Characterisation of Gas Vesicles as Cavitation Nuclei for Ultrasound Therapy using Passive Acoustic Mapping.

Genetically encodable gas filled particles known as gas vesicles (GVs) have shown promise as a biomolecular contrast agent for ultrasound imaging and have the potential to be used as cavitation nuclei for ultrasound therapy. In this study, we used passive acoustic mapping techniques to characterize GV-seeded cavitation, utilizing 0.5 and 1.6 MHz ultrasound over peak rarefactional pressures ranging from 100 to 2200 kPa. We found that GVs produce cavitation for the duration of the first applied pulse, up to at least 5000 cycles, but that bubble activity diminishes rapidly over subsequent pulses. At 0.5 MHz the frequency content of cavitation emissions was predominantly broadband in nature, whilst at 1.6 MHz narrowband content at harmonics of the main excitation frequency dominated. Simulations and high-speed camera imaging suggest that the received cavitation emissions come not from individual GVs but instead from the coalescence of GV-released gas into larger bubbles during the applied ultrasound pulse. These results will aid the future development of GVs as cavitation nuclei in ultrasound therapy.

Microbubble-laden aerosols improve post-nasal aerosol penetration efficiency in a preterm neonate model.

Nebulized lung surfactant therapy has been a neonatology long-pursued goal. Nevertheless, many clinical trials have yet to show a clear clinical efficacy of nebulized surfactant, which, in part, is due to the technical challenges of delivering aerosols to the lungs of preterm neonates. The study aimed to test microbubbles for improving lung deposition in preterm neonates. An in vitro testing method was developed to replicate the clinical environment; it used a 3D-printed preterm neonate model, connected to a high-flow nasal cannula (HFNC) and a vibrating mesh nebulizer. The flow rate of the HFNC mirrored that used in the clinics (i.e., 4, 6, and 8 L/min). Followingly, the lung penetrations of aerosols with and without microbubbles were compared. The aerodynamic diameter of aerosols with microbubbles (MMAD=1.75 µm) was lower than that of the counterpart (MMAD=2.25 µm). Microbubble-laden aerosols had a significantly higher number of microbubbles that were below 1.0 µm. Microbubble-laden aerosols had dramatically higher lung penetration in the preterm model; lung penetration efficiencies were 30.0, 25.5, and 17.5 % at 4, 6, and 8 L/min, respectively, whereas the lung penetration efficiency for conventionally nebulized aerosols was below 1.25 % in the three flow rates.

Quantitative evaluation of anti-biofilm cavitation activity seeded from microbubbles or protein cavitation nuclei by passive acoustic mapping.

OBJECTIVE: Bacterial biofilms represent a major challenge for effective antibiotic therapy as they confer physical and functional changes that protect bacteria from their surrounding environment. In this work, focused ultrasound in combination with cavitation nuclei was used to disrupt biofilms of Staphylococcus aureus and Pseudomonas aeruginosa, both of which are on the World Health Organization's priority list for new antimicrobial research. Approach: Single species biofilms were exposed to ultrasound (0.5 MHz centre frequency, 0.5-1.5 MPa peak rarefactional pressure, 200 cycle pulses, 5 Hz repetition frequency, 30 s duration), in the presence of two different types of cavitation nuclei. Quantitative passive acoustic mapping (PAM) was used to monitor cavitation emissions during treatment using a calibrated linear array. Main Results: It was observed that the cumulative energy of acoustic emissions during treatment was positively correlated with biofilm disruption, with differences between bacterial species attributed to differences in biofilm morphology. PCaN provided increased biofilm reduction compared to microbubbles due in large part to their persistence over the duration of ultrasound exposure. There was also good correlation between the spatial distribution of cavitation as characterized by PAM and the extent of biofilm disruption observed with microscopy. Significance: Collectively, the results from this work indicate the potential broad applicability of cavitation for eliminating biofilms of priority pathogens and the opportunity presented by Passive Acoustic Mapping for real-time monitoring of antimicrobial processes.

Deep learning-assisted interactive contouring of lung cancer: Impact on contouring time and consistency.

BACKGROUND AND PURPOSE: To evaluate the impact of a deep learning (DL)-assisted interactive contouring tool on inter-observer variability and the time taken to complete tumour contouring. MATERIALS AND METHODS: Nine clinicians contoured the gross tumour volume (GTV) using the PET-CT scans of 10 non-small cell lung cancer (NSCLC) patients, either using DL-assisted or manual contouring tools. After contouring a case using one contouring method, the same case was contoured one week later using the other method. The contours and time taken were compared. RESULTS: Use of the DL-assisted tool led to a statistically significant decrease in active contouring time of 23 % relative to the standard manual segmentation method (p < 0.01). The mean observation time for all clinicians and cases made up nearly 60 % of interaction time for both contouring approaches. On average the time spent contouring per case was reduced from 22 min to 19 min when using the DL-assisted tool. Additionally, the DL-assisted tool reduced contour variability in the parts of tumour where clinicians tended to disagree the most, while the consensus contour was similar whichever of the two contouring approaches was used. CONCLUSIONS: A DL-assisted interactive contouring approach decreased active contouring time and local inter-observer variability when used to delineate lung cancer GTVs compared to a standard manual method. Integration of this tool into the clinical workflow could assist clinicians in contouring tasks and improve contouring efficiency.

Microbubbles bound to drug-eluting beads enable ultrasound imaging and enhanced delivery of therapeutics.

Transarterial chemoembolization (TACE) is an image-guided minimally invasive treatment for liver cancer which involves delivery of chemotherapy and embolic material into tumor-supplying arteries to block blood flow to a liver tumor and to deliver chemotherapy directly to the tumor. However, the released drug diffuses only less than a millimeter away from the beads. To enhance the efficacy of TACE, the development of microbubbles electrostatically bound to the surface of drug-eluting beads loaded with different amounts of doxorubicin (0-37.5 mg of Dox/mL of beads) is reported. Up to 400 microbubbles were bound to Dox-loaded beads (70-150 microns). This facilitated ultrasound imaging of the beads and increased the release rate of Dox upon exposure to high intensity focused ultrasound (HIFU). Furthermore, ultrasound exposure (1 MPa peak negative pressure) increased the distance at which Dox could be detected from beads embedded in a tissue-mimicking phantom, compared with a no ultrasound control.

Mitigating infections in implantable urological continence devices: risks, challenges, solutions, and future innovations. A comprehensive literature review.

PURPOSE OF REVIEW: Stress urinary incontinence is a growing issue in ageing men, often following treatment for prostate cancer or bladder outflow obstruction. While implantable urological devices offer relief, infections are a significant concern. These infections can lead to device removal, negating the benefits and impacting patient outcomes. This review explores the risks and factors contributing to these infections and existing strategies to minimize them. These strategies encompass a multifaceted approach that considers patient-specific issues, environmental issues, device design and surgical techniques. However, despite these interventions, there is still a pressing need for further advancements in device infection prevention. RECENT FINDINGS: Faster diagnostics, such as Raman spectroscopy, could enable early detection of infections. Additionally, biocompatible adjuncts like ultrasound-responsive microbubbles hold promise for enhanced drug delivery and biofilm disruption, particularly important as antibiotic resistance rises worldwide. SUMMARY: By combining advancements in diagnostics, device design, and patient-specific surgical techniques, we can create a future where implantable urological devices offer men a significant improvement in quality of life with minimal infection risk.

Investigation of Ultrasound Mediated Extravasation of a Model Drug by Perfluorobutane Nanodroplets.

OBJECTIVE: Perfluorocarbon nanodroplets (NDs) have been widely investigated as both diagnostic and therapeutic agents. There remains, however, a challenge in generating NDs that do not vaporize spontaneously but can be activated at ultrasound pressures that do not produce unwanted bioeffects. In previous work, it has been shown that phospholipid-coated perfluorobutane (PFB) NDs can potentially overcome this challenge. The aim of this study was to investigate whether these NDs can promote drug delivery. METHODS: A combination of high-speed optical imaging and passive cavitation detection was used to study the acoustic properties of the PFB-NDs in a tissue mimicking phantom. PFB-NDs were exposed to ultrasound at frequencies from 0.5 to 1.5 MHz and peak negative pressures from 0.5 to 3.5 MPa. In addition, the penetration depth of two model drugs (Nile Red and 200 nm diameter fluorescent polymer spheres) into the phantom was measured. RESULTS: PFB NDs were found to be stable in aqueous suspension at both 4°C and 37°C; their size remaining unchanged at 215 ± 11 nm over 24 h. Penetration of both model drugs in the phantom was found to increase with increasing ultrasound peak negative pressure and decreasing frequency and was found to be positively correlated with the energy of acoustic emissions. Extravasation depths >1 mm were observed at 0.5 MHz with pressures <1 MPa. CONCLUSION: The results of the study thus suggest that PFB NDs can be used both as drug carriers and as nuclei for cavitation to enhance drug delivery without the need for high intensity ultrasound.

Targeted opening of the blood-brain barrier using VCAM-1 functionalised microbubbles and "whole brain" ultrasound.

Metastatic tumours in the brain now represent one of the leading causes of death from cancer. Current treatments are largely ineffective owing to the combination of late diagnosis and poor delivery of therapies across the blood-brain barrier (BBB). Conjugating magnetic resonance imaging (MRI) contrast agents with a monoclonal antibody for VCAM-1 (anti-VCAM1) has been shown to enable detection of micrometastases, two to three orders of magnitude smaller in volume than those currently detectable clinically. The aim of this study was to exploit this targeting approach to enable localised and temporary BBB opening at the site of early-stage metastases using functionalised microbubbles and ultrasound. Methods: Microbubbles functionalised with anti-VCAM1 were synthesised and shown to bind to VCAM-1-expressing cells in vitro. Experiments were then conducted in vivo in a unilateral breast cancer brain metastasis mouse model using Gadolinium-DTPA (Gd-DTPA) enhanced MRI to detect BBB opening. Following injection of Gd-DTPA and targeted microbubbles, the whole brain volume was simultaneously exposed to ultrasound (0.5 MHz, 10% duty cycle, 0.7 MPa peak negative pressure, 2 min treatment time). T1-weighted MRI was then performed to identify BBB opening, followed by histological confirmation via immunoglobulin G (IgG) immunohistochemistry. Results: In mice treated with targeted microbubbles and ultrasound, statistically significantly greater extravasation of Gd-DTPA and IgG was observed in the left tumour-bearing hemisphere compared to the right hemisphere 5 min after treatment. No acute adverse effects were observed. There was no investigation of longer term bioeffects owing to the nature of the study. Conclusion: The results demonstrate the feasibility of using targeted microbubbles in combination with low intensity ultrasound to localise opening of the BBB to metastatic sites in the brain. This approach has potential application in the treatment of metastatic tumours whose location cannot be established a priori with conventional imaging methods.

Modelling Drug Delivery to the Small Airways: Optimization Using Response Surface Methodology.

AIM: The aim of this in silico study was to investigate the effect of particle size, flow rate, and tidal volume on drug targeting to small airways in patients with mild COPD. METHOD: Design of Experiments (DoE) was used with an in silico whole lung particle deposition model for bolus administration to investigate whether controlling inhalation can improve drug delivery to the small conducting airways. The range of particle aerodynamic diameters studied was 0.4 - 10 µm for flow rates between 100 - 2000 mL/s (i.e., low to very high), and tidal volumes between 40 - 1500 mL. RESULTS: The model accurately predicted the relationship between independent variables and lung deposition, as confirmed by comparison with published experimental data. It was found that large particles (~ 5 µm) require very low flow rate (~ 100 mL/s) and very small tidal volume (~ 110 mL) to target small conducting airways, whereas fine particles (~ 2 µm) achieve drug targeting in the region at a relatively higher flow rate (~ 500 mL/s) and similar tidal volume (~ 110 mL). CONCLUSION: The simulation results indicated that controlling tidal volume and flow rate can achieve targeted delivery to the small airways (i.e., > 50% of emitted dose was predicted to deposit in the small airways), and the optimal parameters depend on the particle size. It is hoped that this finding could provide a means of improving drug targeting to the small conducting airways and improve prognosis in COPD management.

pH-sensitive release of nitric oxide gas using peptide-graphene co-assembled hybrid nanosheets.

Nitric oxide (NO) donating drugs such as organic nitrates have been used to treat cardiovascular diseases for more than a century. These donors primarily produce NO systemically. It is however sometimes desirable to control the amount, location, and time of NO delivery. We present the design of a novel pH-sensitive NO release system that is achieved by the synthesis of dipeptide diphenylalanine (FF) and graphene oxide (GO) co-assembled hybrid nanosheets (termed as FF@GO) through weak molecular interactions. These hybrid nanosheets were characterised by using X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, zeta potential measurements, X-ray photoelectron spectroscopy, scanning and transmission electron microscopies. The weak molecular interactions, which include electrostatic, hydrogen bonding and π-π stacking, are pH sensitive due to the presence of carboxylic acid and amine functionalities on GO and the dipeptide building blocks. Herein, we demonstrate that this formulation can be loaded with NO gas with the dipeptide acting as an arresting agent to inhibit NO burst release at neutral pH; however, at acidic pH it is capable of releasing NO at the rate of up to 0.6 µM per minute, comparable to the amount of NO produced by healthy endothelium. In conclusion, the innovative conjugation of dipeptide with graphene can store and release NO gas under physiologically relevant concentrations in a pH-responsive manner. pH responsive NO-releasing organic-inorganic nanohybrids may prove useful for the treatment of cardiovascular diseases and other pathologies.

Understanding the effects of microbubble concentration on localization accuracy in super-resolution ultrasound imaging.

Super-resolution ultrasound (SRUS) through localising and tracking of microbubbles (MBs) can achieve sub-wavelength resolution for imaging microvascular structure and flow dynamics in deep tissuein vivo. The technique assumes that signals from individual MBs can be isolated and localised accurately, but this assumption starts to break down when the MB concentration increases and the signals from neighbouring MBs start to interfere. The aim of this study is to gain understanding of the effect of MB-MB distance on ultrasound images and their localisation. Ultrasound images of two MBs approaching each other were synthesised by simulating both ultrasound field propagation and nonlinear MB dynamics. Besides the distance between MBs, a range of other influencing factors including MB size, ultrasound frequency, transmit pulse sequence, pulse amplitude and localisation methods were studied. The results show that as two MBs approach each other, the interference fringes can lead to significant and oscillating localisation errors, which are affected by both the MB and imaging parameters. When modelling a clinical linear array probe operating at 6 MHz, localisation errors between 20 and 30µm (∼1/10 wavelength) can be generated when MBs are ∼500µm (2 wavelengths or ∼1.7 times the point spread function (PSF)) away from each other. When modelling a cardiac probe operating at 1.5 MHz, the localisation errors were as high as 200µm (∼1/5 wavelength) even when the MBs were more than 10 wavelengths apart (2.9 times the PSF). For both frequencies, at smaller separation distances, the two MBs were misinterpreted as one MB located in between the two true positions. Cross-correlation or Gaussian fitting methods were found to generate slightly smaller localisation errors than centroiding. In conclusion, caution should be taken when generating and interpreting SRUS images obtained using high agent concentration with MBs separated by less than 1.7 to 3 times the PSF, as significant localisation errors can be generated due to interference between neighbouring MBs.

Use of oxygen-loaded nanobubbles to improve tissue oxygenation: Bone-relevant mechanisms of action and effects on osteoclast differentiation.

Gas-loaded nanobubbles have potential as a method of oxygen delivery to increase tumour oxygenation and therapeutically alleviate tumour hypoxia. However, the mechanism(s) whereby oxygen-loaded nanobubbles increase tumour oxygenation are unknown; with their calculated oxygen-carrying capacity being insufficient to explain this effect. Intra-tumoural hypoxia is a prime therapeutic target, at least partly due to hypoxia-dependent stimulation of the formation and function of bone-resorbing osteoclasts which establish metastatic cells in bone. This study aims to investigate potential mechanism(s) of oxygen delivery and in particular the possible use of oxygen-loaded nanobubbles in preventing bone metastasis via effects on osteoclasts. Lecithin-based nanobubbles preferentially interacted with phagocytic cells (monocytes, osteoclasts) via a combination of lipid transfer, clathrin-dependent endocytosis and phagocytosis. This interaction caused general suppression of osteoclast differentiation via inhibition of cell fusion. Additionally, repeat exposure to oxygen-loaded nanobubbles inhibited osteoclast formation to a greater extent than nitrogen-loaded nanobubbles. This gas-dependent effect was driven by differential effects on the fusion of mononuclear precursor cells to form pre-osteoclasts, partly due to elevated potentiation of RANKL-induced ROS by nitrogen-loaded nanobubbles. Our findings suggest that oxygen-loaded nanobubbles could represent a promising therapeutic strategy for cancer therapy; reducing osteoclast formation and therefore bone metastasis via preferential interaction with monocytes/macrophages within the tumour and bone microenvironment, in addition to known effects of directly improving tumour oxygenation.

3D Acoustic Wave Sparsely Activated Localization Microscopy With Phase Change Contrast Agents.

OBJECTIVE: The aim of this study is to demonstrate 3-dimensional (3D) acoustic wave sparsely activated localization microscopy (AWSALM) of microvascular flow in vivo using phase change contrast agents (PCCAs). MATERIALS AND METHODS: Three-dimensional AWSALM using acoustically activable PCCAs was evaluated on a crossed tube microflow phantom, the kidney of New Zealand White rabbits, and the brain of C57BL/6J mice through intact skull. A mixture of C 3 F 8 and C 4 F 10 low-boiling-point fluorocarbon gas was used to generate PCCAs with an appropriate activation pressure. A multiplexed 8-MHz matrix array connected to a 256-channel ultrasound research platform was used for transmitting activation and imaging ultrasound pulses and recording echoes. The in vitro and in vivo echo data were subsequently beamformed and processed using a set of customized algorithms for generating 3D super-resolution ultrasound images through localizing and tracking activated contrast agents. RESULTS: With 3D AWSALM, the acoustic activation of PCCAs can be controlled both spatially and temporally, enabling contrast on demand and capable of revealing 3D microvascular connectivity. The spatial resolution of the 3D AWSALM images measured using Fourier shell correlation is 64 µm, presenting a 9-time improvement compared with the point spread function and 1.5 times compared with half the wavelength. Compared with the microbubble-based approach, more signals were localized in the microvasculature at similar concentrations while retaining sparsity and longer tracks in larger vessels. Transcranial imaging was demonstrated as a proof of principle of PCCA activation in the mouse brain with 3D AWSALM. CONCLUSIONS: Three-dimensional AWSALM generates volumetric ultrasound super-resolution microvascular images in vivo with spatiotemporal selectivity and enhanced microvascular penetration.

A Double-Blind, Randomized, Placebo-Controlled Pilot Study examining an Oxygen Nanobubble Beverage for 16.1-km Time Trial and Repeated Sprint Cycling Performance.

There is growing interest of ergogenic aids that deliver supplemental oxygen during exercise and recovery, however, breathing supplemental oxygen via specialist facemasks is often not feasible. Therefore, this study investigated the effect of an oxygen-nanobubble beverage during submaximal and repeated sprint cycling. In a double-blind, randomized, placebo-controlled study, 10 male cyclists (peak aerobic capacity, 56.9 ± 6.1 mL·kg-1·min-1; maximal aerobic power, 385 ± 25 W) completed submaximal or maximal exercise after consuming an oxygen-nanobubble (O2) or placebo (PLA) beverage. Submaximal trials comprised 30-min of steady-state cycling at 60% peak aerobic capacity and 16.1-km time-trial (TT). Maximal trials involved 4 × 30 s Wingate tests interspersed by 4-min recovery. Time-to-completion during the 16.1-km TT was 2.4% faster after O2 compared with PLA (95% CI = 0.7-4.0%, p = 0.010, d = 0.41). Average power for the 16.1-km TT was 4.1% higher for O2 vs. PLA (95% CI = 2.1-7.3%, p = 0.006, d = 0.28). Average peak power during the repeated Wingate tests increased by 7.1% for O2 compared with PLA (p = 0.002, d = 0.58). An oxygen-nanobubble beverage improves performance during submaximal and repeated sprint cycling, therefore may provide a practical and effective ergogenic aid for competitive cyclists.

A Review of Ultrasound-Mediated Checkpoint Inhibitor Immunotherapy.

Over the past decade, immunotherapy has emerged as a major modality in cancer medicine. However, despite its unprecedented success, immunotherapy currently benefits only a subgroup of patients, may induce responses of limited duration and is associated with potentially treatment-limiting side effects. In addition, responses to immunotherapeutics are sometimes diminished by the emergence of a complex array of resistance mechanisms. The efficacy of immunotherapy depends on dynamic interactions between tumour cells and the immune landscape in the tumour microenvironment. Ultrasound, especially in conjunction with cavitation-promoting agents such as microbubbles, can assist in the uptake and/or local release of immunotherapeutic agents at specific target sites, thereby increasing treatment efficacy and reducing systemic toxicity. There is also increasing evidence that ultrasound and/or cavitation may themselves directly stimulate a beneficial immune response. In this review, we summarize the latest developments in the use of ultrasound and cavitation agents to promote checkpoint inhibitor immunotherapy.

Protein-Decorated Microbubbles for Ultrasound-Mediated Cell Surface Manipulation.

Delivering cargo to the cell membranes of specific cell types in the body is a major challenge for a range of treatments, including immunotherapy. This study investigates employing protein-decorated microbubbles (MBs) and ultrasound (US) to "tag" cellular membranes of interest with a specific protein. Phospholipid-coated MBs were produced and functionalized with a model protein using a metallochelating complex through an NTA(Ni) and histidine residue interaction. Successful "tagging" of the cellular membrane was observed using microscopy in adherent cells and was promoted by US exposure. Further modification of the MB surface to enable selective binding to target cells was then achieved by functionalizing the MBs with a targeting protein (transferrin) that specifically binds to a receptor on the target cell membrane. Attachment and subsequent transfer of material from MBs functionalized with transferrin to the target cells significantly increased, even in the absence of US. This work demonstrates the potential of these MBs as a platform for the noninvasive delivery of proteins to the surface of specific cell types.

Sonosensitive Cavitation Nuclei-A Customisable Platform Technology for Enhanced Therapeutic Delivery.

Ultrasound-mediated cavitation shows great promise for improving targeted drug delivery across a range of clinical applications. Cavitation nuclei-sound-sensitive constructs that enhance cavitation activity at lower pressures-have become a powerful adjuvant to ultrasound-based treatments, and more recently emerged as a drug delivery vehicle in their own right. The unique combination of physical, biological, and chemical effects that occur around these structures, as well as their varied compositions and morphologies, make cavitation nuclei an attractive platform for creating delivery systems tuned to particular therapeutics. In this review, we describe the structure and function of cavitation nuclei, approaches to their functionalization and customization, various clinical applications, progress toward real-world translation, and future directions for the field.

Drug delivery strategies for antibiofilm therapy.

Although new antibiofilm agents have been developed to prevent and eliminate pathogenic biofilms, their widespread clinical use is hindered by poor biocompatibility and bioavailability, unspecific interactions and insufficient local concentrations. The development of innovative drug delivery strategies can facilitate penetration of antimicrobials through biofilms, promote drug dispersal and synergistic bactericidal effects, and provide novel paradigms for clinical application. In this Review, we discuss the potential benefits of such emerging techniques for improving the clinical efficacy of antibiofilm agents, as well as highlighting the existing limitations and future prospects for these therapies in the clinic.

High-Speed Imaging of Microsphere Transport by Cavitation Activity in a Tissue-Mimicking Phantom.

OBJECTIVE: Ultrasound-mediated cavitation has been harnessed to improve the delivery of various therapeutics, including the extravasation of small molecule drugs and nanoparticles (<1 µm) into soft tissue. This study investigated whether cavitation could also enhance the extravasation of larger (>10 µm) therapeutic particles, representative of radio- or chemo-embolic particles, in a tissue-mimicking phantom. METHODS: High-speed (103-106 frames/s) optical imaging was used to observe the motion of glass microspheres with diameters of 15-32 or 105-107 µm in an agar phantom under exposure to high-intensity focused ultrasound (0.5 MHz) at a range of peak negative pressures (1.9-2.8 MPa) in the presence of SonoVue microbubbles. RESULTS: In contrast to the microstreaming reported to be responsible for nanoparticle transport, the formation and translation of bubble clouds were found to be primarily responsible for the motion of glass microspheres. The bubble clouds were seen both to create channels in the phantom and to travel along them under the action of primary acoustic radiation force, either propelling or entraining microspheres with them. Collisions between microspheres were also seen to promote cloud formation and cavitation activity. CONCLUSION: Ultrasound-mediated cavitation can promote the transport of solid microparticles in tissue-mimicking material. Further work is needed to understand the influence of tissue mechanical properties and ultrasound exposure parameters on the extent and uniformity of particle distribution that can be achieved.