The molecular impact of sonoporation: A transcriptomic analysis of gene regulation profile.

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.

Evaluating ECM stiffness and liver cancer radiation response via shear-wave elasticity in 3D culture models.

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 (ß-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.

Manipulating long-term fates of sonoporated cells by regulating intracellular calcium for improving sonoporation-based delivery.

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.

Calcium ion delivery by microbubble-assisted sonoporation stimulates cell death in human gastrointestinal cancer cells.

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.

Biomodulatory Effects of Molecular Delivery in Human T Cells Using 3D-Printed Acoustofluidic Devices.

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.

Shear stress preconditioning and microbubble flow pattern modulate ultrasound-assisted plasma membrane permeabilization.

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.

Computational analysis of the simultaneous application of ultrasound and electric fields in a lipid bilayer.

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.

Echogenic Gold Nanorod Incorporated Hybrid Poly(2-oxazoline) Nanocapsules for Real-Time Ultrasound/Fluorescent Imaging and Targeted Cancer Theranostics.

In recent years, various nanocarrier systems have been explored to enhance the targeting of cancer cells by improving the ligand-receptor interactions between the nanocarrier and cancer cells for selective cancer cell imaging and targeted delivery of anticancer drugs. Herein, we report multifunctional hydrogen-bonded multilayer nanocapsules functionalized with both folic acid-derived quantum dots (FAQDs) and gold nanorods (AuNRs) for targeted cancer therapy and cancer cell imaging using fluorescence microscopy and medical-range ultrasound imaging systems. The encapsulation efficiency of nanocapsules was found to be 49% for 5-fluorouracil (5-FU). The release percentage reached a plateau at 37% after 1 h at pH 7.4 and increased to 57% after 3 h when the release pH was decreased to pH 5.5 (i.e., the pH of the tumor environment). Under ultrasound irradiation, the release was significantly accelerated, with a total release of 52% and 68% after only 6 min at pH 7.4 and pH 5.5, respectively. While the sonoporation process plays an important role in anticancer activity experiments under ultrasound exposure by generating temporary pores, the targeting ability of FAQDs brings the capsules closer to the cell membrane and improves the cellular uptake of the released drug, thereby increasing local drug concentration. In vitro cytotoxicity experiments with HCT-116 and HEp-2 cells demonstrated anticancer activities of 96% and 98%, respectively. The nanocapsules showed enhanced ultrasound scattering signal intensity and bright spots under ultrasound exposure, most likely caused by high scattering ability and internal reflections of preloaded AuNRs in the interior structure of the nanocapsules. Hence, the demonstrated nanocapsule system not only has the potential to be used as an integrated system for early- stage detection and treatment of cancer cells but also has the ability for live tracking and imaging of cancer cells while undergoing treatment with chemotherapy and radiation therapy.

Analysis of Microbubble-Blood cell system Oscillation/Cavitation influenced by ultrasound Forces: Conjugate applications of FEM and LBM.

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 comparison of the dynamics of Ca2+ and bleomycin intracellular delivery after cell sonoporation and electroporation in vitro.

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.

A T-Cell Inspired Sonoporation System Enhances Low-Dose X-Ray-Mediated Pyroptosis and Radioimmunotherapy Efficacy by Restoring Gasdermin-E Expression.

Genome editing has the potential to improve the unsatisfactory therapeutic effect of antitumor immunotherapy. However, the cell plasma membrane prevents the entry of almost all free genome-manipulation agents. Therefore, a system can be spatiotemporally controlled and can instantly open the cellular membrane to allow the entry of genome-editing agents into target cells is needed. Here, inspired by the ability of T cells to deliver cytotoxins to cancer cells by perforation, an ultrasound (US)-controlled perforation system (UPS) is established to enhance the delivery of free genome-manipulating agents. The UPS can perforate the tumor cell membrane while maintaining cell viability via a controllable lipid peroxidation reaction. In vitro, transmembrane-incapable plasmids can enter cells and perform genome editing with the assistance of UPS, achieving an efficiency of up to 90%. In vivo, the UPS is biodegradable, nonimmunogenic, and tumor-targeting, enabling the puncturing of tumor cells under US. With the application of UPS-assisted genome editing, gasdermin-E expression in 4T1 tumor-bearing mice is successfully restored, which leads to pyroptosis-mediated antitumor immunotherapy via low-dose X-ray irradiation. This study provides new insights for designing a sonoporation system for genome editing. Moreover, the results demonstrate that restoring gasdermin expression by genome editing significantly improves the efficacy of radioimmunotherapy.

Three-dimensional array of microbubbles sonoporation of cells in microfluidics.

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%.

From concept to early clinical trials: 30 years of microbubble-based ultrasound-mediated drug delivery research.

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.

Delivery of Anticancer Drugs Using Microbubble-Assisted Ultrasound in a 3D Spheroid Model.

Tumor spheroids are promising three-dimensional (3D) in vitro tumor models for the evaluation of drug delivery methods. The design of noninvasive and targeted drug methods is required to improve the intratumoral bioavailability of chemotherapeutic drugs and reduce their adverse off-target effects. Among such methods, microbubble-assisted ultrasound (MB-assisted US) is an innovative modality for noninvasive targeted drug delivery. The aim of the present study is to evaluate the efficacy of this US modality for the delivery of bleomycin, doxorubicin, and irinotecan in colorectal cancer (CRC) spheroids. MB-assisted US permeabilized the CRC spheroids to propidium iodide, which was used as a drug model without affecting their growth and viability. Histological analysis and electron microscopy revealed that MB-assisted US affected only the peripheral layer of the CRC spheroids. The acoustically mediated bleomycin delivery induced a significant decrease in CRC spheroid growth in comparison to spheroids treated with bleomycin alone. However, this US modality did not improve the therapeutic efficacy of doxorubicin and irinotecan on CRC spheroids. In conclusion, this study demonstrates that tumor spheroids are a relevant approach to evaluate the efficacy of MB-assisted US for the delivery of chemotherapeutics.

Ultrasound-responsive microbubbles and nanodroplets: A pathway to targeted drug delivery.

Ultrasound-responsive agents have shown great potential as targeted drug delivery agents, effectively augmenting cell permeability and facilitating drug absorption. This review focuses on two specific agents, microbubbles and nanodroplets, and provides a sequential overview of their drug delivery process. Particular emphasis is given to the mechanical response of the agents under ultrasound, and the subsequent physical and biological effects on the cells. Finally, the state-of-the-art in their pre-clinical and clinical implementation are discussed. Throughout the review, major challenges that need to be overcome in order to accelerate their clinical translation are highlighted.

Focused ultrasound-assisted delivery of immunomodulating agents in brain cancer.

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.

Efficient ultrasound-mediated drug delivery to orthotopic liver tumors - Direct comparison of doxorubicin-loaded nanobubbles and microbubbles.

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 C3F8 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.

Microbubble-assisted ultrasound for inner ear drug delivery.

Treating pathologies of the inner ear is a major challenge. To date, a wide range of procedures exists for administering therapeutic agents to the inner ear, with varying degrees of success. The key is to deliver therapeutics in a way that is minimally invasive, effective, long-lasting, and without adverse effects on vestibular and cochlear function. Microbubble-assisted ultrasound ("sonoporation") is a promising new modality that can be adapted to the inner ear. Combining ultrasound technology with microbubbles in the middle ear can increase the permeability of the round window, enabling therapeutic agents to be delivered safely and effectively to the inner ear in a targeted manner. As such, sonoporation is a promising new approach to treat hearing loss and vertigo. This review summarizes all studies on the delivery of therapeutic molecules to the inner ear using sonoporation.

Evaluation of Doxorubicin-loaded Echogenic Macroemulsion for Targeted Drug Delivery.

BACKGROUND: The latest technology trend in targeted drug delivery highlights stimuliresponsive particles that can release an anticancer drug in a solid tumor by responding to external stimuli. OBJECTIVE: This study aims to design, fabricate, and evaluate an ultrasound-responsive drug delivery vehicle for an ultrasound-mediated drug delivery system. METHODS: The drug-containing echogenic macroemulsion (eME) was fabricated by an emulsification method using the three phases (aqueous lipid solution as a shell, doxorubicin (DOX) contained oil, and perfluorohexane (PFH) as an ultrasound-responsive agent). The morphological structure of eMEs was investigated using fluorescence microscopy, and the size distribution was analyzed by using DLS. The echogenicity of eME was measured using a contrast-enhanced ultrasound device. The cytotoxicity was evaluated using a breast cancer cell (MDA-MB-231) via an in vitro cell experiment. RESULTS: The obtained eME showed an ideal morphological structure that contained both DOX and PFH in a single particle and indicated a suitable size for enhancing ultrasound response and avoiding complications in the blood vessel. The echogenicity of eME was demonstrated via an in vitro experiment, with results showcasing the potential for targeted drug delivery. Compared to free DOX, enhanced cytotoxicity and improved drug delivery efficiency in a cancer cell were proven by using DOX-loaded eMEs and ultrasound. CONCLUSION: This study established a platform technology to fabricate the ultrasound-responsive vehicle. The designed drug-loaded eME could be a promising platform with ultrasound technology for targeted drug delivery.

Development of an In Vitro Model to Study Mechanisms of Ultrasound-Targeted Microbubble Cavitation-Mediated Blood-Brain Barrier Opening.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.