Mechanisms Of Sonoporation Research Articles

Sonoporation, a Novel Frontier for Cancer Treatment: A Review of the Literature

Sonoporation has garnered significant attention for its potential to temporarily permeabilize cell membranes through the application of ultrasound waves, thus enabling an efficient cellular uptake of molecules. Despite its promising applications, the precise control of sonoporation remains a complex and evolving challenge in the field of cellular and molecular biology. This review aims to address two key aspects central to advancing our understanding of sonoporation. Firstly, it underscores the necessity for the establishment of a standardized methodology to validate and quantify the successful entry of molecules into target cells. This entails a critical examination of existing techniques and the identification of best practices to ensure accurate, reliable, and reproducible results. By establishing a common framework for assessing sonoporation outcomes, researchers can enhance the reliability and comparability of their experiments, paving the way for more robust findings. Secondly, the review places particular emphasis on the detailed analysis of various acoustic parameters as reported in the papers selected from the literature. Among these parameters, acoustic intensity (specifically, ISPTA) emerges as a pivotal factor in sonoporation studies. Furthermore, this review delves into the exploration of the elastic modulus and its significance in sonoporation mechanisms and associated challenges. This knowledge can inform the development of more effective strategies to optimize sonoporation protocols. In summary, this review not only highlights the pressing need for a standardized approach to verify molecule entry into cells but also delves into the search for an effective frequency and acoustic intensity for in vivo and in vitro applications.

In Vitro Radioenhancement Using Ultrasound-Stimulated Microbubbles: A Comparison of Suspension and Adherent Cell States

Background: Ultrasound-stimulated microbubbles (USMB) have shown potential for enhancing radiation treatment via cavitation and sonoporation mechanisms. However, in vitro studies have produced inconsistent results, with adherent cells demonstrating no radioenhancement. This study aims to investigate the effect of cell adherence on in vitro radioenhancement using USMB and radiation. Method: Lung metastases of follicular thyroid carcinoma cells (FTC-238) and non-small cell lung carcinoma cells (NCI-H727) were treated, both when adhered and in suspension, using 1.6% (v/v) Definity™ microbubbles, ~90 s of 2 MHz ultrasound with mechanical index 0.9, and either 3 Gy or 6 Gy of megavoltage (MV) X-rays. The cell viability was measured using an MTS assay 72 h post-treatment, and statistical analysis was conducted using a three-way analysis of variance. Results: Statistically significant differences were observed for cells treated when adherent compared to suspended. An additive effect was detected in NCI-H727 cells treated in suspension, but not while adherent, while no enhancement was observed for FTC-238 cells in either culture state. Conclusions: To the best of our knowledge, this is the first study to directly compare the effect of cell adherence on the radioenhancement potential of USMB in vitro, and the first to do so using a metastatic cell line.

Advances and Prospects of Ultrasound Targeted Drug Delivery Systems Using Biomaterial-modified Micro/Nanobubbles for Tumor Therapy.

The incidence of malignant tumors is rising rapidly and tends to be in the younger, which has been one of the most important factors endangering the safety of human life. Ultrasound micro/nanobubbles, as a noninvasive and highly specific antitumor strategy, can reach and destroy tumor tissue through their effects of cavitation and acoustic perforation under the guidance of ultrasound. Meanwhile, micro/nanobubbles are now used as a novel drug carrier, releasing drugs at a target region, especially on the prospects of biomaterial-modified micro/nanobubbles as a dual modality for drug delivery and therapeutic monitoring. Successful evaluation of the sonoporation mechanism(s), ultrasound parameters, drug type, and dose will need to be addressed before translating this technology for clinical use. Therefore, this paper collects the literature on the experimental and clinical studies of ultrasound biomaterial-modified micro/nanobubbles therapy in vitro and in vivo in recent years.

Ultrasound-Mediated Drug Delivery: Sonoporation Mechanisms, Biophysics, and Critical Factors.

Sonoporation, or the use of ultrasound in the presence of cavitation nuclei to induce plasma membrane perforation, is well considered as an emerging physical approach to facilitate the delivery of drugs and genes to living cells. Nevertheless, this emerging drug delivery paradigm has not yet reached widespread clinical use, because the efficiency of sonoporation is often deemed to be mediocre due to the lack of detailed understanding of the pertinent scientific mechanisms. Here, we summarize the current observational evidence available on the notion of sonoporation, and we discuss the prevailing understanding of the physical and biological processes related to sonoporation. To facilitate systematic understanding, we also present how the extent of sonoporation is dependent on a multitude of factors related to acoustic excitation parameters (ultrasound frequency, pressure, cavitation dose, exposure time), microbubble parameters (size, concentration, bubble-to-cell distance, shell composition), and cellular properties (cell type, cell cycle, biochemical contents). By adopting a science-backed approach to the realization of sonoporation, ultrasound-mediated drug delivery can be more controllably achieved to viably enhance drug uptake into living cells with high sonoporation efficiency. This drug delivery approach, when coupled with concurrent advances in ultrasound imaging, has potential to become an effective therapeutic paradigm.

Sonoporation: Past, Present, and Future.

A surge of research in intracellular delivery technologies is underway with the increased innovations in cell-based therapies and cell reprogramming. Particularly, physical cell membrane permeabilization techniques are highlighted as the leading technologies because of their unique features, including versatility, independence of cargo properties, and high-throughput delivery that is critical for providing the desired cell quantity for cell-based therapies. Amongst the physical permeabilization methods, sonoporation holds great promise and has been demonstrated for delivering a variety of functional cargos, such as biomolecular drugs, proteins, and plasmids, to various cells including cancer, immune, and stem cells. However, traditional bubble-based sonoporation methods usually require special contrast agents. Bubble-based sonoporation methods also have high chances of inducing irreversible damage to critical cell components, lowering the cell viability, and reducing the effectiveness of delivered cargos. To overcome these limitations, several novel non-bubble-based sonoporation mechanisms are under development. This review will cover both the bubble-based and non-bubble-based sonoporation mechanisms being employed for intracellular delivery, the technologies being investigated to overcome the limitations of traditional platforms, as well as perspectives on the future sonoporation mechanisms, technologies, and applications.

A Setup for Microscopic Studies of Ultrasounds Effects on Microliters Scale Samples: Analytical, Numerical and Experimental Characterization.

Sonoporation is the process of cell membrane permeabilization, due to exposure to ultrasounds. There is a lack of consensus concerning the mechanisms of sonoporation: Understanding the mechanisms of sonoporation refines the choice of the ultrasonic parameters to be applied on the cells. Cells’ classical exposure systems to ultrasounds have several drawbacks, like the immersion of the cells in large volumes of liquid, the nonhomogeneous acoustic pressure in the large sample, and thus, the necessity for magnetic stirring to somehow homogenize the exposure of the cells. This article reports the development and characterization of a novel system allowing the exposure to ultrasounds of very small volumes and their observation under the microscope. The observation under a microscope imposes the exposure of cells and Giant Unilamellar Vesicles under an oblique incidence, as well as the very unusual presence of rigid walls limiting the sonicated volume. The advantages of this new setup are not only the use of a very small volume of cells culture medium/microbubbles (MB), but the presence of flat walls near the sonicated region that results in a more homogeneous ultrasonic pressure field, and thus, the control of the focal distance and the real exposure time. The setup presented here comprises the ability to survey the geometrical and dynamical aspects of the exposure of cells and MB to ultrasounds, if an ultrafast camera is used. Indeed, the setup thus fulfills all the requirements to apply ultrasounds conveniently, for accurate mechanistic experiments under an inverted fluorescence microscope, and it could have interesting applications in photoacoustic research.

Effects of extracellular matrix rigidity on sonoporation facilitated by targeted microbubbles: Bubble attachment, bubble dynamics, and cell membrane permeabilization.

In this study, we investigated the effects of extracellular matrix rigidity, an important physical property of microenvironments regulating cell morphology and functions, on sonoporation facilitated by targeted microbubbles, highlighting the role of microbubbles. We conducted mechanistic studies at the cellular level on physiologically relevant soft and rigid substrates. By developing a unique imaging strategy, we first resolved details of the 3D attachment configurations between targeted microbubbles and cell membrane. High-speed video microscopy then unveiled bubble dynamics driven by a single ultrasound pulse. Finally, we evaluated the cell membrane permeabilization using a small molecule model drug. Our results demonstrate that: (1) stronger targeted microbubble attachment was formed for cells cultured on the rigid substrate, while six different attachment configurations were revealed in total; (2) more violent bubble oscillation was observed for cells cultured on the rigid substrate, while one third of bubbles attached to cells on the soft substrate exhibited deformation shortly after ultrasound was turned off; (3) higher acoustic pressure was needed to permeabilize the cell membrane for cells on the soft substrate, while under the same ultrasound condition, acoustically-activated microbubbles generated larger pores as compared to cells cultured on the soft substrate. The current findings provide new insights to understand the underlying mechanisms of sonoporation in a physiologically relevant context and may be useful for the clinical translation of sonoporation.

New insights on the role of ROS in the mechanisms of sonoporation-mediated gene delivery.

Reactive oxygen species (ROS) are hypothesized to play a role in the sonoporation mechanisms. Nevertheless, the acoustical phenomenon behind the ROS production as well as the exact mechanisms of ROS action involved in the increased cell membrane permeability are still not fully understood. Therefore, we investigated the key processes occurring at the molecular level in and around microbubbles subjected to ultrasound using computational chemistry methods. To confirm the molecular simulation predictions, we measured the ROS production by exposing SonoVue® microbubbles (MBs) to ultrasound using biological assays. To investigate the role of ROS in cell membrane permeabilization, cells were subjected to ultrasound in presence of MBs and plasmid encoding reporter gene, and the transfection level was assessed using flow cytometry. The molecular simulations showed that under sonoporation conditions, ROS can form inside the MBs. These radicals could easily diffuse through the MB shell toward the surrounding aqueous phase and participate in the permeabilization of nearby cell membranes. Experimental data confirmed that MBs favor spontaneous formation of a host of free radicals where HO was the main ROS species after US exposure. The presence of ROS scavengers/inhibitors during the sonoporation process decreased both the production of ROS and the subsequent transfection level without significant loss of cell viability. In conclusion, the exposure of MBs to ultrasound might be the origin of chemical effects, which play a role in the cell membrane permeabilization and in the in vitro gene delivery when generated in its proximity.

Molecular mechanisms of sonoporation in pancreatic ductal adenocarcinoma (PDAC)

Molecular mechanisms of sonoporation in pancreatic ductal adenocarcinoma (PDAC)

High-speed observation for a better understanding of bubble dynamics in an in vivo -like situation of sonoporation

Sonoporation is a technique to temporally increase the permeability of a biological cell membrane by ultrasound exposure and to transduce a foreign gene or drug that normally has no permeability. The mechanisms of sonoporation have not been fully elucidated yet; however, it is widely accepted that microbubbles oscillating adjacent to cells play an important role to induce membrane damage. Dynamics of microbubbles has strong dependence not only on exposure parameters of ultrasound but also on surrounding conditions of the bubbles. To obtain a better understanding of cell-bubble interactions affected by the surrounding conditions, high-speed observation was carried out. A newly designed chamber was used for observation from a lateral direction, enabling visualization of translational movements of bubbles beside a scaffold and of cell-bubble interaction without hiding the interacting point by the oscillating bubble itself. It was observed that a bubble on a soft scaffold surface moves translationally in a direction separating from the surface, whereas a bubble on a hard scaffold surface remains on the surface. Adhesion of a bubble to the top of a cell caused a considerable decrease in bubble oscillation amplitude, but membrane damage was confirmed in all of the observed cells with attached bubbles, indicating the importance of taking into account the conditions surrounding a bubble in order to understand the mechanisms of sonoporation.

High-Speed In Situ Observation System for Sonoporation of Cells With Size- and Position-Controlled Microbubbles.

A high-speed in situ microscopic observation system developed for basic studies on mechanisms of sonoporation is introduced in this paper. The main part of the system is an inverted-type fluorescence microscope, and a high-speed camera of 20 MHz in a maximum framing rate was used to visualize the dynamics of cavitation bubbles that causes a sonoporation effect. Differential interference contrast and fluorescence techniques were used for sensitive visualization of cell changes during sonoporation. The system is also equipped with optical tweezers that can move a microbubble of several microns in size by using a donut-shaped light beam. In situ microscopic observation of sonoporation was carried out using a cell with a size- and position-controlled microbubble. The experimental results showed that the ability of cells to repair sonoporation-induced damage depends on their membrane tension, indicating the usefulness of the observation system as a basic tool for the investigation of sonoporation phenomena.

Biophysical insight into mechanisms of sonoporation

This study presents a unique approach to understanding the biophysical mechanisms of ultrasound-triggered cell membrane disruption (i.e., sonoporation). We report direct correlations between ultrasound-stimulated encapsulated microbubble oscillation physics and the resulting cellular membrane permeability by simultaneous microscopy of these two processes over their intrinsic physical timescales (microseconds for microbubble dynamics and seconds to minutes for local macromolecule uptake and cell membrane reorganization). We show that there exists a microbubble oscillation-induced shear-stress threshold, on the order of kilopascals, beyond which endothelial cellular membrane permeability increases. The shear-stress threshold exhibits an inverse square-root relation to the number of oscillation cycles and an approximately linear dependence on ultrasound frequency from 0.5 to 2 MHz. Further, via real-time 3D confocal microscopy measurements, our data provide evidence that a sonoporation event directly results in the immediate generation of membrane pores through both apical and basal cell membrane layers that reseal along their lateral area (resealing time of ∼<2 min). Finally, we demonstrate the potential for sonoporation to indirectly initiate prolonged, intercellular gaps between adjacent, confluent cells (∼>30-60 min). This real-time microscopic approach has provided insight into both the physical, cavitation-based mechanisms of sonoporation and the biophysical, cell-membrane-based mechanisms by which microbubble acoustic behaviors cause acute and sustained enhancement of cellular and vascular permeability.

Reparable Cell Sonoporation in Suspension: Theranostic Potential of Microbubble.

The conjunction of low intensity ultrasound and encapsulated microbubbles can alter the permeability of cell membrane, offering a promising theranostic technique for non-invasive gene/drug delivery. Despite its great potential, the biophysical mechanisms of the delivery at the cellular level remains poorly understood. Here, the first direct high-speed micro-photographic images of human lymphoma cell and microbubble interaction dynamics are provided in a completely free suspension environment without any boundary parameter defect. Our real-time images and theoretical analyses prove that the negative divergence side of the microbubble's dipole microstreaming locally pulls the cell membrane, causing transient local protrusion of 2.5 µm in the cell membrane. The linear oscillation of microbubble caused microstreaming well below the inertial cavitation threshold, and imposed 35.3 Pa shear stress on the membrane, promoting an area strain of 0.12%, less than the membrane critical areal strain to cause cell rupture. Positive transfected cells with pEGFP-N1 confirm that the interaction causes membrane poration without cell disruption. The results show that the overstretched cell membrane causes reparable submicron pore formation, providing primary evidence of low amplitude (0.12 MPa at 0.834 MHz) ultrasound sonoporation mechanism.

Image-guided sonoporation in an ex vivo machine perfused porcine liver

Sonoporation is the transient and reversible cell membrane permeability change induced with ultrasound and microbubbles. It allows for the uptake of normally impermeable macromolecules and has been suggested for improving drug delivery. The exact sonoporation mechanism and the optimal ultrasound parameters are still under investigation. Ex vivo machine perfused porcine livers are an excellent platform for investigating the sonoporation parameters and specifically the interaction of ultrasound driven microbubbles with the capillaries. Our objective was to identify the ultrasound parameters that are capable of causing detectable perfusion changes in the sonoporation area. Three types of perfusion changes were considered: large mechanical damage void of perfusion, reduced perfusion due to capillary destruction, and unaltered perfusion.

Theoretical study of microbubble dynamics in sonoporation

Sonoporation is a promising technology for promoting the transfer of drug or gene into cells using ultrasound-mediated microbubbles that transiently break up the cell membrane. In this article, a model is established to analyze the dynamics of ultrasound-mediated microbubble near the cell membrane, which may be especially useful for understanding the mechanisms of sonoporation. In the model, the velocity potential of fluid on the microbubble surface and on the cell membrane is obtained by the unsteady Bernoulli equations, and it is solved by using the boundary integral equations. By numerically analyzing the model, the typical microbubble dynamics near the cell membrane are enumerated, which may be mainly governed by mechanical index. The model also established the connections among the parameters of ultrasound exposure, microbubble characteristics, and cell membrane properties in sonoporation.

Bleomycin delivery into cancer cells in vitro with ultrasound and SonoVue® or BR14® microbubbles

Background: Cell exposure to ultrasound (US) in the presence of contrast agent microbubbles (MBs) can result in cell sonoporation that can be exploited for drug or gene delivery. Anticancer drug bleomycin (BLM), used in sonoporation, can effectively eliminate tumor cells in vitro and in vivo. Nevertheless, sonoporation mechanism is not known, thus different US parameters and MB types are used. Recently, we proposed that efficiency of cell sonoporation can be related to the efficiency of MB sonodestruction.Purpose: We analyzed human tumor cells viability in response to BLM, US and MB treatment.Methods: Human glioblastoma astrocytoma (U-87 MG) or colon cancer (HCT-116) cells were exposed to US in the presence of BLM and either SonoVue® or BR14® MBs. MB sonodestruction was evaluated according to US signal attenuation.Results: Both HCT-116 and U-87 MG cell viability following US exposure decreased up to 30%. Decrease in cell viability followed similar tendency as MB sonodestruction, which suggests direct relationship between MB sonodestruction and BLM intracellular delivery.Conclusion: Sonoporation is a feasible method to deliver BLM in to several types of human cancer cell lines. Efficiency of cell sonoporation correlated well with MB sonodestruction, providing a possibility to optimize US parameters by measuring MB sonodestruction.

Microbubble attenuation and destruction: are they involved in sonoporation efficiency?

This technical note investigates the involvement of microbubble attenuation and destruction in sonoporation mechanisms. First, we evaluate sonoporation efficiency using Vevo Micromarker, and a comparison is made with BR14 and SonoVue microbubbles. Then, the acoustical properties of the microbubbles are measured to gain insight into the sonoporation mechanisms using a green fluorescent protein as a marker. Using glioblastoma cells, an unprecedented transfection rate of 70% is reached with Vevo Micromarker, corresponding to a 1.5-fold increase compared with the rate achieved with the other microbubbles. Moreover, attenuation and destruction were shown to be two key parameters in sonoporation efficiency.

Ultrasound and microbubble-assisted Gene Delivery: Recent Advances and Ongoing Challenges

Having first been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble--cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

TH‐C‐217BCD‐07: Ultrasound Contrast Agents as Facilitator for Intracellular Transport of Drugs and Genes

Purpose: The non‐invasive feature of ultrasound applications and the unique capability of ultrasound interaction with biological systems offer a beneficial platform for innovative biomedical applications. The effective and concentrated nature of ultrasound interaction with gaseous particles, has prompted the development of microbubble‐based ultrasound contrast agents to enhance ultrasound imaging capability. Particularly, microbubble ultrasound contrast agents have been exploited as a vehicle for intracellular delivery of cell impermeant drugs and genes. Ultrasound excitation of microbubbles, accompanied by rapid radial oscillation and/or collapse, has shown promise as an advantageous non‐viral strategy to generate transient increase of cell membrane permeability (sonoporation), thereby allowing uptake of exogenous agents into the cell cytoplasm. However, little is known about the basic processes supporting ultrasound‐induced transport into and within the cytoplasm of living cells at the single cell level. Our research aims to gain understanding of the mechanism of sonoporation and to uncover the physical, chemical, and cellular factors affecting the sonoporation process and outcome, in order to successfully develop and translate sonoporation technology for targeted, safe delivery of drugs and genes. Methods: We developed a set of novel techniques to control microbubble cavitation in reference to single cells. We used simultaneous whole‐cell patch clamp recording and fluorescence microscopy for real time monitoring of sonoporation. Results: We obtained measurements regarding the rapid formation and resealing of ultrasound‐induced membrane pores. Based on sonoporation‐mediated influx of an intercalating fluorescent dye, we developed a model to measure the intracellular diffusion coefficient of cytoplasmic RNA/DNA and estimate the pore size generated during sonoporation. We demonstrated spatiotemporally controlled subcellular delivery and calcium signaling in targeted cells. Conclusion: Through controlling of ultrasound excitation of microbubbles near single cells, we obtained results that might reveal key information of sonoporation mechanisms and processes, that can guide the development of sonoporation at as an effective and robust delivery technology.

Bursting Bubbles and Bilayers

This paper discusses various interactions between ultrasound, phospholipid monolayer-coated gas bubbles, phospholipid bilayer vesicles, and cells. The paper begins with a review of microbubble physics models, developed to describe microbubble dynamic behavior in the presence of ultrasound, and follows this with a discussion of how such models can be used to predict inertial cavitation profiles. Predicted sensitivities of inertial cavitation to changes in the values of membrane properties, including surface tension, surface dilatational viscosity, and area expansion modulus, indicate that area expansion modulus exerts the greatest relative influence on inertial cavitation. Accordingly, the theoretical dependence of area expansion modulus on chemical composition - in particular, poly (ethylene glyclol) (PEG) - is reviewed, and predictions of inertial cavitation for different PEG molecular weights and compositions are compared with experiment. Noteworthy is the predicted dependence, or lack thereof, of inertial cavitation on PEG molecular weight and mole fraction. Specifically, inertial cavitation is predicted to be independent of PEG molecular weight and mole fraction in the so-called mushroom regime. In the “brush” regime, however, inertial cavitation is predicted to increase with PEG mole fraction but to decrease (to the inverse 3/5 power) with PEG molecular weight. While excellent agreement between experiment and theory can be achieved, it is shown that the calculated inertial cavitation profiles depend strongly on the criterion used to predict inertial cavitation. This is followed by a discussion of nesting microbubbles inside the aqueous core of microcapsules and how this significantly increases the inertial cavitation threshold. Nesting thus offers a means for avoiding unwanted inertial cavitation and cell death during imaging and other applications such as sonoporation. A review of putative sonoporation mechanisms is then presented, including those involving microbubbles to deliver cargo into a cell, and those - not necessarily involving microubbles - to release cargo from a phospholipid vesicle (or reverse sonoporation). It is shown that the rate of (reverse) sonoporation from liposomes correlates with phospholipid bilayer phase behavior, liquid-disordered phases giving appreciably faster release than liquid-ordered phases. Moreover, liquid-disordered phases exhibit evidence of two release mechanisms, which are described well mathematically by enhanced diffusion (possibly via dilation of membrane phospholipids) and irreversible membrane disruption, whereas liquid-ordered phases are described by a single mechanism, which has yet to be positively identified. The ability to tune release kinetics with bilayer composition makes reverse sonoporation of phospholipid vesicles a promising methodology for controlled drug delivery. Moreover, nesting of microbubbles inside vesicles constitutes a truly “theranostic” vehicle, one that can be used for both long-lasting, safe imaging and for controlled drug delivery.