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Cavitation-mediated extravasation and transtumoral drug delivery by microstreaming: What role do the gas nuclei and the physical properties of the therapeutic play?
For effective cancer therapy, therapeutic agents must extravasate from the blood stream into the tumour mass, then overcome the elevated intratumoural pressure and dense extracellular matrix to reach each and every cancer cell. Several recent studies have suggested that inertial cavitation in the absence or presence of artificial cavitation nuclei can significantly enhance the delivery, penetration, and distribution of small-molecule or nanoparticulate therapeutic agents into tumors. We first present a comparison of the reported enhancements in delivery achieved for a range of frequencies and therapeutic sizes without or with pre-seeding of cavitation, with particular emphasis on the potential role of cavitation persistence and spatial distribution. With microstreaming hypothesized to be the dominant transport mechanism for drug delivery, the likely benefit of using sub-micron cavitation nucleation agents capable of extravasating alongside the therapeutic, rather than microbubbles confined to the blood pool, is then investigated. Lastly, potential physical and biological modification strategies capable of enhancing the transport of therapeutics by cavitation-microstreaming are discussed, and compared in terms of their relative delivery efficacies in vivo.
Giant vesicles as cell models to quantify bio-effects in ultrasound mediated drug delivery
The biophysical mechanisms that underpin the interaction between microbubbles and cells in the context of ultrasound-mediated drug delivery are still poorly understood. To aid the identification of these mechanisms, giant unilamellar vesicles (GUVs) were used as cell models to quantify changes in membrane properties as a result of the interaction with ultrasound (1 MHz, 150 kPa, and 60 s continuous wave) and phospholipid-shelled microbubbles (DSPC-PEG40S 9:1 molar ratio), either alone or in combination. The spatial quantification of the vesicle lipid order was performed via spectral microscope imaging, by measuring the contours of generalized polarisation (GP) from the emission spectrum of c-Laurdan, a polarity-sensitive dye. Preliminary data show synergistic mechanical and chemical effects on membranes: ultrasound exposure and shear flow alone generally decrease the vesicle lipid packing, while exposures involving microbubbles reveal contrasting effects depending on the initial vesicle composition and acoustic regime. Results from the present mechanistic study provide an insight into the mechanisms of microbubble-membrane interactions, potentially benefitting the design of effective and predictable microbubble-based ultrasound treatments.
High-throughput production of microbubble contrast agents using a sonofluidic device
The response of microbubbles to a given sound field is determined by their size and coating. These, in turn, depend on their chemical formulation and the production technique. Sonication is the most commonly employed method and can generate high concentrations of microbubbles rapidly but with a broad size distribution and poor reproducibility. Microfluidic devices provide excellent control over size, but the small-scale architectures required are often challenging to manufacture, offer low production rates, and are prone to clogging. Microbubbles may also have inferior surface characteristics and stability compared to those produced by sonication. In this study we investigate a hybrid technique in which monodisperse microbubbles of ~200μm diameter are produced at high flow rates in a simple T-junction and then undergo controlled fragmentation by exposure to ultrasound via an integrated transducer operating between 71-73kHz. Microbubbles were prepared using the device or a standard sonication protocol and compared in terms of their size, size distribution, concentration, stability, acoustic response, and surface molecular concentration using quantitative fluorescence microscopy. The characteristics of the microbubbles produced by the device were found to be equivalent in terms of production rate, stability and acoustic response but with a narrower size distribution and tunable mean size.
Ultrasound-enhanced thrombolysis: Mechanistic observations
Ultrasound and microbubbles have been widely demonstrated to accelerate the breakdown of blood clots, but the mechanisms of treatment require further investigation. In particular, there is a need to clarify the effect on the fibrin matrix—the insoluble polymer mesh that determines a clot’s integrity and mechanical properties. The objective of this in vitro study was to observe in real-time the mechanisms of microbubble-enhanced sonothrombolysis at the microscale. Fluorescently labeled porcine plasma clots were prepared on a glass coverslip and exposed to different types of microbubbles with or without the fibrinolytic agent recombinant tissue plasminogen activator. A 1 mm thick piezoelectric element was coupled with the glass substrate and driven at the resonant frequency of the system (1.9 MHz), with a duty cycle of 5% and a 0.1 Hz pulse repetition frequency. The acoustic field within the clot was characterized using a fiber optic hydrophone. Changes in the fiber network were monitored for 30 min by confocal microscopy.
Acoustofluidic manipulation of biological bodies: Applications in medical and environmental diagnosis
Ultrasound-based external forcing of biological bodies in microfluidics has emerged as a contactless way of manipulating cells and particles for a range of applications, including sample enrichment, filtration, and sorting. Recently, acoustic radiation forces have shown potential for manipulating pathogenic organisms in biological assays. In this presentation, we demonstrate the development of acoustofluidic systems designed for high-throughput manipulation and capturing of biological bodies in applications ranging from medical to environmental diagnosis. Specifically, we apply our acoustofluidic systems to the detection of (i) cancer and immune cells in the early-stage diagnosis of blood malignancies and allergies, and (ii) bacterial microorganisms, spores, and planktonic cells for screening of environmental and industrial samples.
Acoustofluidic manipulation of biological bodies: Generation, visualization, and stimulation of cellular constructs
Ultrasound-based external manipulation of biological bodies in microfluidics has emerged as a contactless way of manipulating cells and particles for a range of applications, including sample enrichment, filtration, and sorting. Furthermore, it has been recently utilized to drive cells to form multi-cellular architectures, including clusters and planar sheets, by appropriately designing the resonant ultrasound field within the acoustofluidic device. In this presentation, we demonstrate the development of ultrasonic bioreactors for generating 3D, scaffold-free tissue constructs. We apply this technology to the generation of neocartilage grafts and examine their potential for repair chondral defects, and to the generation of co-culture models of the mucosal airway. Furthermore, we illustrate how the ultrasonic standing wave field can be designed to generate and modulate different stress regimes on suspended cells, for activating mechanotransductive pathways or for enhancing intracellular delivery of bioactive compounds. Integration of acoustofluidic systems with advanced microscopy techniques for quantifying biophysical effects of ultrasound on single cells or cellular constructs is also discussed.
Quantifying Ultrasonic Deformation of Cell Membranes with Ultra-High-Speed Imaging
We present a new method for controllable loading of cell models in an ultrasonic (20 kHz) regime. The protocol is based on the inertial-based ultrasonic shaking test and allows to deform cells in the range of few mm/m to help understand potential consequences of repeated loading characteristic of ultrasonic cutting.
Flow dynamics in stented ureter
Urinary flow is governed by the principles of fluid mechanics. Urodynamic studies have revealed the fundamental kinematics and dynamics of urinary flow in various physiological and pathological conditions, which are cornerstones for future development of diagnostic knowledge and innovative devices. There are three primary approaches to study the fluid mechanical characteristics of urinary flow: reduced order, computational, and experimental methods. Reduced-order methods exploit the disparate length scales inherent in the system to reveal the key dominant physics. Computational models can simulate fully three-dimensional, time-dependent flows in physiologically-inspired anatomical domains. Finally, experimental models provide an excellent counterpart to reduced and computational models by providing physical tests under various physiological and pathological conditions. While the interdisciplinary approaches to date have provided a wealth of insight into the fluid mechanical properties of the stented ureter, the next challenge is to develop new theoretical, computational and experimental models to capture the complex interplay between the fluid dynamics in stented ureters and biofilm/encrustation growth. Such studies will (1) enable identification of clinically relevant scenarios to improve patients’ treatment, and (2) provide physical guidelines for next-generation stent design.
Preventing biofilm formation and encrustation on urinary implants: (bio)molecular and physical research approaches
Stents and catheters are used to facilitate urine drainage within the urinary system. When such sterile implants are inserted into the urinary tract, ions, macromolecules and bacteria from urine, blood or underlying tissues accumulate on their surface. We presented a brief but comprehensive overview of future research strategies in the prevention of urinary device encrustation with an emphasis on biodegradability, molecular, microbiological and physical research approaches. The large and strongly associated field of stent coatings and tissue engineering is outlined elsewhere in this book. There is still plenty of room for future investigations in the fields of material science, surface science, and biomedical engineering to improve and create the most effective urinary implants. In an era where material science, robotics and artificial intelligence have undergone great progress, futuristic ideas may become a reality. These ideas include the creation of multifunctional programmable intelligent urinary implants (core and surface) capable to adapt to the complex biological and physiological environment through sensing or by algorithms from artificial intelligence included in the implant. Urinary implants are at the crossroads of several scientific disciplines, and progress will only be achieved if scientists and physicians collaborate using basic and applied scientific approaches.
Development of a nanodroplet formulation for triggered release of BIO for bone fracture healing
Impaired fracture healing impacts patients’ quality of life and imposes a financial burden on healthcare services. Up to 10% of bone fractures result in delayed/non-union fractures, for which new treatments are urgently required. However, systemic delivery of bone anabolic molecules is often sub-optimal and can lead to significant side effects. In this study, we developed ultrasound (US) responsive nano-sized vehicles in the form of perfluorocarbon nanodroplets (NDs), as a means of targeting delivery of drugs to localised tissues. We tested the hypothesis that NDs could stably encapsulate BIO (GSK-3β inhibitor), which could then be released upon US stimulation to activate Wnt signalling and induce ossification. NDs (~280 nm) were prepared from phospholipids and liquid perfluorocarbon and their stability and drug loading was studied by NTA (Nano Tracking Analysis) and HPLC. ND cytotoxicity was assessed in patient-derived bone marrow stromal cells (BMSCs) with Alamar Blue (24 h), and in vitro bioactivity of BIO-NDs was evaluated in a 3T3 Wnt-pathway reporter cell line with luciferase readout. To investigate the acoustic behaviour of NDs, 2% agarose (LM) containing NDs was injected into a bespoke bone fracture model (Sawbones) of various geometries and stimulated by US (1 MHz, 5% duty cycle, 1 MPa, 30 s), allowing the simultaneous capture of optical images and acoustic emissions. Femoral bone hole defects (1–2 mm) were made in WT-MF1 mice (age: 8–12 wks) and DiR-labelled NDs (100 µL, 109 NDs/mL, i.v.) were injected post-fracture to determine biodistribution by IVIS imaging. NDs were stable (4 and 37 °C) and retained >90% BIO until US was applied, which caused ~100% release. ND exposure up to a concentration of 109 NDs/mL showed no cytotoxicity (24 h). BIO-loaded NDs induced Wnt pathway activation in a dose dependent manner. Biodistribution of DiR-NDs in a femoral bone hole defect model in mice demonstrated increased localisation at the fracture site (~2-fold relative to that found in healthy mice or contralateral femurs at 48 h).
Scaleable production of microbubbles using an ultrasound-modulated microfluidic device
Surfactant-coated gas microbubbles are widely used as contrast agents in ultrasound imaging and increasingly for therapeutic applications. It has been shown that the acoustic response of microbubbles is determined by their size and coating properties and hence depends upon both their chemical composition and the manufacturing technique used to produce them. We have previously presented a hybrid device consisting of a simple microfluidic T-junction with an integrated ultrasound transducer that provides superior production rates and microbubble stability compared with conventional microfluidic systems but with significantly better microbubble uniformity than standard emulsification techniques. The maximum production rate was, however, still limited compared to industrial methods. In the present study a new device was developed that enables production of >108 microbubbles per second using a single device with a mean bubble diameter of 1.4 μm without degrading microbubble uniformity. Production rates of >109 microbubbles per second can be achieved through parallel operation of multiple channels within a single device; comparable with bulk emulsification but without the risk of contamination and/or degradation of sensitive components.
A physical model to investigate the acoustic behaviour of microbubbles and nanodroplets within a bone fracture
Impaired fracture healing is a major financial burden for healthcare services; 5%–10% of bone fractures result in non-unions, and there is no clinically approved systemic therapy. This study characterises acoustically stimulated microbubbles (MBs) and nanodroplets (NDs) as non-invasive ultrasound responsive vehicles for the targeted delivery of osteogenic compounds. A microscope-compatible water-tank incorporating a passive cavitation detector was developed to study the acoustic behaviour of MBs and NDs within physical models of bone fractures (gap: 3.5–5.5 mm, angles: 0 deg and 90 deg). The device was designed using COMSOL Multiphysics (Burlington, MA) and tested in-vitro. The bone was simulated using a material with comparable acoustic impedance (Sawbones, WA). Numerical simulations showed that the developed experimental set-up generated a relatively uniform acoustic field at a target plane. It could be operated at either 1 or 2 MHz US frequency, at an acoustic pressure in the range 0–1 MPa. The inclusion of a fracture model caused perturbations to the acoustic field, which were dependent on the architecture of the fracture (i.e., relative to the incident US field). Ongoing studies are investigating how these perturbations affect ND/MB response in-vitro. Further studies will investigate the relationship between MB/ND acoustic response and the release of biologically active compounds.