometer level holds the promise for noninvasive cancer therapy. However, the small size of nanobubbles limits the obtained bioeffects as a result of ultrasound cavitation, when operating near the nanobubble resonance frequency. Here we show that coupled with low energy insonation at a frequency of 80 kHz, well below the resonance frequency of these agents, nanobubbles serve as noninvasive therapeutic warheads that trigger potent mechanical effects in tumors following a systemic injection. We demonstrate these capabilities in tissue mimicking phantoms, where a comparison of the acoustic response of micro- and nano-bubbles after insonation at a frequency of 250 or 80 kHz revealed that higher pressures were needed to implode the nanobubbles compared to microbubbles. Complete nanobubble destruction was achieved at a mechanical index of 2.6 for the 250 kHz insonation vs. 1.2 for the 80 kHz frequency. Thus, the 80 kHz insonation complies with safety regulations that recommend operation below a mechanical index of 1.9. In vitro in breast cancer tumor cells, the cell viability was reduced to 17.3 ± 1.7% of live cells. In vivo, in a breast cancer tumor mouse model, nanobubble tumor distribution and accumulation were evaluated by high frequency ultrasound imaging. Finally, nanobubble-mediated low frequency insonation of breast cancer tumors resulted in effective mechanical tumor ablation and tumor tissue fractionation. This approach provides a unique theranostic platform for safe, noninvasive and low energy tumor mechanotherapy.

Optimized Simultaneous Axial Multifocal Imaging via Frequency Multiplexed Focusing.

Simultaneous axial multifocal imaging (SAMI) using a single acoustical transmission was developed to enhance the depth of field. This technique transmits a superposition of axial multifoci waveforms in a single transmission, thus increasing the frame rate. However, since all the waveforms are transmitted at a constant center frequency, there is a tradeoff between attenuation and lateral resolution when choosing a constant frequency for all the axial depths. In this work, we developed an optimized SAMI method by adding frequency dependence to each axial multifocus. By gradually increasing the frequency as a function of the focal depth, this method makes it possible to compensate for the gradually increasing F-number in order to achieve constant lateral resolution across the entire field of view. Alternatively, by gradually decreasing the axial multifoci frequencies as a function of depth, enhanced penetration depth and contrast are obtained. This method, termed frequency multiplexed SAMI (FM-SAMI), is described analytically and validated by resolution and contrast experiments performed on resolution targets, tissue-mimicking phantoms, and ex vivo biological samples. This is the first real-time implementation of a frequency multiplexing approach for axial multifoci imaging that facilitates high-quality imaging at an increased frame rate.

Ultrasound Frequency Mixing for Enhanced Contrast Harmonic Imaging of Microbubbles.

Microbubbles (MBs) serve as contrast agents in diagnostic ultrasound (US) imaging. Contrast harmonic imaging (CHI) of MBs takes advantage of their nonlinear properties that generate additional harmonic frequencies in the received spectrum. However, CHI suffers from limitations in terms of contrast, the signal-to-noise ratio, and artifacts. This article presents an enhanced, real-time, nonlinear imaging technique based on the excitation of MBs with a dual frequency waveform. The MBs trigger a frequency mixing effect that generates additional frequency components in the received spectrum; i.e., difference and sum frequencies, in addition to the standard harmonics, thus amplifying the MB's nonlinear response and enhancing image contrast. In this real-time approach, two single frequency waveforms are superpositioned into a dual frequency transmission. The dual frequency waveform is incorporated into a standard pulse-inversion (PI) sequence and is transmitted by an array transducer using an arbitrary waveform generator (AWG) in a programmable US system. Upon receive, standard dynamic receive beamforming is used, without additional post processing. Numerical simulations using the Marmottant model are used to confirm the generation of the difference frequency in the MB's backscattered echoes. The resulting image quality enhancement is demonstrated in a tissue-mimicking phantom containing MBs' suspension. A maximal contrast improvement of 3.43 dB compared to standard PI was achieved, along with a reduction by 4.5 fold in the mechanical index (MI).

Nanodroplet-Mediated Low-Energy Mechanical Ultrasound Surgery.

Mechanical ultrasound surgery methods use short, high-intensity pulses to fractionate tissues. This study reports the development of a two-step technology for low-energy mechanical ultrasound surgery of tissues using nanodroplets to reduce the pressure threshold. Step 1 consists of vaporizing the nanodroplets into gaseous microbubbles via megahertz ultrasound excitation. Then, low-frequency ultrasound is applied to the microbubbles, which turns them into therapeutic warheads that trigger potent mechanical effects in the surrounding tissue. The use of nanoscale nanodroplets coupled with low-frequency ultrasound reduces the pressure threshold required for mechanical ultrasound surgery by an order of magnitude. In addition, their average diameter of 300 nm can overcome challenges associated with the size of microbubbles. Optimization experiments were performed to determine the ultrasound parameters for nanodroplet vaporization and the subsequent microbubble implosion processes. Optimal vaporization was obtained when transmitting a 2-cycle excitation pulse at a center frequency of 5 MHz and a peak negative pressure of 4.1 MPa (mechanical index = 1.8). Low-frequency insonation of the generated microbubbles at a center frequency of 850, 250 or 80 kHz caused enhanced contrast reduction at a center frequency of 80 kHz, compared with the other frequencies, while operating at the same mechanical index of 0.9. Nanodroplet-mediated insonation of ex vivo chicken liver samples generated mechanical damage. Low-frequency treatment at a mechanical index of 0.9 and a center frequency of 80 kHz induced the largest lesion area (average of 0.59 mm2) compared with 250- and 850-kHz treatments with the same mechanical index (average lesions areas of 0.29 and 0.19 mm2, respectively, p < 0.001). The two-step approach makes it possible to conduct both the vaporization and implosion stages at mechanical indices below 1.9, thus avoiding undesired mechanical damage. The findings indicate that coupled with low-frequency ultrasound, nanodroplets can be used for low-energy mechanical ultrasound surgery.

Acoustically Detonated Microbubbles Coupled with Low Frequency Insonation: Multiparameter Evaluation of Low Energy Mechanical Ablation.

Noninvasive ultrasound surgery can be achieved using focused ultrasound to locally affect the targeted site without damaging intervening tissues. Mechanical ablation and histotripsy use short and intense acoustic pulses to destroy the tissue via a purely mechanical effect. Here, we show that coupled with low-frequency excitation, targeted microbubbles can serve as mechanical therapeutic warheads that trigger potent mechanical effects in tumors using focused ultrasound. Upon low frequency excitation (250 kHz and below), high amplitude microbubble oscillations occur at substantially lower pressures as compared to higher MHz ultrasonic frequencies. For example, inertial cavitation was initiated at a pressure of 75 kPa for a center frequency of 80 kHz. Low frequency insonation of targeted microbubbles was then used to achieve low energy tumor cell fractionation at pressures below a mechanical index of 1.9, and in accordance with the Food and Drug Administration guidelines. We demonstrate these capabilities in vitro and in vivo. In cell cultures, cell viability was reduced to 16% at a peak negative pressure of 800 kPa at the 250 kHz frequency (mechanical index of 1.6) and to 10% at a peak negative pressure of 250 kPa at a frequency of 80 kHz (mechanical index of 0.9). Following an intratumoral injection of targeted microbubbles into tumor-bearing mice, and coupled with low frequency ultrasound application, significant tumor debulking and cancer cell death was observed. Our findings suggest that reducing the center frequency enhances microbubble-mediated mechanical ablation; thus, this technology provides a unique theranostic platform for safe low energy tumor fractionation, while reducing off-target effects.

Enhanced HIFU Theranostics with Dual-Frequency-Ring Focused Ultrasound and Activatable Perfluoropentane-Loaded Polymer Nanoparticles.

High-intensity focused ultrasound (HIFU) has been widely used in tumor ablation in clinical settings. Meanwhile, there is great potential to increase the therapeutic efficiency of temporary cavitation due to enhanced thermal effects and combined mechanical effects from nonlinear vibration and collapse of the microbubbles. In this study, dual-frequency (1.1 and 5 MHz) HIFU was used to produce acoustic droplet vaporization (ADV) microbubbles from activatable perfluoropentane-loaded polymer nanoparticles (PFP@Polymer NPs), which increased the therapeutic outcome of the HIFU and helped realize tumor theranostics with ultrasound contrast imaging. Combined with PFP@Polymer NPs, dual-frequency HIFU changed the shape of the damage lesion and reduced the acoustic intensity threshold of thermal damage significantly, from 216.86 to 62.38 W/cm2. It produced a nearly 20 °C temperature increase in half the irradiation time and exhibited a higher tumor inhibition rate (84.5% ± 3.4%) at a low acoustic intensity (1.1 MHz: 23.77 W/cm2; 5 MHz: 0.35 W/cm2) in vitro than the single-frequency HIFU (60.2% ± 11.9%). Moreover, compared with the traditional PFP@BSA NDs, PFP@Polymer NPs showed higher anti-tumor efficacy (81.13% vs. 69.34%; * p < 0.05) and better contrast-enhanced ultrasound (CEUS) imaging ability (gray value of 57.53 vs. 30.67; **** p < 0.0001), probably benefitting from its uniform and stable structure. It showed potential as a highly efficient tumor theranostics approach based on dual-frequency HIFU and activatable PFP@Polymer NPs.

Immune modulation resulting from MR-guided high intensity focused ultrasound in a model of murine breast cancer.

High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65 °C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1ß and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.