Pulsed Power Applications for Protein Conformational Change and the Permeabilization of Agricultural Products.

Pulsed electric fields (PEFs), which are generated by pulsed power technologies, are being tested for their applicability in food processing through protein conformational change and the poration of cell membranes. In this article, enzyme activity change and the permeabilization of agricultural products using pulsed power technologies are reviewed as novel, nonthermal food processes. Compact pulsed power systems have been developed with repetitive operation and moderate output power for application in food processing. Firstly, the compact pulsed power systems for the enzyme activity change and permeabilization are outlined. Exposure to electric fields affects hydrogen bonds in the secondary and tertiary structures of proteins; as a result, the protein conformation is induced to be changed. The conformational change induces an activity change in enzymes such as α-amylase and peroxidase. Secondly, the conformational change in proteins and the induced protein functional change are reviewed. The permeabilization of agricultural products is caused through the poration of cell membranes by applying PEFs produced by pulsed discharges. The permeabilization of cell membranes can be used for the extraction of nutrients and health-promoting agents such as polyphenols and vitamins. The electrical poration can also be used as a pre-treatment for food drying and blanching processes. Finally, the permeabilization of cell membranes and its applications in food processing are reviewed.

Enhanced Vascular Permeability by Microbubbles and Ultrasound in Drug Delivery.

Ultrasound and microbubbles, an ultrasound contrast agent, have recently increased attention to developing novel drug delivery systems. Ultrasound exposure can induce mechanical effects derived from microbubbles behaviors such as an expansion, contraction, and collapse depending on ultrasound conditions. These mechanical effects induce several biological effects, including enhancement of vascular permeability. For drug delivery, one promising approach is enhancing vascular permeability using ultrasound and microbubbles, resulting in improved drug transport to targeted tissues. This approach is applied to several tissues and drugs to cure diseases. This review describes the enhancement of vascular permeability by ultrasound and microbubbles and its therapeutic application, including our recent study. We also discuss the current situation of the field and its potential future perspectives.

Effect of 1-MHz ultrasound on the proinflammatory interleukin-6 secretion in human keratinocytes.

Keratinocytes, the main cell type of the skin, are one of the most exposed cells to environmental factors, providing a first defence barrier for the host and actively participating in immune response. In fact, keratinocytes express pattern recognition receptors that interact with pathogen associated molecular patterns and damage associated molecular patterns, leading to the production of cytokines and chemokines, including interleukin (IL)-6. Herein, we investigated whether mechanical energy transported by low intensity ultrasound (US) could generate a mechanical stress able to induce the release of inflammatory cytokine such IL-6 in the human keratinocyte cell line, HaCaT. The extensive clinical application of US in both diagnosis and therapy suggests the need to better understand the related biological effects. Our results point out that US promotes the overexpression and secretion of IL-6, associated with the activation of nuclear factor-κB (NF-κB). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability, paving the way for further investigating the molecular mechanisms related to US exposure.

Inhibition of EphA2 by Dasatinib Suppresses Radiation-Induced Intestinal Injury.

Radiation-induced multiorgan dysfunction is thought to result primarily from damage to the endothelial system, leading to a systemic inflammatory response that is mediated by the recruitment of leukocytes. The Eph-ephrin signaling pathway in the vascular system participates in various disease developmental processes, including cancer and inflammation. In this study, we demonstrate that radiation exposure increased intestinal inflammation via endothelial dysfunction, caused by the radiation-induced activation of EphA2, an Eph receptor tyrosine kinase, and its ligand ephrinA1. Barrier dysfunction in endothelial and epithelial cells was aggravated by vascular endothelial-cadherin disruption and leukocyte adhesion in radiation-induced inflammation both in vitro and in vivo. Among all Eph receptors and their ligands, EphA2 and ephrinA1 were required for barrier destabilization and leukocyte adhesion. Knockdown of EphA2 in endothelial cells reduced radiation-induced endothelial dysfunction. Furthermore, pharmacological inhibition of EphA2-ephrinA1 by the tyrosine kinase inhibitor dasatinib attenuated the loss of vascular integrity and leukocyte adhesion in vitro. Mice administered dasatinib exhibited resistance to radiation injury characterized by reduced barrier leakage and decreased leukocyte infiltration into the intestine. Taken together, these data suggest that dasatinib therapy represents a potential approach for the protection of radiation-mediated intestinal damage by targeting the EphA2-ephrinA1 complex.

Investigating the optimum size of nanoparticles for their delivery into the brain assisted by focused ultrasound-induced blood-brain barrier opening.

The blood-brain barrier (BBB) has hampered the efficiency of nanoparticle delivery into the brain via conventional strategies. The widening of BBB tight junctions via focused ultrasound (FUS) offers a promising approach for enhancing the delivery of nanoparticles into the brain. However, there is currently an insufficient understanding of how nanoparticles pass through the opened BBB gaps. Here we investigated the size-dependence of nanoparticle delivery into the brain assisted by FUS-induced BBB opening, using gold nanoparticles (AuNPs) of 3, 15, and 120 nm diameter. For 3- and 15-nm AuNPs, FUS exposure significantly increased permeation across an in vitro BBB model by up to 9.5 times, and the permeability was higher with smaller diameter. However, in vivo transcranial FUS exposure in mice demonstrated that smaller particles were not necessarily better for delivery into the brain. Medium-sized (15 nm) AuNPs showed the highest delivery efficiency (0.22% ID), compared with 3- and 120-nm particles. A computational model suggested that this optimum size was determined by the competition between their permeation through opened BBB gaps and their excretion from blood. Our results would greatly contribute to designing nanoparticles for their delivery into the brain for the treatment of central nervous system diseases.

The Long-Term Fate of the Sonoporated Pancreatic Cancer Cells is Uncorrelated With the Degree of Model Molecular Loading.

Studies have determined that ultrasound-activated microbubbles can increase the membrane permeability of tumor cells by triggering membrane perforation (sonoporation) to improve drug loading. However, because of the distinct cavitation events adjacent to each cell, the degree of drug loading appeared to be heterogeneous. The relationship between the long-term fate trend and the degree of drug loading remains unclear. To investigate the time-lapse viability of diversity loading cells, fluorescein isothiocyanate-dextran (FITC-dextrans) was used as a molecular model mixed with 2% v/v SonoVue microbubbles (Bracco, Milan, Italy) and exposed to various peak negative pressures (0.25 MPa, 0.6 MPa, 1.2 MPa), 1 MHz frequency and 300 µs pulse duration. To select a suitable parameter, the cavitation activity was measured, and the cell analysis was performed by flow cytometry under these acoustic pressures. The sonoporated cells were then categorized into 3 sub-groups by flow cytometry according to the various fluorescence intensity distributions to analyze their long-term fate. We observed that the stable cavitation occurred at 0.25 MPa and microbubbles underwent ultra-harmonic emission, and obvious broadband signals were observed at 0.6 MPa and 1.2 MPa, suggesting the occurs of inertial cavitation. The cell analysis further showed the maximum delivery efficiency and cell viability at 0.6 MPa, and it was selected for the following experiment. The categorization displayed that the fluorescence intensity of FITC-dextrans in sub-groups 2 and 3 were approximate 5.62-fold and 19.53-fold higher than that in sub-group 1, respectively. After separation of these sub-groups, the apoptosis and necrosis ratios in all 3 sub-groups of sonoporated cells gradually increased with increasing culture time and displayed no significant difference in either the apoptosis (p > 0.05) or necrosis (p > 0.05) ratio after 6 h and 24 h of culture, respectively. Further analysis using Western blot verified that the long-term fate of sonoporated cells involves the mitochondrial signaling proteins. These results provide better insight into the role of cavitation-enhanced permeability and a critical guide for acoustic cavitation designs.

Probing Multidimensional Mechanical Phenotyping of Intracellular Structures by Viscoelastic Spectroscopy.

Mechanical phenotyping of complex cellular structures gives insight into the process and function of mechanotransduction in biological systems. Several methods have been developed to characterize intracellular elastic moduli, while direct viscoelastic characterization of intracellular structures is still challenging. Here, we develop a needle tip viscoelastic spectroscopy method to probe multidimensional mechanical phenotyping of intracellular structures during a mini-invasive penetrating process. Viscoelastic spectroscopy is determined by magnetically driven resonant vibration (about 15 kHz) with a tiny amplitude. It not only detects the unique dynamic stiffness, damping, and loss tangent of the cell membrane-cytoskeleton and nucleus-nuclear lamina but also bridges viscoelastic parameters between the mitotic phase and interphase. Self-defined dynamic mechanical ratios of these two phases can identify two malignant cervical cancer cell lines (HeLa-HPV18+, SiHa-HPV16+) whose membrane or nucleus elastic moduli are indistinguishable. This technique provides a quantitative method for studying mechanosensation, mechanotransduction, and mechanoresponse of intracellular structures from a dynamic mechanical perspective. This technique has the potential to become a reliable quantitative measurement method for dynamic mechanical studies of intracellular structures.

Enhancing Nucleic Acid Delivery with Ultrasound and Microbubbles.

For gene therapy to work in vivo, nucleic acids need to reach the target cells without causing major side effects to the patient. In many cases the gene only has to reach a subset of cells in the body. Therefore, targeted delivery of genes to the desired tissue is a major issue in gene delivery. Many different possibilities of targeted gene delivery have been studied. A physical approach to target nucleic acids and other drugs to specific regions in the body is the use of ultrasound and microbubbles. Microbubbles are gas filled spheres with a stabilizing lipid, protein, or polymer shell. When these microbubbles enter an ultrasonic field, they start to oscillate. The bubbles' expansion and compression are inversely related to the pressure phases in the ultrasonic field. When microbubbles are exposed to high-intensity ultrasound the microbubbles will eventually implode and fragment. This generates shockwaves and microjets which can temporarily permeate cell membranes and blood vessels. Nucleic acids or (non)viral vectors can as a result gain direct access to either the cytoplasm of neighboring cells, or extravasate to the surrounding tissue. The nucleic acids can either be mixed with the microbubbles or loaded on the microbubbles. Nucleic acid loaded microbubbles can be obtained by coupling nucleic acid-containing particles (i.e., lipoplexes) to the microbubbles. Upon ultrasound-mediated implosion of the microbubbles, the nucleic acid-containing particles will be released and will deliver their nucleic acids in the ultrasound-targeted region.

Microbubbles for Nucleic Acid Delivery in Liver Using Mild Sonoporation.

Ultrasound-mediated gene delivery is an interesting approach, which could help in increasing gene transfer in deep tissues. Moreover, it allows for performing experiments guided by the image to determine which elements are required. Microbubbles complexed with a eukaryotic expression cassette are excellent agents as they are responsive to ultrasounds and, upon oscillation, can destabilize membranes to enhance gene transfer. Here, we describe the preparation of positively charged microbubbles, plasmid free of antibiotic resistance marker, their combination and the conditions of ultrasound-mediated liver transfection post-systemic administration in mice. This association allowed us to obtain a superior liver gene expression at least over 8 months after a single injection.

Enhanced Production of Hypocrellin A in Submerged Cultures of Shiraia bambusicola by Red Light.

Hypocrellin A (HA), a promising photosensitizer for anticancer photodynamic therapy (PDT), is a fungal perylenequinone pigment from the fruiting body of Shiraia bambusicola, a traditional Chinese medicine for treating skin diseases. The mycelial cultures are becoming a biotechnological alternative for HA production. In this study, light of different wavelengths was investigated to develop an effective eliciting strategy for HA production in the cultures. Under red LED light (627 nm) at 200 lux, the maximum HA production (175.53 mg L-1 ) in mycelium cultures was reached after 8 days, about 3.82-fold of the dark control. Red light not only promoted HA biosynthesis in mycelia (intracellular HA), but also stimulated HA secretion into the medium (extracellular HA). We found 14 of 310 transcripts differentially expressed under red light treatment were possible candidate genes for HA biosynthetic pathway. Gene ontology (GO) analysis revealed that red light treatment could change the gene expressions responsible for HA biosynthesis and the transmembrane activity, suggesting both intracellular HA and its secretion could contribute to the enhancement of total HA production in the cultures. The results provided new insights of red light elicitation and effective strategy for HA production in mycelium cultures.

Nitrobenzoxadiazole-Appended Cell Membrane Modifiers for Efficient Optoporation with Noncoherent Light.

FLNBD-BAMPEG2k, bearing a nitrobenzoxadiazole (NBD) unit and an oleyl terminus conjugated via a poly(ethylene glycol) (PEG) spacer ( Mn = 2,000), was designed to fluorescently label cell membranes by docking its hydrophobic oleyl terminus. During laser scanning microscopy in a minimal essential medium (MEM), human hepatocellular carcinoma Hep3B cells labeled with FLNBD-BAMPEG2k appeared to undergo optoporation at their plasma membrane. We confirmed this unprecedented possibility by a series of cellular uptake experiments using negatively charged and therefore membrane-impermeable quantum dots (QDs; Dh = 4.7 nm). Detailed studies indicated that the photoexcited NBD unit can generate singlet oxygen (1O2), which oxidizes the constituent phospholipids to transiently deteriorate the cell membrane. Reference membrane modifiers FLNBD-Oleyl and FLNBD-BAMPEG8k having shorter or longer hydrophilic spacers between the NBD and oleyl units showed a little or substantially no optoporation. For understanding these results, one must consider the following contradictory factors: (1) The photosensitized 1O2 generation efficiently occurs only when the NBD unit is in aqueous media, and (2) the lifetime of 1O2 in aqueous media is very short (3.0-3.5 µs). As supported experimentally and computationally, the hydrophilic spacer length of FLNBD-BAMPEG2k is optimal for compromising these factors. Further to note, the optoporation using FLNBD-BAMPEG2k is not accompanied by cytotoxicity.

Membrane Permeabilization of Pathogenic Yeast in Alternating Sub-microsecond Electromagnetic Fields in Combination with Conventional Electroporation.

Recently, a novel contactless treatment method based on high-power pulsed electromagnetic fields (PEMF) was proposed, which results in cell membrane permeabilization effects similar to electroporation. In this work, a new PEMF generator based on multi-stage Marx circuit topology, which is capable of delivering 3.3 T, 0.19 kV/cm sub-microsecond pulses was used to permeabilize pathogenic yeast Candida albicans separately and in combination with conventional square wave electroporation (8-17 kV/cm, 100 µs). Bursts of 10, 25, and 50 PEMF pulses were used. The yeast permeabilization rate was evaluated using flow cytometric analysis and propidium iodide (PI) assay. A statistically significant (P < 0.05) combinatorial effect of electroporation and PEMF treatment was detected. Also the PEMF treatment (3.3 T, 50 pulses) resulted in up to 21% loss of yeast viability, and a dose-dependent additive effect with pulsed electric field was observed. As expected, increase of the dB/dt and subsequently the induced electric field amplitude resulted in a detectable effect solely by PEMF, which was not achievable before for yeasts in vitro.

Cell-cycle-specific Cellular Responses to Sonoporation.

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

Important factors for cell-membrane permeabilization by gold nanoparticles activated by nanosecond-laser irradiation.

PURPOSE: Pulsed-laser irradiation of light-absorbing gold nanoparticles (AuNPs) attached to cells transiently increases cell membrane permeability for targeted molecule delivery. Here, we targeted EGFR on the ovarian carcinoma cell line OVCAR-3 with AuNPs. In order to optimize membrane permeability and to demonstrate molecule delivery into adherent OVCAR-3 cells, we systematically investigated different experimental conditions. MATERIALS AND METHODS: AuNPs (30 nm) were functionalized by conjugation of the antibody cetuximab against EGFR. Selective binding of the particles was demonstrated by silver staining, multiphoton imaging, and fluorescence-lifetime imaging. After laser irradiation, membrane permeability of OVCAR-3 cells was studied under different conditions of AuNP concentration, cell-incubation medium, and cell-AuNP incubation time. Membrane permeability and cell viability were evaluated by flow cytometry, measuring propidium iodide and fluorescein isothiocyanate-dextran uptake. RESULTS: Adherently growing OVCAR-3 cells can be effectively targeted with EGFR-AuNP. Laser irradiation led to successful permeabilization, and 150 kDa dextran was successfully delivered into cells with about 70% efficiency. CONCLUSION: Antibody-targeted and laser-irradiated AuNPs can be used to deliver molecules into adherent cells. Efficacy depends not only on laser parameters but also on AuNP:cell ratio, cell-incubation medium, and cell-AuNP incubation time.

Potential applications of low-energy shock waves in functional urology.

A shock wave, which carries energy and can propagate through a medium, is a type of continuous transmitted sonic wave with a frequency of 16 Hz-20 MHz. It is accompanied by processes involving rapid energy transformations. The energy associated with shock waves has been harnessed and used for various applications in medical science. High-energy extracorporeal shock wave therapy is the most successful application of shock waves, and has been used to disintegrate urolithiasis for 30 years. At lower energy levels, however, shock waves have enhanced expression of vascular endothelial growth factor, endothelial nitric oxide synthase, proliferating cell nuclear antigen, chemoattractant factors and recruitment of progenitor cells; shock waves have also improved tissue regeneration. Low-energy shock wave therapy has been used clinically with musculoskeletal disorders, ischemic cardiovascular disorders and erectile dysfunction, through the mechanisms of neovascularization, anti-inflammation and tissue regeneration. Furthermore, low-energy shock waves have been proposed to temporarily increase tissue permeability and facilitate intravesical drug delivery. The present review article provides information on the basics of shock wave physics, mechanisms of action on the biological system and potential applications in functional urology.

64Cu-MM-302 Positron Emission Tomography Quantifies Variability of Enhanced Permeability and Retention of Nanoparticles in Relation to Treatment Response in Patients with Metastatic Breast Cancer.

Purpose: Therapeutic nanoparticles are designed to deliver their drug payloads through enhanced permeability and retention (EPR) in solid tumors. The extent of EPR and its variability in human tumors is highly debated and has been proposed as an explanation for variable responses to therapeutic nanoparticles in clinical studies.Experimental Design: We assessed the EPR effect in patients using a 64Cu-labeled nanoparticle, 64Cu-MM-302 (64Cu-labeled HER2-targeted PEGylated liposomal doxorubicin), and imaging by PET/CT. Nineteen patients with HER2-positive metastatic breast cancer underwent 2 to 3 PET/CT scans postadministration of 64Cu-MM-302 as part of a clinical trial of MM-302 plus trastuzumab with and without cyclophosphamide (NCT01304797).Results: Significant background uptake of 64Cu-MM-302 was observed in liver and spleen. Tumor accumulation of 64Cu-MM-302 at 24 to 48 hours varied 35-fold (0.52-18.5 %ID/kg), including deposition in bone and brain lesions, and was independent of systemic plasma exposure. Computational analysis quantified rates of deposition and washout, indicating peak liposome deposition at 24 to 48 hours. Patients were classified on the basis of 64Cu-MM-302 lesion deposition using a cut-off point that is comparable with a response threshold in preclinical studies. In a retrospective exploratory analysis of patient outcomes relating to drug levels in tumor lesions, high 64Cu-MM-302 deposition was associated with more favorable treatment outcomes (HR = 0.42).Conclusions: These findings provide important evidence and quantification of the EPR effect in human metastatic tumors and support imaging nanoparticle deposition in tumors as a potential means to identify patients well suited for treatment with therapeutic nanoparticles. Clin Cancer Res; 23(15); 4190-202. ©2017 AACR.

Adaptation response of Pseudomonas fragi on refrigerated solid matrix to a moderate electric field.

BACKGROUND: Moderate electric field (MEF) technology is a promising food preservation strategy since it relies on physical properties-rather than chemical additives-to preserve solid cellular foods during storage. However, the effectiveness of long-term MEF exposure on the psychrotrophic microorganisms responsible for the food spoilage at cool temperatures remains unclear. RESULTS: The spoilage-associated psychrotroph Pseudomonas fragi MC16 was obtained from pork samples stored at 7 °C. Continuous MEF treatment attenuated growth and resulted in subsequent adaptation of M16 cultured on nutrient agar plates at 7 °C, compared to the control cultures, as determined by biomass analysis and plating procedures. Moreover, intracellular dehydrogenase activity and ATP levels also indicated an initial effect of MEF treatment followed by cellular recovery, and extracellular ß-galactosidase activity assays indicated no obvious changes in cell membrane permeability. Furthermore, microscopic observations using scanning and transmission electron microscopy revealed that MEF induced sublethal cellular injury during early treatment stages, but no notable changes in morphology or cytology on subsequent days. CONCLUSION: Our study provides direct evidence that psychrotrophic P. fragi MC16 cultured on nutrient agar plates at 7 °C are capable of adapting to MEF treatment.

Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury.

Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of VillinCre;Dclk1f/f mice were hypoplastic and more apoptotic 24 h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.

Chronic activation of the D156A point mutant of Channelrhodopsin-2 signals apoptotic cell death: the good and the bad.

Channelrhodopsin-2 (ChR2) has become a celebrated research tool and is considered a promising potential therapeutic for neurological disorders. While making its way into the clinic, concerns about the safety of chronic ChR2 activation have emerged; in particular as the high-intensity blue light illumination needed for ChR2 activation may be phototoxic. Here we set out to quantify for the first time the cytotoxic effects of chronic ChR2 activation. We studied the safety of prolonged illumination on ChR2(D156A)-expressing human melanoma cells as cancer cells are notorious for their resistance to killing. Three days of illumination eradicated the entire ChR2(D156A)-expressing cell population through mitochondria-mediated apoptosis, whereas blue light activation of non-expressing control cells did not significantly compromise cell viability. In other words, chronic high-intensity blue light illumination alone is not phototoxic, but prolonged ChR2 activation induces mitochondria-mediated apoptosis. The results are alarming for gain-of-function translational neurological studies but open the possibility to optogenetically manipulate the viability of non-excitable cells, such as cancer cells. In a second set of experiments we therefore evaluated the feasibility to put melanoma cell proliferation and apoptosis under the control of light by transdermally illuminating in vivo melanoma xenografts expressing ChR2(D156A). We show clear proof of principle that light treatment inhibits and even reverses tumor growth, rendering ChR2s potential tools for targeted light-therapy of cancers.

Biophysical insight into mechanisms of sonoporation.

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