Ultrasound Insonation Research Articles

Acoustic Droplet Vaporization Efficiency and Oxygen Scavenging in Whole Blood

ObjectiveAcoustic droplet vaporization (ADV) is the liquid-to-gas phase transition of perfluorocarbon (PFC) droplets to microbubbles upon ultrasound insonation. After ADV, gases dissolved in the surrounding fluid diffuse into microbubbles, enabling oxygen scavenging. Characterization of oxygen scavenging and transition efficiency in whole blood has so far been limited. In this work, oxygen scavenging and PFC droplet transition efficiency in a saline buffer and whole bovine blood were evaluated using blood-gas analysis and flow cytometry. MethodsOxygen scavenging from whole blood via ADV was determined using an in vitro flow phantom with droplets comprising a phospholipid shell and either a decafluorobutane (DFB) or a perfluoropentane (PFP) core. Fluorescent droplets were used to determine ADV transition efficiency in whole blood via flow cytometry. Finally, a mathematical model predicting oxygen scavenging from whole blood was developed based on the experimental transition efficiency values. ResultsDFB droplets enabled greater oxygen scavenging and higher transition efficiency when compared to PFP droplets in both buffer and whole blood. Increasing droplet concentration resulted in a greater amount of hemoglobin-bound and dissolved oxygen scavenging from whole blood. ADV of DFB droplets at a concentration of 5 × 10-4 mL/mL yielded a total oxygen reduction of 913 μM. The transition efficiency decreased with increasing droplet concentration in both buffer and whole blood. Experimental oxygen scavenging data in whole blood aligned with the predicted values from the mathematical model. ConclusionIncreased oxygen scavenging and transition efficiency were achieved with DFB droplets relative to PFP droplets.

A Model of Interpolation of Non-thermal Technique with Antibiotics Ameliorates Diffusion within Biofilm and Prediction of its Binding Site through In-silico Approach

This study aims to elucidate the intricate phenomenon of ultrasound-induced antibiotic transport across bacterial membranes, focusing on the synergistic interplay among sonic oscillation, transient retention, and micropore formation within the plasma membrane. A comprehensive approach was undertaken, involving detailed analysis of E. coli biofilms cultivated for 13 and 24 hours and exposed to distinct ultrasonic frequencies (22 and 33 kHz). Antibiotic diffusion assays were meticulously conducted at 15, 30, 45, and 60 minutes at 37°C. Computational exploration was employed to investigate norfloxacin's binding sites on bacterial gyrase through in-silico methods. The investigation revealed a significant fourfold increase in norfloxacin concentration within biofilms under ultrasound insonation compared to non-insonated samples. Sonic oscillation-induced micropore formation and transient retention facilitated complex exchanges of nutrients, waste, and antibiotics, presenting a potential breakthrough in addressing biofilm infections. Computational analysis further enriched mechanistic understanding by unveiling insightful conformational scores (-7.097 and -7.493 kcal/mol) related to norfloxacin's binding sites on bacterial gyrase. This study underscores the potential of ultrasound-enhanced antibiotic transport as a promising strategy for treating biofilm infections, providing novel insights into antibiotic delivery mechanisms.

Color-Changing Reflection Hologram for Quality Assurance of Therapeutic Ultrasound Systems.

The acoustic output of clinical therapeutic ultrasound equipment requires regular quality assurance (QA) testing to ensure the safety and efficacy of the treatment and that any potentially harmful deviations from the expected output power density are detected as soon as possible. A hologram, consisting of a reflection grating fabricated in an acrylate photopolymer film, has been developed to produce an immediate, visible, and permanent change in the color of the reconstructed hologram from red to green in response to incident ultrasound energy. The influence of the therapeutic ultrasound insonation parameters (exposure time, ultrasound power density, and proximity to the point of maximum acoustic pressure) on the hologram's response has been investigated for two types of therapeutic ultrasound systems: a sonoporation system and an ultrasound physiotherapy system. Findings show that, above a switching temperature of 45 °C, the ultrasound-induced temperature rise produces a structural change in the hologram, which manifests as a visible color change. The area of the color change region correlates with the ultrasound exposure conditions. The suitability of the hologram as a simple and quick QA test tool for therapeutic ultrasound systems has been demonstrated. A prototype ultrasound testing unit which facilitates user-friendly, reproducible testing of the holograms in a clinical setting is also reported.

Ultrasounds trigger reversible permeability of the cerebrovascular endothelium in a novel 3D-bioprinted model of the human blood-brain barrier

The blood-brain barrier (BBB) restricts the penetration of therapeutic agents to the brain. Focused ultrasound (FUS) insonation of microbubbles has enhanced drug delivery via a temporary opening of the tight junctions of the BBB. Rodents are often used for testing novel therapeutic strategies. However, biological discrepancies existing between rodents and humans impedes the translation of some results. We investigated the effect of FUS transient disruption of a human microphysiological model of the BBB consisting of human brain endothelial cells cultured within a 3D-bio printed scaffold designed to reconstitute the architecture of a perfused blood vessel. Cavitation activity was detected in the model when perfused with Lumason microbubbles and exposed to 500 kHz ultrasound at 0.4 MPa for 10 ms pulse duration and 2 Hz pulse repetition frequency. An increase in extravasation of a dye beyond the endothelial layer was detected within 2–3 h of ultrasound insonation. Our results support that this system could be used for targeted BBB opening with a human cell culture model. Moreover, this system holds a promise to further the translational study on US-mediated drug delivery and the development of personalized therapies for patients with brain cancer and other neurovascular disorders.

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.

Ultrasound insonation angle and scanning imaging modes for imaging dental implant structures: A benchtop study.

High frequency ultrasound has shown as a promising imaging modality to evaluate peri-implant tissues. It is not known if the ultrasound imaging settings might influence ultrasound's ability to differentiate implant structures. The aim of this benchtop study was to evaluate the dependence of ultrasound on imaging angles and modes to measure implant geometry-related parameters. A clinical ultrasound scanner (ZS3, Mindray) with an intraoral probe (L30-8) offering combinations of harmonic and compound imaging modes was employed for imaging 16 abutments and 4 implants. The samples were mounted to a micro-positioning system in a water tank, which allowed a range of -30 to 30-degree imaging angles in 5-degree increment between the probe and samples. The abutment angle, implant thread pitch and depth were measured on ultrasound, compared to the reference readings. The errors were computed as a function of the image angles and modes. All samples were replicated 3 times for 3 image modes and 11 image angles, thus resulting in 2,340 images. The mean errors of ultrasound to estimate 16 abutment angles, compared to the reference values, were between -1.8 to 2.7 degrees. The root mean squared error (RMSE) ranged from 1.5 to 4.6 degrees. Ultrasound significantly overestimated the thread pitch by 26.1 μm to 36.2 μm. The error in thread depth measurements were in a range of -50.5 μm to 39.6 μm, respectively. The RMSE of thread pitch and depth of the tested 4 implants was in a range of 34.7 to 56.9 μm and 51.0 to 101.8 μm, respectively. In most samples, these errors were independent of the image angle and modes. Within the limitations of this study, high-frequency ultrasound was feasible in imaging abutments and implant fixtures independent of scanning angle within ±30° of normal incidence and for compounding and non-compounding-based imaging modes.

Acoustic cluster therapy (ACT®) for improved treatment of cancer and brain diseases

Systemic injections of chemotherapeutics often deliver only minor fractions of the administered dose to the targeted pathology, resulting in unwanted toxicity towards healthy tissue. In brain tissue, drug delivery is impaired by the highly selective nature of the blood brain barrier impeding the influx of most substances. Acoustic Cluster Therapy (ACT®) is a platform for targeted therapeutic enhancement, facilitating increased local drug transfer. The platform is comprised of ACT® clusters, a mix of microbubbles and microdroplets and low intensity (diagnostic) ultrasound insonation. Intravenously injected ACT® clusters circulate freely through the body before activation by localised insonation at the targeted pathology. In the ultrasound field, microbubbles transfer energy to microdroplets, which undergo a liquid-to-gas phase transition, forming larger ACT® bubbles that transiently deposit in the targeted microvasculature. Further exposure to ultrasound results in controlled volume oscillation of the ACT® bubbles, inducing a range of biomechanical effects. The effects lead to enhanced extravasation across the endothelial barrier, facilitating the transport of co-administered chemotherapeutics to the targeted tissue. Proof of concept studies showed an increased therapeutic efficacy of standard of care drugs, when combined with ACT® treatment. Furthermore, ACT® treatment induced a controlled and temporal opening of the blood brain barrier observed by the extravasation of fluorescent macromolecules into brain tissue.

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.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Lung Ultrasound Surface Wave Elastography for Assessing Patients With Pulmonary Edema.

B-Mode ultrasound insonation of lungs that are dense with extravascular lung water (EVLW) produces characteristic reverberation artifacts termed B-lines. The number of B-lines present demonstrates reasonable correlation to the amount of EVLW. However, analysis of B-line artifacts generated by this modality is semi-quantitative relying on visual interpretation, and as a result, can be subject to inter-observer variability. The purpose of this study was to translate the use of a novel, quantitative lung ultrasound surface wave elastography technique (LUSWE) into the bedside assessment of pulmonary edema in patients admitted with acute congestive heart failure. B-mode lung ultrasound and LUSWE assessment of the lungs were performed using anterior and lateral intercostal spaces in the supine patient. 14 patients were evaluated at admission with reassessment performed 1-2 days after initiation of diuretic therapy. Each exam recorded the total lung B-lines, lung surface wave speeds (at 100, 150, and 200 Hz) and net fluid balance. The patient cohort experienced effective diuresis (average net fluid balance of negative 2.1 liters) with corresponding decrease in pulmonary edema visualized by B-mode ultrasound (average decrease of 13 B-Lines). In addition, LUSWE demonstrated a statistically significant reduction in the magnitude of wave speed from admission to follow-up. The reduction in lung surface wave speed suggests a decrease in lung stiffness (decreased elasticity) mediated by successful reduction of pulmonary edema. In summary, LUSWE is a noninvasive technique for quantifying elastic properties of superficial lung tissue that may prove useful as a diagnostic test, performed at the bedside, for the quantitative assessment of pulmonary edema.

Gas-Stabilizing Sub-100 nm Mesoporous Silica Nanoparticles for Ultrasound Theranostics.

Recent studies have demonstrated that gas-stabilizing particles can generate cavitating micron-sized bubbles when exposed to ultrasound, offering excellent application potential, including ultrasound imaging, drug delivery, and tumor ablation. However, the majority of the reported gas-stabilizing particles are relatively large (>200 nm), and smaller particles require high acoustic pressures to promote cavitation. Here, this paper reports the preparation of sub-100 nm gas-stabilizing nanoparticles (GSNs) that can initiate cavitation at low acoustic intensities, which can be delivered using a conventional medical ultrasound imaging system. The highly echogenic GSNs (F127-hMSN) were prepared by carefully engineering the surfaces of ∼50 nm mesoporous silica nanoparticles. It was demonstrated that the F127-hMSNs could be continuously imaged with ultrasound in buffer or biological solutions or agarose phantoms for up to 20 min. Also, the F127-hMSN can be stored in phosphate-buffered saline for at least a month with no loss in ultrasound responsiveness. The particles significantly degraded when diluted in simulated body fluids, indicating possible biodegradation of the F127-hMSNs in vivo. Furthermore, at ultrasound imaging conditions, F127-hMSNs did not cause detectable cell death, supporting the potential safety of these particles. Finally, strong cavitation activity generation by the F127-hMSNs under high-intensity focused ultrasound insonation was demonstrated and applied to effectively ablate cancer cells.

Theranostic Attributes of Acoustic Cluster Therapy and Its Use for Enhancing the Effectiveness of Liposomal Doxorubicin Treatment of Human Triple Negative Breast Cancer in Mice.

IntroductionAcoustic cluster therapy (ACT) comprises co-administration of a formulation containing microbubble/microdroplet clusters (PS101), together with a regular medicinal drug (e.g., a chemotherapeutic) and local ultrasound (US) insonation of the targeted pathological tissue (e.g., the tumor). PS101 is confined to the vascular compartment and, when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase-shift to produce bubbles with a median diameter of 22 µm when unconstrained by the capillary wall. In vivo these bubbles transiently lodge in the tumor’s microvasculature. Low frequency ultrasound (300 kHz) at a low mechanical index (MI = 0.15) is then applied to drive oscillations of the deposited ACT bubbles to induce a range of biomechanical effects that locally enhance extravasation, distribution, and uptake of the co-administered drug, significantly increasing its therapeutic efficacy.MethodsIn this study we investigated the therapeutic efficacy of ACT with liposomal doxorubicin for the treatment of triple negative breast cancer using orthotopic human tumor xenografts (MDA-MB-231-H.luc) in athymic mice (ICR-NCr-Foxn1nu). Doxil® (6 mg/kg, i.v.) was administered at days 0 and 21, each time immediately followed by three sequential ACT (20 ml/kg PS101) treatment procedures (n = 7–10). B-mode and nonlinear ultrasound images acquired during the activation phase were correlated to the therapeutic efficacy.ResultsResults show that combination with ACT induces a strong increase in the therapeutic efficacy of Doxil®, with 63% of animals in complete, stable remission at end of study, vs. 10% for Doxil® alone (p < 0.02). A significant positive correlation (p < 0.004) was found between B-mode contrast enhancement during ACT activation and therapy response. These observations indicate that ACT may also be used as a theranostic agent and that ultrasound contrast enhancement during or before ACT treatment may be employed as a biomarker of therapeutic response during clinical use.

Therapeutic Dose Response of Acoustic Cluster Therapy in Combination With Irinotecan for the Treatment of Human Colon Cancer in Mice.

Introduction: Acoustic Cluster Therapy (ACT) comprises coadministration of a formulation containing microbubble-microdroplet clusters (PS101) together with a regular medicinal drug and local ultrasound (US) insonation of the targeted pathological tissue. PS101 is confined to the vascular compartment and when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase shift to produce bubbles with a median diameter of 22 µm. Low frequency, low mechanical index US is then applied to drive oscillations of the deposited ACT bubbles to induce biomechanical effects that locally enhance extravasation, distribution, and uptake of the coadministered drug, significantly increasing its therapeutic efficacy. Methods: The therapeutic efficacy of ACT with irinotecan (60 mg/kg i.p.) was investigated using three treatment sessions given on day 0, 7, and 14 on subcutaneous human colorectal adenocarcinoma xenografts in mice. Treatment was performed with three back-to-back PS101+US administrations per session with PS101 doses ranging from 0.40–2.00 ml PS101/kg body weight (n = 8–15). To induce the phase shift, 45 s of US at 8 MHz at an MI of 0.30 was applied using a diagnostic US system; low frequency exposure consisted of 1 or 5 min at 500 kHz with an MI of 0.20. Results: ACT with irinotecan induced a strong, dose dependent increase in the therapeutic effect (R2 = 0.95). When compared to irinotecan alone, at the highest dose investigated, combination treatment induced a reduction in average normalized tumour volume from 14.6 (irinotecan), to 5.4 (ACT with irinotecan, p = 0.002) on day 27. Median survival increased from 34 days (irinotecan) to 54 (ACT with irinotecan, p = 0.002). Additionally, ACT with irinotecan induced an increase in the fraction of complete responders; from 7% to 26%. There was no significant difference in the therapeutic efficacy whether the low frequency US lasted 1 or 5 min. Furthermore, there was no significant difference between the enhancement observed in the efficacy of ACT with irinotecan when PS101+US was administered before or after irinotecan. An increase in early dropouts was observed at higher PS101 doses. Both mean tumour volume (on day 27) and median survival indicate that the PS101 dose response was linear in the range investigated.

Novel acoustic coupling bath using magnetite nanoparticles for MR-guided transcranial focused ultrasound surgery.

Acoustic coupling baths, nominally composed of degassed water, play important roles during transcranial focused ultrasound surgery. However, this large water bolus also degrades the quality of intraoperative magnetic resonance (MR) guidance imaging. In this study, we test the feasibility of using dilute, aqueous magnetite nanoparticle suspensions to suppress these image degradations while preserving acoustic compatibility. We examine the effects of these suspensions on metrics of image quality and acoustic compatibility for two types of transcranial focused ultrasound insonation regimes: low-duty cycle histotripsy procedures and high-duty cycle thermal ablation procedures. Magnetic resonance guidance imaging was used to monitor thermal ablations of in vitro gel targets using a coupling bath composed of various concentrations of aqueous, suspended, magnetite nanoparticles in a clinical transcranial transducer under stationary and flowing conditions. Thermal deposition was monitored using MR thermometry simultaneous to insonation. Then, using normal degassed water as a coupling bath, various concentrations of aqueous, suspended, magnetite nanoparticles were placed at the center of this same transducer and insonated using high-duty cycle pulsing parameters. Passive cavitation detectors recorded cavitation emissions, which were then used to estimate the relative number of cavitation events per insonation (cavitation duty cycle) and thecavitation dose estimates of each nanoparticle concentration. Finally, the nanoparticle mixtures were exposed to low-duty cycle, histotripsy pulses. Passive cavitation detectors monitored cavitation emissions, which were used to estimate cavitation threshold pressures. The nanoparticles reduced the MR signal of the coupling bath by 90% in T2- and T2*-weighted images and also removed almost all imaging artifacts caused by coupling bath motion. The coupling baths caused <5% changes in peak temperature change achieved during sonication, as observed via MR thermometry. At low duty cycleinsonations, the nanoparticles decreased the cavitation threshold pressure by about 15±7% in a manner uncorrelated with nanoparticle concentration. At high duty cycleinsonations, the 0.5 cavitation duty cycle acoustic power threshold varied linearly with nanoparticle concentration. Dilute aqueous magnetite nanoparticle suspensions effectively reduced MR imaging artifacts caused by the acoustic coupling bath. They also attenuated acoustic power deposition by <5%. For low duty cycle insonation regimes, the nanoparticles decreased the cavitation threshold by 15±7%. However, for high-duty cycle regimes, the nanoparticles decreased the threshold for cavitation in proportion to nanoparticle concentration.

In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke

The development of adjuvant techniques to improve thrombolytic efficacy is important for advancing ischemic stroke therapy. We characterized octafluoropropane and recombinant tissue plasminogen activator (rt-PA)-loaded echogenic liposomes (OFP t-ELIP) using differential interference and fluorescence microscopy, attenuation spectroscopy, and electrozone sensing. The loading of rt-PA in OFP t-ELIP was assessed using spectrophotometry. Further, it was tested whether the agent shields rt-PA against degradation by plasminogen activator inhibitor-1 (PAI-1). An in vitro system was used to assess whether ultrasound (US) combined with either Definity or OFP t-ELIP enhances rt-PA thrombolysis. Human whole blood clots were mounted in a flow system and visualized using an inverted microscope. The perfusate consisted of either (1) plasma alone, (2) rt-PA, (3) OFP t-ELIP, (4) rt-PA and US, (5) OFP t-ELIP and US, (6) Definity and US, or (7) rt-PA, Definity, and US (n = 16 clots per group). An intermittent US insonation scheme was employed (220 kHz frequency, and 0.44 MPa peak-to-peak pressures) for 30 min. Microscopic imaging revealed that OFP t-ELIP included a variety of structures such as liposomes (with and without gas) and lipid-shelled microbubbles. OFP t-ELIP preserved up to 76% of rt-PA activity in the presence of PAI-1, whereas only 24% activity was preserved for unencapsulated rt-PA. The use of US with rt-PA and Definity enhanced lytic efficacy (p < 0.05) relative to rt-PA alone. US combined with OFP t-ELIP enhanced lysis over OFP t-ELIP alone (p < 0.01). These results demonstrate that ultrasound combined with Definity or OFP t-ELIP can enhance the lytic activity relative to rt-PA or OFP t-ELIP alone, respectively.

Abstract 3952: Localized nanodelivery combined with immunotherapy promotes curative anti-tumor responses in a murine breast cancer model

Abstract Optimal pairing of chemotherapy with immunotherapy can harness potential synergies and augment tumor immunity in solid tumors with low mutation burden by i) reducing tumor bulk, ii) releasing tumor antigen for cross-presentation and cross-priming, iii) releasing cancer-suppressive cytokines/chemokines, and iv) retaining circulating immune cell populations. Temperature-sensitive liposomes (TSL) have the potential to accomplish these goals as ultrasound (US) insonation or other treatment modalities can release the chemotherapy within bulky solid tumors in response to hyperthermia to maximize the local response and minimize off-target effects. Here we used Doxorubicin (Dox), stabilized within the core of TSL by forming a drug complex with copper (CuDox), to mediate immunogenic cell death (ICD) and release of type I interferons (IFNs). This activatable chemotherapy (CuDox-TSL+US) was combined with immunotherapy using CpG-ODN, a toll-like receptor 9 agonist, and a PD-1 antibody (αPD-1) for checkpoint blockade. We assessed the schedule and sequence of the chemo-immunotherapy protocol to maximize therapeutic outcome. CuDox-TSL were made of DPPC:MPPC:DSPE-PEG2k, 86:10:4 in the presence of copper (II) gluconate and triethanolamine at 0.2 mg-Dox/mg-lipid. Mice with bilateral invasive neu deletion (NDL) tumors (4 mm) were treated with an i.v. administration of CuDox-TSL at 6 mg Dox/kg body weight. One tumor was insonified with a peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz at 42°C for 5 min prior to and 20 min post drug injection with a variable duty cycle. Upon completion of US-hyperthermia, 100 µg of CpG-ODN 1826 was injected intratumorally to the insonified tumor. Three days later αPD-1 (200 µg, i.p.) was administrated. We found that Dox in combination with hyperthermia induced release of ICD markers and type I IFNs in cell culture of various murine cancer cell lines. Following intravenous administration of CuDox-TSL, ultrasonic activation of liposomes in the treated tumors promoted release and cross-presentation of a model tumor antigen by antigen-presenting cells in distant tumors. While a variety of chemo-immunotherapy protocols achieved a complete response in more than 50% of treated mice, the complete response rate was greatest (90%) when one week of immunotherapy priming preceded activatable chemotherapeutic administration. Repeated chemotherapeutic delivery, while effectively reducing viable tumor, also reduced efficacy and the fraction of viable CD8+ T-cells as indicated by flow cytometric analysis. Taken together, the results suggest that a single-dose administration of activatable chemotherapy after immune priming enhances survival in a murine model of breast cancer. In clinical practice, however, assessment of macrophage and T-cell populations over the course of treatment could help inform dose timing and number of treatment repetitions. Citation Format: Azadeh Kheirolomoom, Matthew T. Silvestrini, Elizabeth S. Ingham, Lisa M. Mahakian, Sarah M. Tam, Spencer K. Tumbale, Josquin Foiret, Neil E. Hubbard, Alexander D. Borowsky, William J. Murphy, Katherine W. Ferrara. Localized nanodelivery combined with immunotherapy promotes curative anti-tumor responses in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3952.

Sonoprinting of nanoparticle-loaded microbubbles: Unraveling the multi-timescale mechanism

Ultrasound-triggered microbubble-assisted drug delivery is a promising tool for localized therapy. Several studies have shown the potential of nanoparticle-loaded microbubbles to effectively enhance the delivery of therapeutic agents to target tissue. We recently discovered that nanoparticle-carrying microbubbles can deposit the nanoparticles in patches onto cell membranes, a process which we termed ‘sonoprinting’. However, the biophysical mechanisms behind sonoprinting are not entirely clear. In addition, the question remains how the ultrasound parameters, such as acoustic pressure and pulse duration, influence sonoprinting. Aiming for a better understanding of sonoprinting, this report investigates the behavior of nanoparticle-loaded microbubbles under ultrasound exposure, making use of three advanced optical imaging techniques with frame rates ranging from 5 frames per second to 10 million frames per second, to capture the biophysical cell-bubble interactions that occur on a multitude of timescales. We observed that non-spherically oscillating microbubbles release their nanoparticle payload in the first few cycles of ultrasound insonation. At low acoustic pressures, the released nanoparticles are transported away from the cells by microstreaming, which does not favor uptake of the nanoparticles by the cells. However, higher acoustic pressures (>300 kPa) and longer ultrasound pulses (>100 cycles) lead to rapid translation of the microbubbles, due to acoustic radiation forces. As a result, the released nanoparticles are transported along in the wake of the microbubbles, which eventually leads to the deposition of nanoparticles in elongated patches on the cell membrane, i.e. sonoprinting. We conclude that a sufficiently high acoustic pressure and long pulses are needed for sonoprinting of nanoparticles on cells.

9 超音波照射による生体軟組織の振動応答(一般講演)

9 超音波照射による生体軟組織の振動応答(一般講演)

Tumor ablation using low-intensity ultrasound and sound excitable drug

The cell membrane is a semi-fluid container that defines the boundary of cells, and provides an enclosed environment for vital biological processes. A sound excitable drug (SED) that is non-cytotoxic to cells is developed to disrupt the plasma membrane under gentle ultrasound insonation, 1MHz, 1W/cm2. The frequency and power density of insonation are within the physical therapy and medical imaging windows; thus the applied ultrasound is safe and not harmful to tissues. The insertion of SEDs into the plasma membrane is not toxic to cells; however, the intruding SEDs weaken the membrane's integrity. Under insonation, the ultrasound energy destabilized the SED disrupted membranes, resulting in membrane rupture and eventual cell death. In a xenograft breast tumor model, the SED alone or the ultrasound alone caused little adverse effects to tumor tissue, while the combined treatment triggered necrosis with a brief local insonation of 3min. The described sono-membrane rupture therapy could be a safe alternative to the currently used high-energy tissue ablation technology, which uses X-rays, gamma rays, electron beams, protons, or high-intensity focused ultrasound.