Microbubble-cell Interactions Research Articles

Internalization of targeted microbubbles by endothelial cells and drug delivery by pores and tunnels.

Ultrasound insonification of microbubbles can locally enhance drug delivery by increasing the cell membrane permeability. To aid development of a safe and effective therapeutic microbubble, more insight into the microbubble-cell interaction is needed. In this in vitro study we aimed to investigate the initial 3D morphology of the endothelial cell membrane adjacent to individual microbubbles (n=301), determine whether this morphology was affected upon binding and by the type of ligand on the microbubble, and study its influence on microbubble oscillation and the drug delivery outcome. High-resolution 3D confocal microscopy revealed that targeted microbubbles were internalized by endothelial cells, while this was not the case for non-targeted or IgG1-κ control microbubbles. The extent of internalization was ligand-dependent, since αvβ3-targeted microbubbles were significantly more internalized than CD31-targeted microbubbles. Ultra-high-speed imaging (~17 Mfps) in combination with high-resolution confocal microscopy (n=246) showed that microbubble internalization resulted in a damped microbubble oscillation upon ultrasound insonification (2MHz, 200kPa peak negative pressure, 10cycles). Despite damped oscillation, the cell's susceptibility to sonoporation (as indicated by PI uptake) was increased for internalized microbubbles. Monitoring cell membrane integrity (n=230) showed the formation of either a pore, for intracellular delivery, or a tunnel (i.e. transcellular perforation), for transcellular delivery. Internalized microbubbles caused fewer transcellular perforations and smaller pore areas than non-internalized microbubbles. In conclusion, studying microbubble-mediated drug delivery using a state-of-the-art imaging system revealed receptor-mediated microbubble internalization and its effect on microbubble oscillation and resulting membrane perforation by pores and tunnels.

Sonoprinting liposomes on tumor spheroids by microbubbles and ultrasound

Ultrasound-triggered drug-loaded microbubbles have great potential for drug delivery due to their ability to locally release drugs and simultaneously enhance their delivery into the target tissue. We have recently shown that upon applying ultrasound, nanoparticle-loaded microbubbles can deposit nanoparticles onto cells grown in 2D monolayers, through a process that we termed “sonoprinting”. However, the rigid surfaces on which cell monolayers are typically growing might be a source of acoustic reflections and aspherical microbubble oscillations, which can influence microbubble-cell interactions. In the present study, we aim to reveal whether sonoprinting can also occur in more complex and physiologically relevant tissues, by using free-floating 3D tumor spheroids as a tissue model. We show that both monospheroids (consisting of tumor cells alone) and cospheroids (consisting of tumor cells and fibroblasts, which produce an extracellular matrix) can be sonoprinted. Using doxorubicin-liposome-loaded microbubbles, we show that sonoprinting allows to deposit large amounts of doxorubicin-containing liposomes to the outer cell layers of the spheroids, followed by doxorubicin release into the deeper layers of the spheroids, resulting in a significant reduction in cell viability. Sonoprinting may become an attractive approach to deposit drug patches at the surface of tissues, thereby promoting the delivery of drugs into target tissues.

The Role of Ultrasound-Driven Microbubble Dynamics in Drug Delivery: From Microbubble Fundamentals to Clinical Translation

In the last couple of decades, ultrasound-driven microbubbles have proven excellent candidates for local drug delivery applications. Besides being useful drug carriers, microbubbles have demonstrated the ability to enhance cell and tissue permeability and, as a consequence, drug uptake herein. Notwithstanding the large amount of evidence for their therapeutic efficacy, open issues remain. Because of the vast number of ultrasound- and microbubble-related parameters that can be altered and the variability in different models, the translation from basic research to (pre)clinical studies has been hindered. This review aims at connecting the knowledge gained from fundamental microbubble studies to the therapeutic efficacy seen in in vitro and in vivo studies, with an emphasis on a better understanding of the response of a microbubble upon exposure to ultrasound and its interaction with cells and tissues. More specifically, we address the acoustic settings and microbubble-related parameters (i.e., bubble size and physicochemistry of the bubble shell) that play a key role in microbubble-cell interactions and in the associated therapeutic outcome. Additionally, new techniques that may provide additional control over the treatment, such as monodisperse microbubble formulations, tunable ultrasound scanners, and cavitation detection techniques, are discussed. An in-depth understanding of the aspects presented in this work could eventually lead the way to more efficient and tailored microbubble-assisted ultrasound therapy in the future.

Targeted microbubble opening of cell-cell junctions for vascular drug delivery elucidated with combined confocal microscopy and Brandaris 128 imaging

Ultrasound insonification of microbubbles enhances vascular drug delivery pathways, such as opening of cell-cell junctions and pore formation (sonoporation). However, the underlying mechanism remains unknown. The aim of our study was to elucidate the microbubble-cell interaction using the Brandaris 128 ultra-high speed camera (~17 Mfps), to visualize microbubble oscillation, coupled to a custom-build Nikon confocal microscope, to visualize cellular response. Confluent endothelial cells were evaluated for opening of cell-cell junctions with Cell Mask and for sonoporation with Propidium Iodide (PI). The cellular response of single targeted microbubbles (n = 168) was monitored up to 4 min after ultrasound insonification (2 MHz, 100–400kPa, 10-cycles). Cell-cell junctions opening occurred more often when cells were only partially attached to their neighbors (45%) than when fully attached (15%). Almost all fully attached cells showing cell-cell opening also showed PI uptake (92%). The mean microbubble excursion was larger when a cell was sonoporated (1.0μm) versus non-sonoporated (0.47μm). Additionally, larger microbubble excursion amplitudes correlated with larger pore size coefficients, obtained by fitting the PI uptake profiles to the Fan model [Fan, et al., PNAS, 2012]. In conclusion, using the state-of-the-art imaging system we can now elucidate the relationship between microbubble oscillation behavior and the drug delivery pathways.

Acoustic Characterization of a Vessel-on-a-Chip Microfluidic System for Ultrasound-Mediated Drug Delivery.

Ultrasound in the presence of gas-filled microbubbles can be used to enhance local uptake of drugs and genes. To study the drug delivery potential and its underlying physical and biological mechanisms, an in vitro vessel model should ideally include 3-D cell culture, perfusion flow, and membrane-free soft boundaries. Here, we propose an organ-on-a-chip microfluidic platform to study ultrasound-mediated drug delivery: the OrganoPlate. The acoustic propagation into the OrganoPlate was determined to assess the feasibility of controlled microbubble actuation, which is required to study the microbubble-cell interaction for drug delivery. The pressure field in the OrganoPlate was characterized non-invasively by studying experimentally the well-known response of microbubbles and by simulating the acoustic wave propagation in the system. Microbubble dynamics in the OrganoPlate were recorded with the Brandaris 128 ultrahigh-speed camera (17 million frames/s) and a control experiment was performed in an OptiCell, an in vitro monolayer cell culture chamber that is conventionally used to study ultrasound-mediated drug delivery. When insonified at frequencies between 1 and 2 MHz, microbubbles in the OrganoPlate experienced larger oscillation amplitudes resulting from higher local pressures. Microbubbles responded similarly in both systems when insonified at frequencies between 2 and 4 MHz. Numerical simulations performed with a 3-D finite-element model of ultrasound propagation into the OrganoPlate and the OptiCell showed the same frequency-dependent behavior. The predictable and homogeneous pressure field in the OrganoPlate demonstrates its potential to develop an in vitro 3-D cell culture model, well suited to study ultrasound-mediated drug delivery.

Interaction between cavitation microbubble and cell: A simulation of sonoporation using boundary element method (BEM)

Sonoporation has been widely accepted as a significant tool for gene delivery as well as some bio-effects like hemolysis, bringing in high demands of looking into its underlying mechanism. A two-dimensional (2D) boundary element method (BEM) model was developed to investigate microbubble-cell interaction, especially the morphological and mechanical characteristics around the close-to-bubble point (CP) on cell membrane. Based on time evolution analysis of sonoporation, detailed information was extracted from the model for analysis, including volume expansion ratio of the bubble, areal expansion ratio of the cell, jet velocity and CP displacement. Parametric studies were carried out, revealing the influence of different ultrasound parameters (i.e., driving frequency and acoustic pressure) and geometrical configurations (i.e., bubble-cell distance and initial bubble radius). This model could become a powerful tool not only for understanding bubble-cell interactions, but also for optimizing the strategy of sonoporation, such that it could be safer and of higher efficiency for biological and medical studies especially in clinics.

Cell-cycle-specific Cellular Responses to Sonoporation.

Microbubble-mediated sonoporation has shown its great potential in facilitating intracellular uptake of gene/drugs and other therapeutic agents that are otherwise difficult to enter cells. However, the biophysical mechanisms underlying microbubble-cell interactions remain unclear. Particularly, it is still a major challenge to get a comprehensive understanding of the impact of cell cycle phase on the cellular responses simultaneously occurring in cell membrane and cytoskeleton induced by microbubble sonoporation.Methods: Here, efficient synchronizations were performed to arrest human cervical epithelial carcinoma (HeLa) cells in individual cycle phases. The, topography and stiffness of synchronized cells were examined using atomic force microscopy. The variations in cell membrane permeabilization and cytoskeleton arrangement induced by sonoporation were analyzed simultaneously by a real-time fluorescence imaging system.Results: The results showed that G1-phase cells typically had the largest height and elastic modulus, while S-phase cells were generally the flattest and softest ones. Consequently, the S-Phase was found to be the preferred cycle for instantaneous sonoporation treatment, due to the greatest enhancement of membrane permeability and the fastest cytoskeleton disassembly at the early stage after sonoporation.Conclusion: The current findings may benefit ongoing efforts aiming to pursue rational utilization of microbubble-mediated sonoporation in cell cycle-targeted gene/drug delivery for cancer therapy.

In vitro methods to study bubble-cell interactions: Fundamentals and therapeutic applications

Besides their use as contrast agents for ultrasound imaging, microbubbles are increasingly studied for a wide range of therapeutic applications. In particular, their ability to enhance the uptake of drugs through the permeabilization of tissues and cell membranes shows great promise. In order to fully understand the numerous paths by which bubbles can interact with cells and the even larger number of possible biological responses from the cells, thorough and extensive work is necessary. In this review, we consider the range of experimental techniques implemented in in vitro studies with the aim of elucidating these microbubble-cell interactions. First of all, the variety of cell types and cell models available are discussed, emphasizing the need for more and more complex models replicating in vivo conditions together with experimental challenges associated with this increased complexity. Second, the different types of stabilized microbubbles and more recently developed droplets and particles are presented, followed by their acoustic or optical excitation methods. Finally, the techniques exploited to study the microbubble-cell interactions are reviewed. These techniques operate over a wide range of timescales, or even off-line, revealing particular aspects or subsequent effects of these interactions. Therefore, knowledge obtained from several techniques must be combined to elucidate the underlying processes.

353. New Insights of Microbubbles-Cell Interactions During Sonoporation Process

Upon ultrasound (US) exposure, gas microbubbles (MBs) can be expanded, moved and even destroyed. These properties offer the opportunity of site-specific local drug/gene delivery. Activation of MB under specific US beams induces a transient cell membrane permeabilization with a process known as sonoporation. Transient pores formed at the plasma membrane are supposed to be responsible for the intracellular delivery of molecules but also the outward transport of intracellular molecules. Endocytosis process has been shown to be involved during sonoporation. In the field of gene transfer, several studies including ours have reported an improvement of gene delivery by US assisted MB. A key to success of this technique lies in understanding mechanisms governing microbubble-cell interactions. Improving our knowledge will allow us to fully exploit this method for gene therapy purpose.Here, we investigate how MB and US behave towards cells under optimal conditions that allow an efficient gene transfer. Studies were performed on a specific set-up composed of sonoporation chamber mounted on a fluorescence confocal microscopy coupled to a high speed camera. Results obtained from our real time sonoporation indicate that, three events types could be recorded according to the acoustic parameters applied: 1) oscillating microbubbles that are stuck on the plasma membrane during the ultrasound stimulation corresponding to the cellular massage; 2) microbubbles entering into cells (translation); 3) microbubbles having a violent interaction with cells. All experiments were carried out at 1MHz of frequency. The first event occurs at 100 kPa a condition leading to low gene transfer. The second event is observed at 150 kPa after 600ms ultrasound stimulation, an optimal efficiency of gene transfer was obtained at this sound pressure range. The third event occurs for a sound pressure of 200kPa during which two phenomena were identified: either MBs were driven from the field of view due to excessive radiation force or a violent interaction of MB with the cell was observed. At 200kPa the gene transfer efficiency is ten times lower than at 150 kPa due likely to toxicity. It is tempting to correlate the entry of microbubbles phenomenon and efficiency of gene transfer meaning that the entry of the microbubble could promote the delivery of the plasmid into the cell. MBs were found to attach preferentially on lipid rafts of the plasma membrane. Most interestingly, sonoporation was able to modify chromatin compaction and a link between the plasma membrane and the effect on the nucleus has been investigated.

Efficient Gene Delivery by Sonoporation Is Associated with Microbubble Entry into Cells and the Clathrin-Dependent Endocytosis Pathway

Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble–cell interactions do not occur randomly during sonoporation; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.

Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis

Although promising results are achieved in ultrasound mediated drug delivery, its underlying biophysical mechanisms remain to be elucidated. Pore formation as well as endocytosis has been reported during ultrasound application. Due to the plethora of ultrasound settings used in literature, it is extremely difficult to draw conclusions on which mechanism is actually involved. To our knowledge, we are the first to show that acoustic pressure influences which route of drug uptake is addressed, by inducing different microbubble–cell interactions. To investigate this, FITC-dextrans were used as model drugs and their uptake was analyzed by flow cytometry. In fluorescence intensity plots, two subpopulations arose in cells with FITC-dextran uptake after ultrasound application, corresponding to cells having either low or high uptake. Following separation of the subpopulations by FACS sorting, confocal images indicated that the low uptake population showed endocytic uptake. The high uptake population represented uptake via pores. Moreover, the distribution of the subpopulations shifted to the high uptake population with increasing acoustic pressure. Real-time confocal recordings during ultrasound revealed that membrane deformation by microbubbles may be the trigger for endocytosis via mechanostimulation of the cytoskeleton. Pore formation was shown to be caused by microbubbles propelled towards the cell. These results provide a better insight in the role of acoustic pressure in microbubble–cell interactions and the possible consequences for drug uptake. In addition, it pinpoints the need for a more rational, microbubble behavior based choice of acoustic parameters in ultrasound mediated drug delivery experiments.

A Novel Microfluidic Chip for Assessing Dynamic Adhesion Behavior of Cell-Targeting Microbubbles

The primary aim of this study was to develop a microfluidic chip to study the dynamic adhesion behavior of cell-targeted microbubbles. The microfluidic device is composed of polydimethylsiloxane and is fabricated using the soft lithography technique. Each chamber of the microfluidic chip comprises eight U-shaped microsieves, by which various flow velocity distributions are generated. LyP-1-conjugated microbubbles were prepared by coating the surface of the phospholipid shell of microbubbles with LyP-1 peptides via biotin-avidin linkage. Under static conditions, the resulting targeted microbubbles are able to bind onto the surface of cells on incubation with breast cancer cells. Under dynamic fluid conditions, the cell targeting efficiency of the microbubbles was assessed at various flow velocity distributions in a chamber. Accumulation of targeted microbubbles was strongly influenced by flow velocity. Better retention of targeted microbubbles on cell surfaces was achieved at low mean flow velocities (<0.03 cm/s), in agreement with our computer simulation results. In conclusion, our results indicate that the microfluidic system is a useful platform for studying the microbubble-cell adhesive interaction.

Microbubble-Enhanced Cell Membrane Permeability in High Gravity Field

Cell permeability controls the transportation of extracellular materials through cell membranes, which plays a critical role in drug and gene delivery. This work reports an innovative method to enhance the cell permeability through cell-bubble interactions in a high gravity field. In the presence of microbubbles, the cell membrane permeability of mammalian cells was significantly increased in the high gravity field, and up to 80% THP-1 and 70% MCF-7 cells were permeabilized by using FITC-Dextran with average molecular weight of 40 and 70 kDa as fluorescent markers which were found to locate in both cytoplasm and cell nucleus by using a confocal microscope. Micro-scale pores were detected on the cell membrane by a scanning electron microscope after the cell-microbubble interactions in the high gravity field. The delivery efficiency of FITC-Dextran could be further enhanced in gravity field of higher strength and in solutions with higher volume fraction of microbubbles, though the cell viability would also fall under extreme conditions. A simplified model was proposed to compare the contributions of surface forces (i.e., Derjaguin–Landau–Verwey–Overbeek (DLVO) interaction force and membrane undulation force) with hydrodynamic force associated with cell-bubble interactions in the high gravity field. The hydrodynamic force was found to dominate the cell-bubble interaction while the DLVO force and membrane undulation force only play an important role at small separation (<10 nm) and low relative velocity of approach (i.e., low gravity field strength). The enhanced cell membrane permeability and formation of micro-scale pores are mainly due to the microbubble-cell interactions through collision and/or cavitation effects from the bursting of microbubbles. Our results have important implications in many bioengineering processes which are dependent on cell membrane permeability.

Producing single microbubbles with controlled size using microfiber

Microbubble-mediated pore formation in sonoporation includes a combination of complex processes such as collision or coalescence of translating or collapsing microbubbles with vascular cells. Although understanding the pore formation mechanisms may improve drug delivery efficiency, their details are still poorly understood. In the present study, we describe an experimental model that produces single air bubbles with controllable size. A carbon microfiber in liquids is illuminated by an infrared laser to produce individual bubbles having size comparable to that of the microfiber. The microbubbles can be physically isolated from the fiber for placing at arbitrary positions in the liquids. The lifetime of the bubbles is several tens of minutes depending on the intensity of the laser used. The preparation of the controllable air bubbles may be useful in future investigations of ultrasound-mediated microbubble–cell interactions.

Ultrasound-Induced Calcium Oscillations and Waves in Chinese Hamster Ovary Cells in the Presence of Microbubbles

This study investigated the effects of ultrasound on the intracellular [Ca 2+] of Chinese hamster ovary cells in the presence of albumin-encapsulated Optison microbubbles. Cells were exposed to 1 MHz ultrasound (tone burst of 0.2 s duration, 0.45 MPa peak pressure) while immersed in solution of 0.9 mM Ca 2+. Calcium imaging of the cells was performed using digital video fluorescence microscopy and Ca 2+-indicator dye fura-2AM. Experimental evidence indicated that ultrasound caused a direct microbubble-cell interaction resulting in the breaking and eventual dissolution of the microbubble and concomitant permeabilization of the cells to Ca 2+. These cells exhibited a large influx of Ca 2+ over 3–4 s and did not return to their equilibrium levels. Subsequently, some cells exhibited one or more Ca 2+ oscillations with the onset of oscillations delayed by 10–80 s after the ultrasound pulse. A variety of oscillations were observed including decaying oscillations returning to the baseline value over 35–100 s, oscillations superimposed on a more gradual recovery over 150–200 s, and oscillations continued with increased amplitude caused by a second ultrasound tone burst. The delays in onset appeared to result from calcium waves that propagated across the cells after the application of the ultrasound pulse.