Cavitation microstreaming has been identified as an important mechanism underpinning sonoporation. The reported evidence includes experimental observation of the permeabilization of non-adherent cells or vesicles in microstreaming and theoretical and experimental estimates of the fluid shear stress required to induce pore formation in cell membranes and/or the activation of membrane components such as ion channels. The aim of this study was to assess the relative contribution of microstreaming to sonoporation observed in vitro with adherent cells, as commonly used in drug delivery studies. Streak velocimetry measurements were performed within a microfluidic cell of the microstreaming around a contrast agent microbubble driven at 0.5 and 1MHz and peak negative pressures up to 1MPa. The results suggest that, for the cell densities and microbubble concentrations typically reported in sonoporation studies, only a very small proportion of cells within a typical culture device would be exposed to sufficiently high shear stresses to be permeabilised. This finding is inconsistent with reported results for drug delivery efficiency and suggests that microstreaming may not be the dominant mechanism of sonoporation in this type of experiment.

Microbubbles stabilized by an outer lipid shell have been studied extensively for both diagnostic and therapeutic applications. The shell composition can significantly influence microbubble behavior, but performing quantitative measurements of shell properties is challenging. The aim of this study is to investigate the use of spectral imaging to characterize the surface properties of a range of microbubble formulations representing both commercial and research agents. A lipophilic dye, C-laurdan, whose fluorescence emission varies according to the properties of the local environment, was used to compare the degree and uniformity of the lipid order in the microbubble shell, and these measurements were compared with the acoustic response and stability of the different formulations. Spectral imaging was found to be suitable for performing rapid and hence relatively high throughput measurements of microbubble surface properties. Interestingly, despite significant differences in lipid molecule size and charge, all of the different formulations exhibited highly ordered lipid shells. Measurements of liposomes with the same composition and the debris generated by destroying lipid microbubbles with ultrasound (US) showed that these exhibited a lower and more varied lipid order than intact microbubbles. This suggests that the high lipid order of microbubbles is due primarily to compression of the shell as a result of surface tension and is only minimally affected by composition. This also explains the similarity in acoustic response observed between the formulations, because microbubble dynamics are determined by the diameter and shell viscoelastic properties that are themselves a function of the lipid order. Within each population, there was considerable variability in the lipid order and response between individual microbubbles, suggesting the need for improved manufacturing techniques. In addition, the difference in the lipid order between the shell and lipid debris may be important for therapeutic applications in which shedding of the shell material is exploited, for example, drug delivery.

Sonoporation, the permeabilization of cell membranes following exposure to microbubbles and ultrasound, has considerable potential for therapeutic delivery. To date, engineering of microbubbles for these applications has focused primarily upon optimizing microbubble size and stability, or attachment of targeting species and/or drug molecules. In this work, it is demonstrated that the microbubble coating can also be tailored to directly influence cell permeabilization. Specifically, lipid exchange mechanisms between phospholipid microbubbles and cells can be exploited to significantly increase sonoporation efficiency in vitro. A theoretical analysis of the energy required for pore formation was carried out. From this, it was hypothesized that sonoporation could be promoted by the transfer of lipid molecules with appropriate carbon chain length and/or shape (cylindrical or conical). Spectral imaging with a hydration-sensitive membrane probe (C-Laurdan) was used to measure changes in the membrane lipid order of A-549 cancer cells following exposure to suspensions of different phospholipids. Two candidate lipids were identified, a short-chain-length phospholipid (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) and a medium-chain-length lysolipid (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lyso-PC)). Microbubbles were prepared with matched concentrations, size distributions, and acoustic responses. Confocal microscopy was used to measure cell uptake of a model drug (propidium iodide) with and without ultrasound exposure (1 MHz, 250 kPa peak negative pressure, 1 kHz pulse repetition frequency, 10% duty cycle, 15 s exposure). Despite significantly decreasing the cell membrane lipid order, DLPC did not increase sonoporation. Microbubbles containing 16:0 lyso-PC, however, produced a ∼5-fold increase in sonoporation compared to control microbubbles. Importantly, the lyso-PC molecules were incorporated into the microbubble coating and did not affect cell permeability prior to ultrasound exposure. These findings indicate that microbubbles can be engineered to exploit lipid exchange between microbubble shells and cell membranes to enhance drug delivery, a new optimization route that may lead to enhanced therapeutic efficacy of ultrasound-mediated treatments.

The study of the effects of ultrasound-induced acoustic cavitation on biological structures is an active field in biomedical research. Of particular interest for therapeutic applications is the ability of oscillating microbubbles to promote both cellular and tissue membrane permeabilisation and to improve the distribution of therapeutic agents in tissue through extravasation and convective transport. The mechanisms that underpin the interaction between cavitating agents and tissues are, however, still poorly understood. One challenge is the practical difficulty involved in performing optical microscopy and acoustic emissions monitoring simultaneously in a biologically compatible environment. Here we present and characterise a microfluidic layered acoustic resonator (μLAR) developed for simultaneous ultrasound exposure, acoustic emissions monitoring, and microscopy of biological samples. The μLAR facilitates in vitro ultrasound experiments in which measurements of microbubble dynamics, microstreaming velocity fields, acoustic emissions, and cell-microbubble interactions can be performed simultaneously. The device and analyses presented provide a means of performing mechanistic in vitro studies that may benefit the design of predictable and effective cavitation-based ultrasound treatments.

The permeabilisation of cell membranes following exposure to microbubbles and ultrasound has considerable potential for therapeutic delivery. Recent studies have demonstrated that transfer of material takes place between phospholipid-coated microbubbles and cell membranes. The aim of this study was to investigate the impact of this transfer on membrane properties and cell permeability. Microbubbles were prepared with matched concentrations and size distributions from a series of formulations utilising phospholipids with differing molecular characteristics. Quantitative fluorescence microscopy techniques were used to quantify changes in the molecular packing, viscosity, and permeability to model drugs of cancer cells under mild ultrasound exposure (15 s of 1 MHz centre frequency, 125 kPa peak negative pressure, 1 kHz pulse repetition frequency, and 10% duty cycle). The molecular “packing” of cancer cell membranes was found to be significantly affected by the transfer of both phospholipids and other microbubble coating constituents. This was particularly pronounced in the case of microbubbles containing a specific class of lipids, which promoted a ~4-fold increase in model drug uptake following ultrasound exposure. Similar results were observed in an investigation of permeability in an in vitro blood brain barrier model suggesting that microbubble composition may play a key role in the efficacy of ultrasound mediated delivery.

Blood-brain barrier (BBB) disruption mediated by ultrasound and microbubbles (US-BBBD) is a promising strategy for non-invasive and targeted delivery of therapeutics to the brain. In US-BBBD, treatment control is achieved by externally monitoring acoustic emissions (AE) and adjusting ultrasound parameters in real-time to avoid AE associated with damage. Recent work suggests that AE may also provide insight regarding the extent of BBB opening and BBB recovery time. The mechanisms underlying BBB opening and recovery, however, are largely not understood. To investigate US-BBBD mechanisms with regard to AE, we developed an in vitro platform for monitoring both BBB integrity and AE during US-BBBD. Temporally resolved BBB integrity monitoring was achieved using a microfluidic BBB-on-a-chip device with integrated trans-endothelial electrical resistance (TEER) measurements. Well-characterized ultrasound exposure and AE monitoring were achieved using a focally aligned high-intensity focused ultrasound transducer and passive cavitation detector. In addition to recording TEER and AE data, our platform is compatible with fluorescence microscopy during ultrasound exposure, providing further insight into US-BBBD mechanisms. This work further demonstrates potential for in vitro screening of cavitation agents and/or therapeutics for novel US-BBBD applications and strategies.

The aim of this study was to characterize the behaviour of superparamagnetic particles in magnetic drug targeting (MDT) schemes. A 3-dimensional mathematical model was developed, based on the analytical derivation of the trajectory of a magnetized particle suspended inside a fluid channel carrying laminar flow and in the vicinity of an external source of magnetic force. Semi-analytical expressions to quantify the proportion of captured particles, and their relative accumulation (concentration) as a function of distance along the wall of the channel were also derived. These were expressed in terms of a non-dimensional ratio of the relevant physical and physiological parameters corresponding to a given MDT protocol. The ability of the analytical model to assess magnetic targeting schemes was tested against numerical simulations of particle trajectories. The semi-analytical expressions were found to provide good first-order approximations for the performance of MDT systems in which the magnetic force is relatively constant over a large spatial range. The numerical model was then used to test the suitability of a range of different designs of permanent magnet assemblies for MDT. The results indicated that magnetic arrays that emit a strong magnetic force that varies rapidly over a confined spatial range are the most suitable for concentrating magnetic particles in a localized region. By comparison, commonly used magnet geometries such as button magnets and linear Halbach arrays result in distributions of accumulated particles that are less efficient for delivery. The trajectories predicted by the numerical model were verified experimentally by acoustically focusing magnetic microbeads flowing in a glass capillary channel, and optically tracking their path past a high field gradient Halbach array.

Background: Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internal-ized fluorescent probes. Results: Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internal-ized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is deter-mined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification of the local lateral density of lipids or lipid packing. Conclusions: The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membranes segmentation and no ability in programming required. For re-viewers the software is available at: https://ora.ox.ac.uk/objects/uuid:4375842f-3598-418d-8aa39b31f5023401. It will be further available on the MATLAB file exchange following acceptance for publication. Corresponding Author: Miles Aron University of Oxford UNITED KINGDOM Corresponding Author Secondary Information: Corresponding Author's Institution: University of Oxford Corresponding Author's Secondary Institution: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation First Author: Miles Aron First Author Secondary Information: Order of Authors: Miles Aron Richard Browning Dario Carugo Erdinc Sezgin Jorge Bernardino de la Serna Christian Eggeling Eleanor Stride Order of Authors Secondary Information: Author Comments: For reviewers the software is available at: https://ora.ox.ac.uk/objects/uuid:4375842f3598-418d-8aa3-9b31f5023401. It will be further available on the MATLAB file exchange following acceptance for publication. Response to Reviewers: The authors' response letter has been included as a supplementary file Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation

BACKGROUND:Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internalized fluorescent probes. RESULTS:Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internalized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is determined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di-4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification of the local lateral density of lipids or lipid packing. CONCLUSIONS:The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membrane segmentation and no ability in programming required. The Spectral Imaging Toolbox can be downloaded from https://uk.mathworks.com/matlabcentral/fileexchange/62617-spectral-imaging-toolbox .

Cancer imposes a significant disease burden and constitutes a major societal challenge. Despite being widely used, chemotherapy suffers numerous disadvantages, including off- target drug toxicity, poor tumour penetration, and drug resistance. The use of ultrasound in combination with contrast agents has shown promise in enhancing outcomes in the treatment of cancerous and non-cancerous diseases. However, the underlying biophysical processes that underpin their interactions with tissues remain poorly understood. The aim of the research presented in this thesis is to develop methods to elucidate these processes. Development is hindered by the difficulties involved in isolating cellular parameters, noninvasively quantifying biological features at the molecular scale, and recreating a predictable and repeatable ultrasound field. To overcome these challenges, giant unilamellar vesicles made with 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol were used initially as cell models and exposed to therapeutically relevant conditions in a purpose-built acoustofluidic device. The resulting effects on the hydration and permeability of the vesicle membranes and the dynamics of their intravesicle milieu were characterised in situ using quantitative microscopy. Results show that flow, ultrasound, and microbubbles led to an increase in vesicle membrane hydration, while dehydration was seen only in the presence of microbubbles. An increase in permeability was observed for all exposure conditions, and was accentuated when microbubble shell fragments were incorporated in the vesicle bilayer. These findings thus indicate that ultrasound-mediated delivery is governed by a combination of physical and chemical mechanisms that influence the permeation of molecules through lipid membranes. Further, it was shown that exposure to the same conditions led to the onset of streaming flows within the vesicles’ lumen. An experimental proof-of-concept for extending the study in vitro was also presented. Actuating the motion of cytoplasmic constituents may potentially induce a broad range of underexplored and therapeutically relevant bioeffects.

Background: Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for... Expand documentation

The transfer of material from phospholipid-coated microbubbles to cell membranes has been hypothesized to play a role in ultrasound-mediated drug delivery. In this study, we employed quantitative fluorescence microscopy techniques to investigate this phenomenon in both artificial and biological membrane bilayers in an acoustofluidic system. The results of the present study provide strong evidence for the transfer of material from microbubble coatings into cell membranes. Our results indicate that transfer of phospholipids alters the organization of molecules in cell membranes, specifically the lipid ordering or packing, which is known to be a key determinant of membrane mechanical properties, protein dynamics, and permeability. We further show that polyethylene-glycol, used in many clinical microbubble formulations, also has a major impact on both membrane lipid ordering and the extent of lipid transfer, and that this occurs even in the absence of ultrasound exposure.