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.

BackgroundSpectral 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.ResultsHere 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.ConclusionsThe 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.

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.

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 (LAR) developed for simultaneous ultrasound exposure, acoustic emissions monitoring, and microscopy of biological samples. The LAR facilitates 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 studies that may benefit the design of predictable and effective cavitation-based ultrasound treatments.

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-2hydroxy-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.