Sonoporation-based delivery has great promise for noninvasive drug and gene therapy. After short-term membrane resealing, the long-term function recovery of sonoporated cells affects the efficiency and biosafety of sonoporation-based delivery. It is necessary to identify the key early biological signals that influence cell fate and to develop strategies for manipulating the long-term fates of sonoporated cells. Here, we used a customized experimental platform with a single cavitating microbubble induced by a single ultrasound pulse (frequency: 1.5 MHz, pulse length:13.33 μs, peak negative pressure: ∼0.40 MPa) to elicit single-site reversible sonoporation on a single HeLa cell model. We used a living-cell microscopic imaging system to trace the long-term fates of sonoporated HeLa cells in real-time for 48 h. Fluorescence from intracellular propidium iodide and Fluo-4 was used to evaluate the degree of sonoporation and intracellular calcium fluctuation (ICF), respectively. Changes in cell morphology were used to assess the long-term cell fates (i.e., proliferation, arrest, or death). We found that heterogeneously sonoporated cells had different long-term fates. With increasing degree of sonoporation, the probability of normal (proliferation) and abnormal fates (arrest and death) in sonoporated cells decreased and increased, respectively. We identified ICF as an important early event for triggering different long-term fates. Reversibly sonoporated cells exhibited stronger proliferation and restoration at lower extents of ICF. We then regulated ICF dynamics in sonoporated cells using 2-APB or BAPTA treatment to reduce calcium release from intracellular organelles and enhance intracellular calcium clearance, respectively. This significantly enhanced the proliferation and restoration of sonoporated cells and reduced the occurrence of cell-cycle arrest and death. Finally, we found that the long-term fates of sonoporated cells at multiple sites and neighboring cells were also dependent on the extent of ICF, and that 2-APB significantly enhanced their viability and reduced death. Thus, using a single HeLa cell model, we demonstrated that regulating intracellular calcium can effectively enhance the proliferation and restoration capabilities of sonoporated cells, therefore rescuing the long-term viability of sonoporated cells. These findings add to our understanding of the biophysical process of sonoporation and help design new strategies for improving the efficiency and biosafety of sonoporation-based delivery.

Ultrasound-mediated cell membrane permeabilization - sonoporation, enhances drug delivery directly to tumor sites while reducing systemic side effects. The potential of ultrasound to augment intracellular calcium uptake - a critical regulator of cell death and proliferation - offers innovative alternative to conventional chemotherapy. However, calcium therapeutic applications remain underexplored in sonoporation studies. This research provides a comprehensive analysis of calcium sonoporation (CaSP), which combines ultrasound treatment with calcium ions and SonoVue microbubbles, on gastrointestinal cancer cells LoVo and HPAF-II. Initially, optimal sonoporation parameters were determined: an acoustic wave of 1 MHz frequency with a 50 % duty cycle at intensity of 2 W/cm2. Subsequently, various cellular bioeffects, such as viability, oxidative stress, metabolism, mitochondrial function, proliferation, and cell death, were assessed following CaSP treatment. CaSP significantly impaired cancer cell function by inducing oxidative and metabolic stress, evidenced by increased mitochondrial depolarization, decreased ATP levels, and elevated glucose uptake in a Ca2+ dose-dependent manner, leading to activation of the intrinsic apoptotic pathway. Cellular response to CaSP depended on the TP53 gene\'s mutational status: colon cancer cells were more susceptible to CaSP-induced apoptosis and G1 phase cell cycle arrest, whereas pancreatic cancer cells showed a higher necrotic response and G2 cell cycle arrest. These promising results encourage future research to optimize sonoporation parameters for clinical use, investigate synergistic effects with existing treatments, and assess long-term safety and efficacy in vivo. Our study highlights CaSP\'s clinical potential for improved safety and efficacy in cancer therapy, offering significant implications for the pharmaceutical and biomedical fields.

OBJECTIVE: Cell-based therapies have shown significant promise for treating many diseases, including cancer. Current cell therapy manufacturing processes primarily utilize viral transduction to insert genomic material into cells, which has limitations, including variable transduction efficiency and extended processing times. Non-viral transfection techniques are also limited by high variability or reduced molecular delivery efficiency. Novel 3D-printed acoustofluidic devices are in development to address these challenges by delivering biomolecules into cells within seconds via sonoporation.
METHODS: In this study, we assessed biological parameters that influence the ultrasound-mediated delivery of fluorescent molecules (i.e., calcein and 150 kDa FITC-Dextran) to human T cells using flow cytometry and confocal imaging.
RESULTS: Low cell plating densities (100,000 cells/mL) enhanced molecular delivery compared to higher cell plating densities (p < 0.001), even though cells were resuspended at equal concentrations for acoustofluidic processing. Additionally, cells in the S phase of the cell cycle had enhanced intracellular delivery compared to cells in the G2/M phase (p < 0.001) and G0/G1 phase (p < 0.01), while also maintaining higher viability compared to G0/G1 phase (p < 0.001). Furthermore, the calcium chelator (EGTA) decreased overall molecular delivery levels. Confocal imaging indicated that the actin cytoskeleton had important implications on plasma membrane recovery dynamics after sonoporation. In addition, confocal imaging indicates that acoustofluidic treatment can permeabilize the nuclear membrane, which could enable rapid intranuclear delivery of nucleic acids.
CONCLUSIONS: The results of this study demonstrate that a 3D-printed acoustofluidic device can enhance molecular delivery to human T cells, which may enable improved techniques for non-viral processing of cell therapies.

The recent and exciting success of anti-inflammatory therapies for ischemic heart disease (e.g. atherosclerosis) is hindered by the lack of site-specific and targeted therapeutic deposition. Microbubble-mediated focused ultrasound, which uses circulating, lipid-encapsulated intravascular microbubbles to locally enhance endothelial permeability, offers an exciting approach. Atherosclerotic plaques preferentially develop in regions with disturbed blood flow, and microbubble-endothelial cell membrane interactions under such flow conditions are not well understood. Here, using an acoustically-coupled microscopy system, endothelial cells were sonicated (1 MHz, 20 cycle bursts, 1 ms PRI, 4 s duration, 300 kPa peak-negative pressure) under perfusion with Definity™ bubbles to examine microbubble-mediated endothelial permeabilization under a range of physiological conditions. Endothelial preconditioning under prolonged shear influenced physiology and the secretome, inducing increased expression of pro-angiogenesis analytes, decreasing levels of pro-inflammatory ones, and increasing the susceptibility of ultrasound therapy. Ultrasound treatment efficiency was positively correlated with concentrations of pro-angiogenic cytokines (e.g. VEGF-A, EGF, FGF-2), and negatively correlated with pro-inflammatory chemokines (e.g. MCP-1, GCP-2, SDF-1). Furthermore, ultrasound therapy under non-reversing pulsatile flow (∼4-8 dyne/cm2, 0.5-1 Hz) increased permeabilization up to 2.4-fold compared to shear-matched laminar flow, yet treatment under reversing oscillatory flow resulted in more heterogeneous modulation. This study provides insight into the role of vascular physiology, including endothelial biology, into the design of a localized ultrasound drug delivery system for ischemic heart disease.

Sonoporation is a non-invasive method that uses ultrasound for drug and gene delivery for therapeutic purposes. Here, both Finite Element Method (FEM) and Lattice Boltzmann Method (LBM) are applied to study the interaction physics of microbubble oscillation and collapse near flexible tissue. After validating the Finite Element Method with the nonlinear excited lipid-coated microbubble as well as the Lattice Boltzmann Method with experimental results, we have studied the behavior of a three-dimensional compressible microbubble in the vicinity of tissue. In the FEM phase, the oscillation microbubble with a lipid shell interacts with the boundary. The range of pressure and ultrasound frequency have been considered in the field of therapeutic applications of sonoporation. The viscoelastic and interfacial tension as the coating properties of the microbubble shell have been investigated. The presence of an elastic boundary increases the resonance frequency of the microbubble compared to that of a free microbubble. The increase in pressure leads to an expansion in the range of the microbubble\'s motion, the velocity induced in the fluid, and the shear stress on the boundary walls of tissue. An enhancement in the surface tension of the microbubble can influence fluid flow and reduce the shear stress on the boundary. The multi-pseudo-potential interaction LBM is used to reduce thermodynamic inconsistency and high-density ratio in a two-phase system for modeling the cavitation process. The three-dimensional shape of the microbubble during the collapse stages and the counter of pressure are displayed. There is a time difference between the occurrence of maximum velocity and pressure. All results in detail are presented in the article bodies.

The combined application of electric fields and ultrasonic waves has shown promise in controlling cell membrane permeability, potentially resulting in synergistic effects that can be explored in the biotechnology industry. However, further clarification on how these processes interact is still needed. The objective of the present study was to investigate the atomic-scale effects of these processes on a DPPC lipid bilayer using molecular dynamics simulations. For higher electric fields, capable of independently forming pores, the application of an ultrasonic wave in the absence of cavitation yielded no additional effects on pore formation. However, for lower electric fields, the reduction in bilayer thickness induced by the shock wave catalyzed the electroporation process, effectively shortening the mean path that water molecules must traverse to form pores. When cavitation was considered, synergistic effects were evident only if the wave alone was able to generate pores through the formation of a water nanojet. In these cases, sonoporation acted as a mean to focus the electroporation effects on the initial pore formed by the nanojet. This study contributes to a better understanding of the synergy between electric fields and ultrasonic waves and to an optimal selection of processing parameters in practical applications of these processes.

Ca2+, in combination with SP or EP, induces cell cytotoxicity much faster compared to BLM. The application of BLM in combination with, SP or EP, reaches the level of cell death, induced by similar combination with Ca2+, only after 72 h. The methods of SP and EP were calibrated according to the level of differential cytotoxicity, determined after 6 days (using cell clonogenic assay). The combination of Ca2+ SP induces cell death faster than Ca2+ EP - after Ca2+ SP it increases to a maximum level after 15 min and remains constant for up to 6 days, while the cytotoxic efficiency after Ca2+ EP increases to the level of Ca2+ SP only after 72 h. The combination of BLM SP shows a very similar dynamics to BLM EP - both reach maximal level of cytotoxicity after 48-72 h. Ca2+ and BLM in combination with SP have shown similar levels of cytotoxicity at higher acoustic pressures (≥250 kPa); therefore, Ca2+ SP can be used to induce immediate and maximal level of cytotoxic effect. The faster cytotoxic efficiency of Ca2+ in combination with SP than EP was determined to be due to the involvement of microbubble inertial cavitation.

Sonoporation is a popular membrane disruption technique widely applicable in various fields, including cell therapy, drug delivery, and biomanufacturing. In recent years, there has been significant progress in achieving controlled, high-viability, and high-efficiency cell sonoporation in microfluidics. If the microchannels are too small, especially when scaled down to the cellular level, it still remains a challenge to overcome microchannel clogging, and low throughput. Here, we presented a microfluidic device capable of modulating membrane permeability through oscillating three-dimensional array of microbubbles. Simulations were performed to analyze the effective range of action of the oscillating microbubbles to obtain the optimal microchannel size. Utilizing a high-precision light curing 3D printer to fabricate uniformly sized microstructures in a one-step on both the side walls and the top surface for the generation of microbubbles. These microbubbles oscillated with nearly identical amplitudes and frequencies, ensuring efficient and stable sonoporation within the system. Cells were captured and trapped on the bubble surface by the acoustic streaming and secondary acoustic radiation forces induced by the oscillating microbubbles. At a driving voltage of 30 Vpp, the sonoporation efficiency of cells reached 93.9% ± 2.4%.

Ultrasound mediated drug delivery, a promising therapeutic modality, has evolved remarkably over the past three decades. Initially designed to enhance contrast in ultrasound imaging, microbubbles have emerged as a main vector for drug delivery, offering targeted therapy with minimized side effects. This review addresses the historical progression of this technology, emphasizing the pivotal role microbubbles play in augmenting drug extravasation and targeted delivery. We explore the complex mechanisms behind this technology, from stable and inertial cavitation to diverse acoustic phenomena, and their applications in medical fields. While the potential of ultrasound mediated drug delivery is undeniable, there are still challenges to overcome. Balancing therapeutic efficacy and safety and establishing standardized procedures are essential areas requiring attention. A multidisciplinary approach, gathering collaborations between researchers, engineers, and clinicians, is important for exploiting the full potential of this technology. In summary, this review highlights the potential of using ultrasound mediated drug delivery in improving patient care across various medical conditions.

Focused ultrasound (FUS) combined with intravascularly circulating microbubbles can transiently increase the permeability of the blood-brain barrier (BBB) to enable targeted therapeutic delivery to the brain, the clinical testing of which is currently underway in both adult and pediatric patients. Aside from traditional cancer drugs, this technique is being extended to promote the delivery of immunomodulating therapeutics to the brain, including antibodies, immune cells, and cytokines. In this manner, FUS approaches are being explored as a tool to improve and amplify the effectiveness of immunotherapy for both primary and metastatic brain cancer, a particularly challenging solid tumor to treat. Here, we present an overview of the latest groundbreaking research in FUS-assisted delivery of immunomodulating agents to the brain in pre-clinical models of brain cancer, and place it within the context of the current immunotherapy approaches. We follow this up with a discussion on new developments and emerging strategies for this rapidly evolving approach.