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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. [Display omitted] [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Background: The stiffness of the tumor microenvironment (TME) directly influences cellular behaviors. Radiotherapy (RT) is a common treatment for solid tumors, but the TME can impact its efficacy. In the case of liver cancer, clinical observations have shown that tumors within a cirrhotic, stiffer background respond less to RT, suggesting that the extracellular matrix (ECM) stiffness plays a critical role in the development of radioresistance. Methods: This study explored the effects of ECM stiffness and the inhibition of lysyl oxidase (LOX) isoenzymes on the radiation response of liver cancer in a millimeter-sized three-dimensional (3D) culture. We constructed a cube-shaped ECM-based millimeter-sized hydrogel containing Huh7 human liver cancer cells. By modulating the collagen concentration, we produced two groups of samples with different ECM stiffnesses to mimic the clinical scenarios of normal and cirrhotic livers. We used a single-transducer system for shear-wave-based elasticity measurement, to derive Young's modulus of the 3D cell culture to investigate how the ECM stiffness affects radiosensitivity. This is the first demonstration of a workflow for assessing radiation-induced response in a millimeter-sized 3D culture. Results: Increased ECM stiffness was associated with a decreased radiation response. Moreover, sonoporation-assisted LOX inhibition with BAPN (β-aminopropionitrile monofumarate) significantly decreased the initial ECM stiffness and increased RT-induced cell death. Inhibition of LOX was particularly effective in reducing ECM stiffness in stiffer matrices. Combining LOX inhibition with RT markedly increased radiation-induced DNA damage in cirrhotic liver cancer cells, enhancing their response to radiation. Furthermore, LOX inhibition can be combined with sonoporation to overcome stiffness-related radioresistance, potentially leading to better treatment outcomes for patients with liver cancer. Conclusions: The findings underscore the significant influence of ECM stiffness on liver cancer's response to radiation. Sonoporation-aided LOX inhibition emerges as a promising strategy to mitigate stiffness-related resistance, offering potential improvements in liver cancer treatment outcomes. [ABSTRACT FROM AUTHOR]

Abstract Background The stiffness of the tumor microenvironment (TME) directly influences cellular behaviors. Radiotherapy (RT) is a common treatment for solid tumors, but the TME can impact its efficacy. In the case of liver cancer, clinical observations have shown that tumors within a cirrhotic, stiffer background respond less to RT, suggesting that the extracellular matrix (ECM) stiffness plays a critical role in the development of radioresistance. Methods This study explored the effects of ECM stiffness and the inhibition of lysyl oxidase (LOX) isoenzymes on the radiation response of liver cancer in a millimeter-sized three-dimensional (3D) culture. We constructed a cube-shaped ECM-based millimeter-sized hydrogel containing Huh7 human liver cancer cells. By modulating the collagen concentration, we produced two groups of samples with different ECM stiffnesses to mimic the clinical scenarios of normal and cirrhotic livers. We used a single-transducer system for shear-wave-based elasticity measurement, to derive Young’s modulus of the 3D cell culture to investigate how the ECM stiffness affects radiosensitivity. This is the first demonstration of a workflow for assessing radiation-induced response in a millimeter-sized 3D culture. Results Increased ECM stiffness was associated with a decreased radiation response. Moreover, sonoporation-assisted LOX inhibition with BAPN (β-aminopropionitrile monofumarate) significantly decreased the initial ECM stiffness and increased RT-induced cell death. Inhibition of LOX was particularly effective in reducing ECM stiffness in stiffer matrices. Combining LOX inhibition with RT markedly increased radiation-induced DNA damage in cirrhotic liver cancer cells, enhancing their response to radiation. Furthermore, LOX inhibition can be combined with sonoporation to overcome stiffness-related radioresistance, potentially leading to better treatment outcomes for patients with liver cancer. Conclusions The findings underscore the significant influence of ECM stiffness on liver cancer’s response to radiation. Sonoporation-aided LOX inhibition emerges as a promising strategy to mitigate stiffness-related resistance, offering potential improvements in liver cancer treatment outcomes.

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.

Ultrasound-targeted microbubble cavitation (UTMC)-mediated blood–brain barrier (BBB) opening is being explored as a method to increase drug delivery to the brain. This strategy has progressed to clinical trials for various neurological disorders, but the underlying cellular mechanisms are incompletely understood. In the study described here, a contact co-culture transwell model of the BBB was developed that can be used to determine the signaling cascade leading to increased BBB permeability. This BBB model consists of bEnd.3 cells and C8-D1A astrocytes seeded on opposite sides of a transwell membrane. Pulsed ultrasound (US) is applied to lipid microbubbles (MBs), and the change in barrier permeability is measured via transendothelial electrical resistance and dextran flux. Live cell calcium imaging (Fluo-4 AM) is performed during UTMC treatment. This model exhibits important features of the BBB, including endothelial tight junctions, and is more restrictive than the endothelial cell (EC) monolayer alone. When US is applied to MBs in contact with the ECs, BBB permeability increases in this model by two mechanisms: UTMC induces pore formation in the EC membrane (sonoporation), leading to increased transcellular permeability, and UTMC causes formation of reversible inter-endothelial gaps, which increases paracellular permeability. Additionally, this study determines that calcium influx into ECs mediates the increase in BBB permeability after UTMC in this model. Both transcellular and paracellular permeability can be used to increase drug delivery to the brain. Future studies can use this model to determine how UTMC-induced calcium-mediated signaling increases BBB permeability. [ABSTRACT FROM AUTHOR]

Liver metastasis is a major obstacle in treating aggressive cancers, and current therapeutic options often prove insufficient. To overcome these challenges, there has been growing interest in ultrasound-mediated drug delivery using lipid-shelled microbubbles (MBs) and nanobubbles (NBs) as promising strategies for enhancing drug delivery to tumors. Our previous work demonstrated the potential of Doxorubicin-loaded C 3 F 8 NBs (hDox-NB, 280 ± 123 nm) in improving cancer treatment in vitro using low-frequency unfocused therapeutic ultrasound (TUS). In this study, we investigated the pharmacokinetics and biodistribution of sonicated hDox-NBs in orthotopic rat liver tumors. We compared their delivery and therapeutic efficiency with size-isolated MBs (hDox-MB, 1104 ± 373 nm) made from identical shell material and core gas. Results showed a similar accumulation of hDox in tumors treated with hDox-MBs and unfocused therapeutic ultrasound (hDox-MB + TUS) and hDox-NB + TUS. However, significantly increased apoptotic cell death in the tumor and fewer off-target apoptotic cells in the normal liver were found upon the treatment with hDox-NB + TUS. The tumor-to-liver apoptotic ratio was elevated 9.4-fold following treatment with hDox-NB + TUS compared to hDox-MB + TUS, suggesting that the therapeutic efficacy and specificity are significantly increased when using hDox-NB + TUS. These findings highlight the potential of this approach as a viable treatment modality for liver tumors. By elucidating the behavior of drug-loaded bubbles in vivo , we aim to contribute to developing more effective liver cancer treatments that could ultimately improve patient outcomes and decrease off-target side effects. [Display omitted] • Bubble size is a critical factor in ultrasound-mediated drug delivery to tumors. • Nanobubbles excited with ultrasound deliver more drugs to tumors than microbubbles. • Nanobubbles + ultrasound increase apoptotic cell death in tumors while sparing normal liver. [ABSTRACT FROM AUTHOR]

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. [Display omitted] [ABSTRACT FROM AUTHOR]

Ultrasound-mediated intracellular delivery is one of the popular technologies based on membrane rupture at present. To date, ultrasound directly acts on a large number of cells to achieve cargo delivery and has been widely used in drug delivery, disease therapy and other fields. However, the existing macroscopic methods can no longer meet the requirements of accurate tracking and analysis and are prone to extensive cell damage and even death. With the rapid advancements in microfluidic technologies, the combination of ultrasound and microfluidics (CUM) technology can effectively improve the delivery efficiency and cell survival rates. This new technology has rapidly become a new direction and focus of research. Thus, we analysed the mechanism of sonoporation and the effect of acoustic waves in a microfluidic channel. In addition, we reviewed the application of these new technologies in terms of structure and fabrication of ultrasound transducers and microfluidic devices. As regards our main objective, we hope to help researchers better understand the future developments and the challenges of new technologies. With this review, researchers can promote the development of new technologies to solve the current challenges of intracellular delivery and advance clinical applications. [ABSTRACT FROM AUTHOR]

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 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.

Lipid-coated microbubbles are widely used as an ultrasound contrast agent, as well as drug delivery carriers. However, the two main limitations in ultrasound diagnosis and drug delivery using microbubbles are the short half-life in the blood system, and the difficulty of surface modification of microbubbles for active targeting. The exosome, a type of extracellular vesicle, has a preferentially targeting ability for its original cell. In this study, exosome-fused microbubbles (Exo-MBs) were developed by embedding the exosome membrane proteins into microbubbles. As a result, the stability of Exo-MBs is improved over the conventional microbubbles. On the same principle that under the exposure of ultrasound, microbubbles are cavitated and self-assembled into nano-sized particles, and Exo-MBs are self-assembled into exosome membrane proteins-embedded nanoparticles (Exo-NPs). The Exo-NPs showed favorable targeting properties to their original cells. A photosensitizer, chlorin e6, was loaded into Exo-MBs to evaluate therapeutic efficacy as a drug carrier. Much higher therapeutic efficacy of photodynamic therapy was confirmed, followed by cancer immunotherapy from immunogenic cell death. We have therefore developed a novel ultrasound image-guided drug delivery platform that overcomes the shortcomings of the conventional ultrasound contrast agent and is capable of simultaneous photodynamic therapy and cancer immunotherapy.

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. [ABSTRACT FROM AUTHOR]

Objective: Pulsed ultrasound has been proposed as a tool to enhance ocular drug delivery, but its effects on drug potency are not well understood. Doxorubicin-HCl and cisplatin are two drugs commonly used to treat ocular melanoma. We report the effects of pulsed ultrasound on the cytotoxicity of doxorubicin-HCl and cisplatin in vitro. Methods: Cultured human retinal pigment epithelium cells (ARPE-19) were treated with doxorubicin-HCl or cisplatin in the presence or absence of ultrasound. MTT and Trypan blue assays were performed at 24 and 48 h post treatment to assess cell metabolism and death. Results: Cells treated with ultrasound plus doxorubicin-HCl demonstrated a significant decrease in metabolism compared with cells treated with doxorubicin-HCl alone. In contrast, cells treated with ultrasound plus cisplatin exhibited a significant increase in metabolism compared with cells treated with cisplatin alone for 48 h. Cells treated with cisplatin and pretreated with ultrasound (US–Cis) exhibited a significant decrease in metabolism. Cell death was similar in doxorubicin- and cisplatin-treated cells with and without ultrasound. Conclusion: Pulsed ultrasound enhances the cytotoxicity of doxorubicin-HCl at 24 and 48 h post treatment but abrogates cisplatin toxicity 48 h post treatment. This suggests ultrasound modulates cell–drug interactions in a drug-specific manner. These findings may influence the future development of ultrasound-assisted ocular drug delivery systems. [ABSTRACT FROM AUTHOR]

Lipid-coated microbubbles are widely used as an ultrasound contrast agent, as well as drug delivery carriers. However, the two main limitations in ultrasound diagnosis and drug delivery using microbubbles are the short half-life in the blood system, and the difficulty of surface modification of microbubbles for active targeting. The exosome, a type of extracellular vesicle, has a preferentially targeting ability for its original cell. In this study, exosome-fused microbubbles (Exo-MBs) were developed by embedding the exosome membrane proteins into microbubbles. As a result, the stability of Exo-MBs is improved over the conventional microbubbles. On the same principle that under the exposure of ultrasound, microbubbles are cavitated and self-assembled into nano-sized particles, and Exo-MBs are self-assembled into exosome membrane proteins-embedded nanoparticles (Exo-NPs). The Exo-NPs showed favorable targeting properties to their original cells. A photosensitizer, chlorin e6, was loaded into Exo-MBs to evaluate therapeutic efficacy as a drug carrier. Much higher therapeutic efficacy of photodynamic therapy was confirmed, followed by cancer immunotherapy from immunogenic cell death. We have therefore developed a novel ultrasound image-guided drug delivery platform that overcomes the shortcomings of the conventional ultrasound contrast agent and is capable of simultaneous photodynamic therapy and cancer immunotherapy. Exosome-fused microbubbles (Exo-MBs) were developed and self-assembled into exosome membrane proteins-embedded nanoparticles (Exo-NPs), which became a novel ultrasound image-guided drug delivery platform in simultaneous photodynamic therapy and cancer immunotherapy. [Display omitted] [ABSTRACT FROM AUTHOR]

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%.

Sonoporation is the process where intracellular drug delivery is facilitated by ultrasound-driven microbubble oscillations. Several mechanisms have been proposed to relate microbubble dynamics to sonoporation including shear and normal stress. The present work aims to gain insight into the role of microbubble size on sonoporation and thereby into the relevant mechanism(s) of sonoporation. To this end, we measured the sonoporation efficiency while varying microbubble size using monodisperse microbubble suspensions. Sonoporation experiments were performed in vitro on cell monolayers using a single ultrasound pulse with a fixed frequency of 1 MHz while the acoustic pressure amplitude and pulse length were varied at 250, 500, and 750 kPa, and 10, 100, and 1000 cycles, respectively. Sonoporation efficiency was quantified using flow cytometry by measuring the FITC-dextran (4 kDa and 2 MDa) fluorescence intensity in 10,000 cells per experiment to average out inherent variations in the bioresponse. Using ultra-high-speed imaging at 10 million frames per second, we demonstrate that the bubble oscillation amplitude is nearly independent of the equilibrium bubble radius at acoustic pressure amplitudes that induce sonoporation (≥ 500 kPa). However, we show that sonoporation efficiency is strongly dependent on the equilibrium bubble size and that under all explored driving conditions most efficiently induced by bubbles with a radius of 4.7 μm. Polydisperse microbubbles with a typical ultrasound contrast agent size distribution perform almost an order of magnitude lower in terms of sonoporation efficiency than the 4.7-μm bubbles. We elucidate that for our system shear stress is highly unlikely the mechanism of action. By contrast, we show that sonoporation efficiency correlates well with an estimate of the bubble-induced normal stress. [Display omitted] [ABSTRACT FROM AUTHOR]

We propose an amplitude-modulated pump ultrasound irradiation sequence to generate bubble cavitation for efficient sonoporation. By matching the envelope wavelength of the modulated wave to four times the vessel diameter, we aimed to improve the efficiency of bubble collapse near the vessel wall and extend the duration of collapse during repeated application of the sequence. Using an agarose-based blood vessel phantom, we compared the effects of the amplitude-modulated sequence with those of the normal sinusoidal sequence in which the sound pressure was adjusted so that the acoustic energy in one burst length of each sequence was equal. Compared with the normal irradiation sequence, the amplitude-modulated sequence yielded 2.45- and 1.31-fold greater bubble collapse near the vessel walls proximal and distal, respectively, to the pump ultrasound source, a 1.59-fold longer duration of bubble collapse, and a 1.69-fold larger area of micropores on the channel wall. [ABSTRACT FROM AUTHOR]

Systemic drugs can treat various retinal pathologies such as retinal cancers; however, their ocular diffusion may be limited by the blood–retina barrier (BRB). Sonication corresponds to the use of ultrasound (US) to increase the permeability of cell barriers including in the BRB. The objective was to study the efficacy and safety of sonication using microbubble-assisted low-intensity pulsed US in inducing a transient opening of the BRB. The eyes of C57/BL6J mice were sonicated at different acoustic pressures (0.10 to 0.50 MPa). Efficacy analyses consisted of fluorescein angiography (FA) performed at different timepoints and the size of the leaked molecules was assessed using FITC-marked dextrans. Tolerance was assessed by fundus photographs, optical coherence tomography, immunohistochemistry, RT-qPCR, and electroretinograms. Sonication at 0.15 MPa was the most suitable pressure for transient BRB permeabilization without altering the morphology or function of the retina. It did not increase the expression of inflammation or apoptosis markers in the retina, retinal pigment epithelium, or choroid. The dextran assay suggested that drugs up to 150 kDa in size can cross the BRB. Microbubble-assisted sonication at an optimized acoustic pressure of 0.15 MPa provides a non-invasive method to transiently open the BRB, increasing the retinal diffusion of systemic drugs without inducing any noticeable side-effect. [ABSTRACT FROM AUTHOR]

Despite recent advancements in ultrasound-mediated drug delivery and the remarkable success observed in pre-clinical studies, no delivery platform utilizing ultrasound contrast agents has yet received FDA approval. The sonoporation effect was a game-changing discovery with a promising future in clinical settings. Various clinical trials are underway to assess sonoporation's efficacy in treating solid tumors; however, there are disagreements on its applicability to the broader population due to long-term safety issues. In this review, we first discuss how acoustic targeting of drugs gained importance in cancer pharmaceutics. Then, we discuss ultrasound-targeting strategies that have been less explored yet hold a promising future. We aim to shed light on recent innovations in ultrasound-based drug delivery including newer designs of ultrasound-sensitive particles specifically tailored for pharmaceutical usage. [ABSTRACT FROM AUTHOR]

Pancreatic tumors can be resistant to drug penetration due to high interstitial fluid pressure, dense stroma, and disarrayed vasculature. Ultrasound-induced cavitation is an emerging technology that may overcome many of these limitations. Low-intensity ultrasound, coupled with co-administered cavitation nuclei consisting of gas-stabilizing sub-micron scale SonoTran Particles, is effective at increasing therapeutic antibody delivery to xenograft flank tumors in mouse models. Here, we sought to evaluate the effectiveness of this approach in situ using a large animal model that mimics human pancreatic cancer patients. Immunocompromised pigs were surgically engrafted with human Panc-1 pancreatic ductal adenocarcinoma (PDAC) tumors in targeted regions of the pancreas. These tumors were found to recapitulate many features of human PDAC tumors. Animals were intravenously injected with the common cancer therapeutics Cetuximab, gemcitabine, and paclitaxel, followed by infusion with SonoTran Particles. Select tumors in each animal were targeted with focused ultrasound to induce cavitation. Cavitation increased the intra-tumor concentrations of Cetuximab, gemcitabine, and paclitaxel by 477%, 148%, and 193%, respectively, compared to tumors that were not targeted with ultrasound in the same animals. Together, these data show that ultrasound-mediated cavitation, when delivered in combination with gas-entrapping particles, improves therapeutic delivery in pancreatic tumors under clinically relevant conditions. [ABSTRACT FROM AUTHOR]

Hepatic fibrosis is the common pathway for most chronic liver diseases, characterized by excessive accumulation of extracellular matrix (ECM) proteins. It has been shown that fibrotic ECM significantly hindered passage of nanoparticles. Efforts have been made by decorating degrading enzymes on surfaces of nanosized delivery vehicles to improve drug delivery. However, these strategies are restricted by limiting shelf-life. Inspired by the application of sonoporation in assisting drug delivery through blood-brain barrier and tumor tissues, we investigated whether sonoporation can be an alternative strategy in improving drug delivery for fibrotic diseases. Hydroxycamptothecin (HCPT), a potential drug in treating liver fibrosis, was selected as a model drug to evaluate the drug delivery efficiency and therapeutic effect among three delivery strategies, i.e., (1) injection solution, (2) delivery through liposomes, and (3) delivery via sonoporation. Our study showed that in addition to the improved drug delivery efficiency, the combination of HCPT and sonoporation led to synergistic effect and the mechanisms were investigated. The treatment group of HCPT delivered with sonoporation achieved the most significant attenuation in liver fibrosis among the three delivery strategies. [Display omitted] [ABSTRACT FROM AUTHOR]

The local fluid dynamics experienced by circulating microbubbles vary across different anatomical sites, which can influence ultrasound-mediated therapeutic delivery efficacy. This study aimed to elucidate the effect of fluid flow rate in combination with repeated short-pulse ultrasound on microbubble-mediated endothelial cell permeabilization. Here, a seeded monolayer of human umbilical (HUVEC) or brain endothelial cells (HBEC-5i) was co-perfused with a solution of microbubbles and propidium iodide (PI) at either a flow rate of 5 or 30 ml/min. Using an acoustically coupled inverted microscope, cells were exposed to 1 MHz ultrasound with 20-cycle bursts, 1 ms PRI, and 2 s duration at a peak negative pressure of 305 kPa to assess the role of flow rate on ultrasound-stimulated endothelial cell permeability, as well as Ca2+ modulation. In addition, the effect of inter-pulse delays (∆ t = 1 s) on the resulting endothelial permeability was investigated. Our results demonstrate that under an identical acoustic stimulus, fast-flowing microbubbles resulted in a statistically significant increase in cell membrane permeability, at least by 2.3-fold, for both endothelial cells. Likewise, there was a substantial difference in intracellular Ca2+ levels between the two examined flow rates. In addition, multiple short pulses rather than a single pulse ultrasound, with an equal number of bursts, significantly elevated endothelial cell permeabilization, at least by 1.4-fold, in response to ultrasound-stimulated microbubbles. This study provides insights into the design of optimal, application-dependent pulsing schemes to improve the effectiveness of ultrasound-mediated local therapeutic delivery. [Display omitted] [ABSTRACT FROM AUTHOR]

Microbubble (MB)- and ultrasound (US)-facilitated intracellular Ca2+ delivery, known as sonoporation (SP), is a promising anticancer treatment modality, since it allows a spatio-temporally controllable and side-effect-free alternative to conventional chemotherapy. The current study provides extensive evidence that a 5 mM concentration of Ca2+ in combination with US alone or US and Sonovue MBs can be an alternative to the conventional 20 nM concentration of the anticancer drug bleomycin (BLM). Ca2+ application together with SP induces a similar level of death in Chinese hamster ovary cells to the combination of BLM and SP but does not cause systemic toxicity, as is inherent to conventional anticancer drugs. In addition, Ca2+ delivery via SP alters three vital characteristics essential for viable cells: membrane permeability, metabolic activity and proliferation ability. Most importantly, Ca2+ delivery via SP elicits sudden cell death—occurring within 15 min—which remains similar during 24–72 h and 6 d periods. The extensive study of US waves side-scattered by MBs led to the quantification of the cavitation dose (CD) separately for subharmonics, ultraharmonics, harmonics and broadband noise (up to 4 MHz). The CD was suitable for the prognostication of the cytotoxic efficiency of both anticancer agents, Ca2+ and BLM, as was indicated by an overall high (R2 ≥ 0.8) correlation (22 pairs in total). These extensive analytical data imply that a broad range of frequencies are applicable for the feedback-loop control of the process of US-mediated Ca2+ or BLM delivery, successively leading to the eventual standardization of the protocols for the sonotransfer of anticancer agents as well as the establishment of a universal cavitation dosimetry model. [ABSTRACT FROM AUTHOR]

Currently available cytotoxic treatments have limited effect on pancreatic ductal adenocarcinoma (PDAC) because desmoplastic stroma limits drug delivery. Efforts have been made to overcome these barriers by drug targeting the tumor microenvironment. Results so far are promising, but without clinical impact. Our aim was to investigate whether ultrasound and microbubbles could improve the uptake and therapeutic response of conventional chemotherapy. Orthotopic pancreatic tumors growing in mice were treated with commercially available FOLFIRINOX (fluorouracil, irinotecan, oxaliplatin and calcium folinate) and SonoVue microbubbles combined with focused ultrasound. Tumor uptake of platinum (Pt) was measured by inductively coupled plasma mass spectroscopy (ICP-MS), and tumor volumes were measured by ultrasound imaging. Uptake of Pt, the active ingredient of oxaliplatin, was significantly increased after ultrasound treatment of orthotopic PDAC tumors. Multiple injections with FOLFIRONOX increased the amount of Pt in tumors. However, the enhanced accumulation did not improve therapeutic response. Increased uptake of Pt confirms that ultrasound and microbubbles have potential in clinical practice with existing drugs. The lack of therapeutic response, despite increased uptake in tumor tissue, emphasizes the importance of studying how to overcome stromal barriers. [ABSTRACT FROM AUTHOR]

In this study, we developed an efficient mRNA delivery vehicle by optimizing a lyophilization method for preserving human serum albumin-based nanobubbles (HSA-NBs), bypassing the need for artificial stabilizers. The morphology of the lyophilized material was verified using scanning electron microscopy, and the concentration, size, and mass of regenerated HSA-NBs were verified using flow cytometry, nanoparticle tracking analysis, and resonance mass measurements, and compared to those before lyophilization. The study also evaluated the response of HSA-NBs to 1 MHz ultrasound irradiation and their ultrasound (US) contrast effect. The functionality of the regenerated HSA-NBs was confirmed by an increased expression of intracellularly transferred Gluc mRNA, with increasing intensity of US irradiation. The results indicated that HSA-NBs retained their structural and functional integrity markedly, post-lyophilization. These findings support the potential of lyophilized HSA-NBs, as efficient imaging, and drug delivery systems for various medical applications. [ABSTRACT FROM AUTHOR]

Surface tension provides microbubbles (MB) with a perfect spherical shape. Here, we demonstrate that MB can be engineered to be nonspherical, endowing them with unique features for biomedical applications. Anisotropic MB were generated via one-dimensionally stretching spherical poly(butyl cyanoacrylate) MB above their glass transition temperature. Compared to their spherical counterparts, nonspherical polymeric MB displayed superior performance in multiple ways, including i) increased margination behavior in blood vessel–like flow chambers, ii) reduced macrophage uptake in vitro, iii) prolonged circulation time in vivo, and iv) enhanced blood–brain barrier (BBB) permeation in vivo upon combination with transcranial focused ultrasound (FUS). Our studies identify shape as a design parameter in the MB landscape, and they provide a rational and robust framework for further exploring the application of anisotropic MB for ultrasound-enhanced drug delivery and imaging applications. [ABSTRACT FROM AUTHOR]

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.

Sonoporation using microbubble-assisted ultrasound increases the permeability of a biological barrier to therapeutic molecules. Application of this method to the round window membrane could improve the delivery of therapeutics to the inner ear. The aim of this study was to assess the safety of sonoporation of the round window membrane in a sheep model. To achieve this objective, we assessed auditory function and cochlear heating, and analysed the metabolomics profiles of perilymph collected after sonoporation, comparing them with those of the control ear in the same animal. Six normal-hearing ewes were studied, with one sonoporation ear and one control ear for each. A mastoidectomy was performed on both ears. On the sonoporation side, Vevo MicroMarker® microbubbles (MBs; VisualSonics—Fujifilm, Amsterdam, The Netherlands) at a concentration of 2 × 108 MB/mL were locally injected into the middle ear and exposed to 1.1 MHz sinusoidal ultrasonic waves at 0.3 MPa negative peak pressure with 40% duty cycle and 100 μs interpulse period for 1 min; this was repeated three times with 1 min between applications. The sonoporation protocol did not induce any hearing impairment or toxic overheating compared with the control condition. The metabolomic analysis did not reveal any significant metabolic difference between perilymph samples from the sonoporation and control ears. The results suggest that sonoporation of the round window membrane does not cause damage to the inner ear in a sheep model. [ABSTRACT FROM AUTHOR]

Continuous-flow acoustofluidic technologies can potentially improve processing of T lymphocytes for cell therapies by addressing the limitations with viral and non-viral delivery methods. The objective of this study was to assess the intracellular delivery efficiency with acoustofluidic treatment compared with that of static ultrasound treatment. Optimization of parameters in acoustofluidic and static configurations was performed by assessing intracellular delivery of a fluorescent compound (calcein) in viable human Jurkat T lymphocytes. Ultrasound pressure and the concentration of cationic phospholipid-coated microbubbles influenced calcein delivery in both systems. In the static system, a treatment time of 45 s increased molecular delivery compared with 0-30 s (p < 0.01). Refined parameters were used to assess molecular delivery of small and large compounds (0.6-kDa calcein and 150-kDa fluorescein isothiocyanate-dextran, respectively) after ultrasound treatment with the acoustofluidic or static systems. Molecular delivery was similar with refined parameters for acoustofluidic treatment and static treatment (p > 0.05), even though acoustofluidic treatment had lower microbubble concentration (24 μg/mL vs. 94 μg/mL) and shorter treatment time (∼2-3 s vs. 45 s). This study indicates that the acoustofluidic system can significantly enhance intracellular molecular delivery, which could potentially enable acoustofluidic cell transfection during continuous flow processing for manufacture of cell therapies or other applications. [ABSTRACT FROM AUTHOR]

In endothelial cells, microRNA-126 (miR-126) promotes angiogenesis, and modulating the intracellular levels of this gene could suggest a method to treat cardiovascular diseases such as ischemia. Novel ultrasound-stimulated microbubbles offer a means to deliver therapeutic payloads to target cells and sites of disease. The purpose of this study was to investigate the feasibility of gene delivery by stimulating miR-126-decorated microbubbles using gentle acoustic conditions (stable cavitation). A cationic DSTAP microbubble was formulated and characterized to carry 6 µg of a miR-126 payload per 109 microbubbles. Human umbilical vein endothelial cells (HUVECs) were treated at 20–40% duty cycle with miR-126-conjugated microbubbles in a custom ultrasound setup coupled with a passive cavitation detection system. Transfection efficiency was assessed by RT-qPCR, Western blotting, and endothelial tube formation assay, while HUVEC viability was monitored by MTT assay. With increasing duty cycle, the trend observed was an increase in intracellular miR-126 levels, up to a 2.3-fold increase, as well as a decrease in SPRED1 (by 33%) and PIK3R2 (by 46%) expression, two salient miR-126 targets. Under these ultrasound parameters, HUVECs maintained >95% viability after 96 h. The present work describes the delivery of a proangiogenic miR-126 using an ultrasound-responsive cationic microbubble with potential to stimulate therapeutic angiogenesis while minimizing endothelial damage. [ABSTRACT FROM AUTHOR]

Introduction: Coaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection.Phospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl2 crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression.Coaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments.Our results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine.Methods: Coaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection.Phospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl2 crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression.Coaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments.Our results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine.Results: Coaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection.Phospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl2 crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression.Coaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments.Our results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine.Conclusions: Coaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection.Phospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl2 crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression.Coaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments.Our results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine. [ABSTRACT FROM AUTHOR]

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. • sonoporation combined with Ca2+ is cytotoxic to gastrointestinal cancer cells. • CaSP is associated with intense oxidative stress and mitochondria disturbance. • CaSP leads to induction of intrinsic apoptotic pathway and altered proliferation. • slight cytotoxic effect of CaSP on healthy cells. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Computational analysis of electric fields and ultrasound sinergy in lipid membranes. • Ultrasonic waves had no effect on pore formation under high electric fields. • The waves thinned the bilayer, catalyzing electroporation in lower electric fields. • Cavitation enhances electroporation by directing its effects with a water nanojet. [ABSTRACT FROM AUTHOR]

Ultrasound insonation of microbubbles can be used to form pores in cell membranes and facilitate the local trans-membrane transport of drugs and genes. An important factor in efficient delivery is the size of the delivered target compared to the generated membrane pores. Large molecule delivery remains a challenge, and can affect the resulting therapeutic outcomes. To facilitate large molecule delivery, large pores need to be formed. While ultrasound typically uses megahertz frequencies, it was recently shown that when microbubbles are excited at a frequency of 250 kHz (an order of magnitude below the resonance frequency of these agents), their oscillations are significantly enhanced as compared to the megahertz range. Here, to promote the delivery of large molecules, we suggest using this low frequency and inducing large pore formation through the high-amplitude oscillations of microbubbles. We assessed the impact of low frequency microbubble-mediated sonoporation on breast cancer cell uptake by optimizing the delivery of 4 fluorescent molecules ranging from 1.2 to 70 kDa in size. The optimal ultrasound peak negative pressure was found to be 500 kPa. Increasing the pressure did not enhance the fraction of fluorescent cells, and in fact reduced cell viability. For the smaller molecule sizes, 1.2 kDa and 4 kDa, the groups treated with an ultrasound pressure of 500 kPa and MB resulted in a fraction of 58% and 29% of fluorescent cells respectively, whereas delivery of 20 kDa and 70 kDa molecules yielded 10% and 5%, respectively. These findings suggest that low-frequency (e.g., 250 kHz) insonation of microbubbles results in high amplitude oscillation in vitro that increase the uptake of large molecules. Successful ultrasound-mediated molecule delivery requires the careful selection of insonation parameters to maximize the therapeutic effect by increasing cell uptake.

Targeted delivery of therapeutics to specific tissues is critically important for reducing systemic toxicity and optimizing therapeutic efficacy, especially in the case of cytotoxic drugs. Many strategies currently exist for targeting systemically administered drugs, and ultrasound-controlled targeting is a rapidly advancing strategy for externally-stimulated drug delivery. In this non-invasive method, ultrasound waves penetrate through tissue and stimulate gas-filled microbubbles, resulting in bubble rupture and biophysical effects that power delivery of attached cargo to surrounding cells. Drug delivery capabilities from ultrasound-sensitive microbubbles are greatly expanded when nanocarrier particles are attached to the bubble surface, and cargo loading is determined by the physicochemical properties of the nanoparticles. This review serves to highlight and discuss current microbubble–nanoparticle complex component materials and designs for ultrasound-mediated drug delivery. Nanocarriers that have been complexed with microbubbles for drug delivery include lipid-based, polymeric, lipid–polymer hybrid, protein, and inorganic nanoparticles. Several schemes exist for linking nanoparticles to microbubbles for efficient nanoparticle delivery, including biotin–avidin bridging, electrostatic bonding, and covalent linkages. When compared to unstimulated delivery, ultrasound-mediated cargo delivery enables enhanced cell uptake and accumulation of cargo in target organs and can result in improved therapeutic outcomes. These ultrasound-responsive delivery complexes can also be designed to facilitate other methods of targeting, including bioactive targeting ligands and responsivity to light or magnetic fields, and multi-level targeting can enhance therapeutic efficacy. Microbubble–nanoparticle complexes present a versatile platform for controlled drug delivery via ultrasound, allowing for enhanced tissue penetration and minimally invasive therapy. Future perspectives for application of this platform are also discussed in this review. [ABSTRACT FROM AUTHOR]

Sonoporation is the process of transient pore formation in the cell membrane triggered by ultrasound (US). Numerous studies have provided us with firm evidence that sonoporation may assist cancer treatment through effective drug and gene delivery. However, there is a massive gap in the body of literature on the issue of understanding the complexity of biophysical and biochemical sonoporation-induced cellular effects. This study provides a detailed explanation of the US-triggered bioeffects, in particular, cell compartments and the internal environment of the cell, as well as the further consequences on cell reproduction and growth. Moreover, a detailed biophysical insight into US-provoked pore formation is presented. This study is expected to review the knowledge of cellular effects initiated by US-induced sonoporation and summarize the attempts at clinical implementation. [ABSTRACT FROM AUTHOR]

Placental dysregulation frequently results in pregnancy complications that impact fetal well-being and potentially predispose the infant to diseases later in life. Thus, efforts to understand the molecular mechanisms underlying placental disorders are crucial to aid the development of effective treatments to restore placental function. Currently, the most common methods used for trophoblast-specific gene modulation in the laboratory are transgenic animals and lentiviral trophectoderm transduction. The generation of transgenic animal lines is costly and requires a considerable amount of time to generate and maintain, while the integration preference of lentiviruses, actively transcribed genes, may result in genotoxicity. Therefore, there is much interest in the development of non-viral in vivo transfection techniques for use in both research and clinical settings. Herein, we describe a non-viral, minimally invasive method for in vivo placental gene modulation through sonoporation, an ultrasound-mediated transfection technique wherein the application of ultrasound on target tissues is used to direct the uptake of DNA vectors. In this method, plasmids are bound to lipid microbubbles, which are then injected into the maternal bloodstream and ultimately delivered to the placenta when subjected to low-frequency ultrasound. Syncytiotrophoblasts are directly exposed to maternal blood and, therefore highly accessible to therapeutic agents in the maternal circulation. This technique can be used to modulate gene expression and, subsequently, the function of the placenta, circumventing the requirement to generate transgenic animals. Sonoporation also offers a safer alternative to existing viral techniques, making it not only an advantageous research tool but also a potentially adaptable technique in clinical settings., Competing Interests: Declaration of competing interest I Lance G. A. Nunes, declare that I substantially contributed to the conception, design and preparation of the manuscript. I have seen and approved the final version. I have no conflicts of interest. I Fredrick J Rosario, declare that I substantially contributed to the conception, design and preparation of the manuscript. I have seen and approved the final version. I have no conflicts of interest. I Johann Urschitz, declare that I substantially contributed to the conception, design and preparation of the manuscript. I have seen and approved the final version. I have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sonoporation has long been known to disrupt intracellular signaling, yet the involved molecules and pathways have not been identified with clarity. In this study, we employed whole transcriptome shotgun sequencing (RNA-seq) to profile sonoporation-induced gene responses after membrane resealing has taken place. Sonoporation was achieved by microbubble-mediated ultrasound (MB-US) exposure in the form of 1 MHz ultrasound pulsing (0.50 MPa peak negative pressure, 10 % duty cycle, 30 s exposure period) in the presence of microbubbles (1:1 cell-to-bubble ratio). Using propidium iodide (PI) and calcein respectively as cell viability and cytoplasmic uptake labels, post-exposure flow cytometry was performed to identify three viable cell populations: 1) unsonoporated cells, 2) sonoporated cells with low uptake, and 3) sonoporated cells with high uptake. Fluorescence-activated cell sorting was then conducted to separate the different groups followed by RNA-seq analysis of the gene expressions in each group of cells. We found that sonoporated cells with low or high calcein uptake showed high similarity in the gene responses, including the activation of multiple heat shock protein (HSP) genes and immediate early response genes mediating apoptosis and transcriptional regulation. In contrast, unsonoporated cells exhibited a more extensive gene expression alteration that included the activation of more HSP genes and the upregulation of diverse apoptotic mediators. Four oxidative stress-related and three immune-related genes were also differentially expressed in unsonoporated cells. Our results provided new information for understanding the intracellular mobilization in response to sonoporation at the molecular level, including the identification of new molecules in the sonoporation-induced apoptosis regulatory network. Our data also shed light on the innovative therapeutic strategy which could potentially leverage the responses of viable unsonoporated cells as a synergistic effector in the microenvironment to favor tumor treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Systemic drugs can treat various retinal pathologies such as retinal cancers; however, their ocular diffusion may be limited by the blood–retina barrier (BRB). Sonication corresponds to the use of ultrasound (US) to increase the permeability of cell barriers including in the BRB. The objective was to study the efficacy and safety of sonication using microbubble-assisted low-intensity pulsed US in inducing a transient opening of the BRB. The eyes of C57/BL6J mice were sonicated at different acoustic pressures (0.10 to 0.50 MPa). Efficacy analyses consisted of fluorescein angiography (FA) performed at different timepoints and the size of the leaked molecules was assessed using FITC-marked dextrans. Tolerance was assessed by fundus photographs, optical coherence tomography, immunohistochemistry, RT-qPCR, and electroretinograms. Sonication at 0.15 MPa was the most suitable pressure for transient BRB permeabilization without altering the morphology or function of the retina. It did not increase the expression of inflammation or apoptosis markers in the retina, retinal pigment epithelium, or choroid. The dextran assay suggested that drugs up to 150 kDa in size can cross the BRB. Microbubble-assisted sonication at an optimized acoustic pressure of 0.15 MPa provides a non-invasive method to transiently open the BRB, increasing the retinal diffusion of systemic drugs without inducing any noticeable side-effect.

Despite recent advancements in ultrasound-mediated drug delivery and the remarkable success observed in pre-clinical studies, no delivery platform utilizing ultrasound contrast agents has yet received FDA approval. The sonoporation effect was a game-changing discovery with a promising future in clinical settings. Various clinical trials are underway to assess sonoporation’s efficacy in treating solid tumors; however, there are disagreements on its applicability to the broader population due to long-term safety issues. In this review, we first discuss how acoustic targeting of drugs gained importance in cancer pharmaceutics. Then, we discuss ultrasound-targeting strategies that have been less explored yet hold a promising future. We aim to shed light on recent innovations in ultrasound-based drug delivery including newer designs of ultrasound-sensitive particles specifically tailored for pharmaceutical usage.

The aim of this study was to investigate whether sonoporation of cancer cells using ultrasound (US) technology could enhance the anticancer effects of cold atmospheric plasma. US‐induced transient sonoporation of cancer cells with little to no cytotoxicity observed on the cell lines tested. Synergistic effects of US were observed when combined with both direct and indirect cold atmospheric plasma. These cytotoxic effects were dependent on reactive species production. To the best of our knowledge that is the first time that the effects of those two nonthermal technologies were evaluated in cancer cells, demonstrating a promising combined strategy for cancer therapies, particularly for those with penetration limitations, such as glioblastoma. [ABSTRACT FROM AUTHOR]

Microbubble-mediated ultrasound (MB-US) can be used to realize sonoporation and, in turn, facilitate the transfection of leukocytes in the immune system. Nevertheless, the bio-effects that can be induced by MB-US exposure on leukocytes have not been adequately studied, particularly for different leukocyte lineage subsets with distinct cytological characteristics. Here, we describe how that same set of MB-US exposure conditions would induce heterogeneous bio-effects on the three main leukocyte subsets: lymphocytes, monocytes and granulocytes. MB-US exposure was delivered by applying 1-MHz pulsed ultrasound (0.50-MPa peak negative pressure, 10% duty cycle, 30-s exposure period) in the presence of microbubbles (1:1 cell-to-bubble ratio); sonoporated and non-viable leukocytes were respectively labeled using calcein and propidium iodide. Flow cytometry was then performed to classify leukocytes into their corresponding subsets and to analyze each subset's post-exposure viability, sonoporation rate, uptake characteristics and morphology. Results revealed that, when subjected to MB-US exposure, granulocytes experienced the highest loss of viability (64.0 ± 11.0%) and the lowest sonoporation rate (6.3 ± 2.5%), despite maintaining their size and granularity. In contrast, lymphocytes exhibited the lowest loss of viability (20.9 ± 7.0%), while monocytes had the highest sonoporation rate (24.1 ± 13.6%). For these two sonoporated leukocyte subsets, their cell size and granularity were found to be reduced. Also, they exhibited graded levels of calcein uptake, whereas sonoporated granulocytes achieved only mild calcein uptake. These heterogeneous bio-effects should be accounted for when using MB-US and sonoporation in immunomodulation applications. [ABSTRACT FROM AUTHOR]

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 α v β 3 -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 (2 MHz, 200 kPa peak negative pressure, 10 cycles). 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. [Display omitted] • Targeted microbubbles can be internalized by endothelial cells. • Internalized microbubbles exhibit damped oscillation upon ultrasound insonification. • Microbubble internalization increases the cell's susceptibility to sonoporation. • Microbubble-mediated drug delivery can occur via pores and tunnels. • Transcellular perforations can persist after intracellular drug uptake ends. [ABSTRACT FROM AUTHOR]

Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that sonobactericide can lead to bacterial dispersion or sonoporation (i.e., cell membrane permeabilization); however, real-time observations distinguishing individual bacteria during and directly after insonification are missing. Therefore, in this study, we investigated, in real-time and at high-resolution, the effects of ultrasound-induced microbubble oscillation on Staphylococcus aureus biofilms, without or with an antibiotic (oxacillin, 1 μg/mL). Biofilms were exposed to ultrasound (2 MHz, 100–400 kPa, 100–1000 cycles, every second for 30 s) during time-lapse confocal microscopy recordings of 10 min. Bacterial responses were quantified using post hoc image analysis with particle counting. Bacterial dispersion was observed as the dominant effect over sonoporation, resulting from oscillating microbubbles. Increasing pressure and cycles both led to significantly more dispersion, with the highest pressure leading to the most biofilm removal (up to 83.7%). Antibiotic presence led to more variable treatment responses, yet did not significantly impact the therapeutic efficacy of sonobactericide, suggesting synergism is not an immediate effect. These findings elucidate the direct effects induced by sonobactericide to best utilize its potential as a biofilm treatment strategy. [ABSTRACT FROM AUTHOR]

Ultrasound has previously been demonstrated to non-invasively cause tissue disruption. Small animal studies have demonstrated that this effect can be enhanced by contrast microbubbles and has the potential to be clinically beneficial in techniques such as targeted drug delivery or enhancing liquid biopsies when a physical biopsy may be inappropriate. Cavitating microbubbles in close proximity to cells increases membrane permeability, allowing small intracellular molecules to leak into the extracellular space. This study sought to establish whether cavitating microbubbles could liberate cell-specific miRNAs, augmenting biomarker detection for non-invasive liquid biopsies. Insonating human polarized renal proximal tubular epithelial cells (RPTECs), in the presence of SonoVue microbubbles, revealed that cellular health could be maintained while achieving the release of miRNAs, miR-21, miR-30e, miR-192 and miR-194 (respectively, 10.9-fold, 7.17-fold, 5.95-fold and 5.36-fold). To examine the mechanism of release, RPTECs expressing enhanced green fluorescent protein were generated and the protein successfully liberated. Cell polarization, cellular phenotype and cell viability after sonoporation were measured by a number of techniques. Ultrastructural studies using electron microscopy showed gap-junction disruption and pore formation on cellular surfaces. These studies revealed that cell-specific miRNAs can be non-specifically liberated from RPTECs by sonoporation without a significant decrease in cell viability. [ABSTRACT FROM AUTHOR]

The use of nanobubbles (NBs) for ultrasound-mediated gene therapy has recently attracted much attention. However, few studies have evaluated the effect of different NB size distribution to the efficiency of gene delivery into cells. In this study, various size of albumin stabilized sub-micron bubbles were examined in an in vitro ultrasound (1 MHz) irradiation setup in the aim to compare and optimize gene transfer efficiency. Results with pDNA showed that gene transfer efficiency in the presence of NB size of 254.7 ± 3.8 nm was 2.5 fold greater than those with 187.3 ± 4.8 nm. Similarly, carrier-free mRNA transfer efficiency increased in the same conditions. It is suggested that NB size greater than 200 nm contributed more to the delivery of genes into the cytoplasm with ultrasound. Although further experiments are needed to understand the underlying mechanism for this phenomenon, the present results offer valuable information in optimizing of NB for future ultrasound-mediate gene therapy. [ABSTRACT FROM AUTHOR]