Preliminary Exploration of the Biophysical Mechanisms of Pulsed Magnetic Field- Induced Cell Permeabilization.

Pulsed magnetic field treatment can enhance cell membrane permeability, allowing large molecular substances that normally cannot pass through the cell membrane to enter the cell. This research holds significant prospects for biomedical applications. However, the mechanism underlying pulsed magnetic field-induced cell permeabilization remains unclear, impeding further progress in research related to pulsed magnetic field. Currently, hypotheses about the mechanism are struggling to explain experimental results. Therefore, this study developed a parameter-adjustable pulsed magnetic field generator and designed experiments. Starting from the widely accepted hypothesis of "induced electric fields by pulsed magnetic field," we conducted a preliminary exploration of the biophysical mechanisms underlying pulsed magnetic field-induced cell permeabilization. Finally, we have arrived at an intriguing conclusion: under the current technical parameters, the impact of the pulsed magnetic field itself is the primary factor influencing changes in cell membrane permeability, rather than the induced electric field. This conclusion holds significant implications for understanding the biophysical mechanisms behind pulsed magnetic field therapy and its potential biomedical applications.

On the molecular mechanisms implicated in the bipolar cancellation of membrane electroporation.

Bipolar cancellation is the phenomenon in which the permeability of cell membranes subjected to high intensity short pulsed electric field (ns-µs range) is reduced or eliminated when the system is subjected to bipolar instead of monopolar pulses. Although several studies have tried to explain bipolar cancellation, the underlying mechanisms remain unclear. Very few articles study bipolar cancellation by means of molecular dynamics (MD) simulation. In this paper, we investigated the molecular mechanisms underlying the difference in electroporation induced by bipolar and monopolar picosecond electric pulses (EPs) using MD simulation. The electric field gradients and electric forces on water molecules of the two pulses were analyzed in detail for the first time. For a certain pulse width, when the field intensity is relatively small, the direction of bipolar electric force on the interfacial water molecule reverses as the bipolar EPs reverse, while the electric force on interfacial water molecules of the cathode side remains in the same direction as that of applied monopolar EPs. The bipolar electric force reversal delays the water protrusion and increases the pore formation time. Therefore, this phenomenon could correspond to bipolar cancellation. When the field intensity is relatively large, although the bipolar electric force direction still reverses, half of the total time of the monopolar EPs has no electric fields. The electric forces of monopolar no-field half-cycles are much smaller than those of the bipolar EPs. Therefore, the pore formation time of bipolar EPs reduces, and this phenomenon is called bipolar enhancement. The occurrence of bipolar cancellation or bipolar enhancement depends on conditions such as the width and intensity of the pulse.

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.

Enhanced Sensitivity of Salmonella to Antimicrobial Blue Light Caused by Inactivating rfaC Gene Involved in Lipopolysaccharide Biosynthesis.

Salmonella is a global foodborne pathogen that causes human diseases ranging from mild gastroenteritis to severe systemic infections. Recently, antimicrobial blue light (aBL) showed effective bactericidal activity against a variety of bacteria (e.g., Salmonella) with varying efficiency. However, the antimicrobial mechanism of aBL has not been fully elucidated. Our previous report showed that the outer membrane (OM) is a key target of aBL. The major component of the OM, lipopolysaccharide (LPS), may play a role in aBL bactericidal effect. Therefore, the influence of LPS truncation on the sensitivity of Salmonella Typhimurium SL1344 to aBL was investigated for the first time. First, the rfaC gene in the SL1344 strain likely involved in linking lipid A to the core region of LPS was inactivated and the influence on LPS structure was verified in the mutant strain SL1344ΔrfaC. SL1344ΔrfaC showed a significant increase in sensitivity to aBL, and the bactericidal efficiency exceeded 8 log CFU at an aBL dose of 383 J/cm2, while that of its parental SL1344 strain approached 4 log CFU. To discover the possible mechanism of higher sensitivity, the permeability of OM was determined. Compared to SL1344, SL1344ΔrfaC showed 2.7-fold higher permeability of the OM at 20 J/cm2, this may explain the higher vulnerability of the OM to aBL. Furthermore, the fatty acid profile was analyzed to reveal the detailed changes in the OM and inner membrane of the mutant. Results showed that the membrane lipids of SL1344ΔrfaC were markedly different to SL1344, indicating that change in fatty acid profile might mediate the enhancement of OM permeability and the increased sensitivity to aBL in SL1344ΔrfaC. Hence, we concluded that disruption of rfaC in Salmonella Typhimurium led to the formation of truncated LPS and thus enhanced the permeability of the OM, which contributed to the increased sensitivity to aBL.

The Effect of an Anthropogenic Magnetic Field on the Early Developmental Stages of Fishes-A Review.

The number of sources of anthropogenic magnetic and electromagnetic fields generated by various underwater facilities, industrial equipment, and transferring devices in aquatic environment is increasing. These have an effect on an array of fish life processes, but especially the early developmental stages. The magnitude of these effects depends on field strength and time of exposure and is species-specific. We review studies on the effect of magnetic fields on the course of embryogenesis, with special reference to survival, the size of the embryos, embryonic motor function, changes in pigment cells, respiration hatching, and directional reactions. We also describe the effect of magnetic fields on sperm motility and egg activation. Magnetic fields can exert positive effects, as in the case of the considerable extension of sperm capability of activation, or have a negative influence in the form of a disturbance in heart rate or developmental instability in inner ear organs.

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.

Transendothelial Perforations and the Sphere of Influence of Single-Site Sonoporation.

Acoustically driven gas bubble cavitation locally concentrates energy and can result in physical phenomena including sonoluminescence and erosion. In biomedicine, ultrasound-driven microbubbles transiently increase plasma membrane permeability (sonoporation) to promote drug/gene delivery. Despite its potential, little is known about cellular response in the aftermath of sonoporation. In the work described here, using a live-cell approach, we assessed the real-time interplay between transendothelial perforations (∼30-60 s) up to 650 µm2, calcium influx, breaching of the local cytoskeleton and sonoporation resealing upon F-actin recruitment to the perforation site (∼5-10 min). Through biophysical modeling, we established the critical role of membrane line tension in perforation resealing velocity (10-30 nm/s). Membrane budding/shedding post-sonoporation was observed on complete perforation closure, yet successful pore repair does not mark the end of sonoporation: protracted cell mobility from 8 µs of ultrasound is observed up to 4 h post-treatment. Taken holistically, we established the biophysical context of endothelial sonoporation repair with application in drug/gene delivery.

Impact of pulsed electric fields and mechanical compressions on the permeability and structure of Chlamydomonas reinhardtii cells.

Current research findings clearly reveal the role of the microalga's cell wall as a key obstacle to an efficient and optimal compound extraction. Such extraction process is therefore closely related to the microalga species used. Effects of electrical or mechanical constraints on C. reinhardtii's structure and particularly its cell wall and membrane, is therefore investigated in this paper using a combination of microscopic tools. Membrane pores with a radius between 0.77 and 1.59 nm were determined for both reversible (5 kV∙cm-1) and irreversible (7 kV∙cm-1) electroporation with a 5 µs pulse duration. Irreversible electroporation with longer pulses (10 µs) lead to the entry of large molecules (at least 5.11 nm). Additionally, for the first time, the effect of pulsed electric fields on the cell wall was observed. The combined electrical and mechanical treatment showed a significant impact on the cell wall structure as observed under Transmission Electron Microscopy. This treatment permits the penetration of larger molecules (at least 5.11 nm) within the cell, shown by tracking the penetration of dextran molecules. For the first time, the size of pores on the cell membrane and the structural changes on the microalgae cell wall induced by electrical and mechanical treatments is reported.

Role of ultrasound in assisted fermentation technologies for process enhancements.

Biological molecules are widely produced by fermentation technology using bacteria, fungi or yeast. Fermentation is a biochemical process wherein the rate of bioconversion is governed by the organisms involved. The growth of the organism is mainly limited by mass transfer rates of nutrients and gases that directly affect the product formation in fermentation. Attempts have been made to enhance the growth rate and yield using mutational, recombinant strain development approach at microbial level as well as fed batch and continuous processing approach at bioprocess level in the past. The growth rate of microbes can be accelerated by increased mass transfer rates and cell wall permeability with the use of controlled low frequency ultrasound irradiation. The present review provides insights into the application of acoustic cavitation in process intensification of fermentation approaches and the role of various factors involved are highlighted with typical examples.

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.

Detection of intracellular biomarkers in viable cells using millisecond pulsed electric fields.

Reversible electroporation is a temporary permeabilization of cell membrane through the formation of transient pores created by short high voltage electric pulses. This method has numerous applications in biology and biotechnology and has become an important technique in molecular medicine. Reversible electroporation is usually used to transfer macromolecules into the cells. However, the delivery of large molecules such as proteins into cells without loss of cell viability remains a challenge. In our study, we investigated whether electroporation can be used for this purpose. The study was performed with the primary mouse splenocytes and Jurkat cell line. The electroporation efficacy was evaluated by flow cytometry. We used the reversible electroporation for intracellular marker detection investigating antibody and fluorescein-conjugated dextran transfer efficiency, cell viability and metabolic activity. We have found that reversible electroporation parameters can be optimized for efficient transfer of large molecules such as antibodies/proteins into live cells without a significant loss of cell viability. We conclude that a well-established and relatively easy method of reversible electroporation can be adjusted to detect intracellular biomarkers in viable cells. This is a new approach on how electroporation could be utilised in medicine and biological research to detect rare subpopulations of cells that produce specific markers and to keep cells viable. This would allow the use of these rare subpopulations of isolated cells for further research and personalized medicine.

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.

Stimulation or Cancellation of Ca2+ Influx by Bipolar Nanosecond Pulsed Electric Fields in Adrenal Chromaffin Cells Can Be Achieved by Tuning Pulse Waveform.

Exposing adrenal chromaffin cells to single 150 to 400 ns electric pulses triggers a rise in intracellular Ca2+ ([Ca2+]i) that is due to Ca2+ influx through voltage-gated Ca2+ channels (VGCC) and plasma membrane electropores. Immediate delivery of a second pulse of the opposite polarity in which the duration and amplitude were the same as the first pulse (a symmetrical bipolar pulse) or greater than the first pulse (an asymmetrical bipolar pulse) had a stimulatory effect, evoking larger Ca2+ responses than the corresponding unipolar pulse. Progressively decreasing the amplitude of the opposite polarity pulse while also increasing its duration converted stimulation to attenuation, which reached a maximum of 43% when the positive phase was 150 ns at 3.1 kV/cm, and the negative phase was 800 ns at 0.2 kV/cm. When VGCCs were blocked, Ca2+ responses evoked by asymmetrical and even symmetrical bipolar pulses were significantly reduced relative to those evoked by the corresponding unipolar pulse under the same conditions, indicating that attenuation involved mainly the portion of Ca2+ influx attributable to membrane electropermeabilization. Thus, by tuning the shape of the bipolar pulse, Ca2+ entry into chromaffin cells through electropores could be attenuated while preserving Ca2+ influx through VGCCs.

Untargeted metabolomics unveil alterations of biomembranes permeability in human HaCaT keratinocytes upon 60 GHz millimeter-wave exposure.

A joint metabolomic and lipidomic workflow is used to account for a potential effect of millimeter waves (MMW) around 60 GHz on biological tissues. For this purpose, HaCaT human keratinocytes were exposed at 60.4 GHz with an incident power density of 20 mW/cm², this value corresponding to the upper local exposure limit for general public in the context of a wide scale deployment of MMW technologies and devices. After a 24h-exposure, endo- and extracellular extracts were recovered to be submitted to an integrative UPLC-Q-Exactive metabolomic and lipidomic workflow. R-XCMS data processing and subsequent statistical treatment led to emphasize a limited number of altered features in lipidomic sequences and in intracellular metabolomic analyses, whatever the ionization mode (i.e 0 to 6 dysregulated features). Conversely, important dysregulations could be reported in extracellular metabolomic profiles with 111 and 99 frames being altered upon MMW exposure in positive and negative polarities, respectively. This unexpected extent of modifications can hardly stem from the mild changes that could be reported throughout transcriptomics studies, leading us to hypothesize that MMW might alter the permeability of cell membranes, as reported elsewhere.

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.

Lipid Unsaturation Properties Govern the Sensitivity of Membranes to Photoinduced Oxidative Stress.

Unsaturated lipid oxidation is a fundamental process involved in different aspects of cellular bioenergetics; dysregulation of lipid oxidation is often associated with cell aging and death. To study how lipid oxidation affects membrane biophysics, we used a chlorin photosensitizer to oxidize vesicles of various lipid compositions and degrees of unsaturation in a controlled manner. We observed different shape transitions that can be interpreted as an increase in the area of the targeted membrane followed by a decrease. These area modifications induced by the chemical modification of the membrane upon oxidation were followed in situ by Raman tweezers microspectroscopy. We found that the membrane area increase corresponds to the lipids' peroxidation and is initiated by the delocalization of the targeted double bonds in the tails of the lipids. The subsequent decrease of membrane area can be explained by the formation of cleaved secondary products. As a result of these area changes, we observe vesicle permeabilization after a time lag that is characterized in relation with the level of unsaturation. The evolution of photosensitized vesicle radius was measured and yields an estimation of the mechanical changes of the membrane over oxidation time. The membrane is both weakened and permeabilized by the oxidation. Interestingly, the effect of unsaturation level on the dynamics of vesicles undergoing photooxidation is not trivial and thus carefully discussed. Our findings shed light on the fundamental dynamic mechanisms underlying the oxidation of lipid membranes and highlight the role of unsaturations on their physical and chemical properties.