Performance evaluation of ultrasonic transducer in a mist bioreactor by different nutrient media.

The mist bioreactor has been proved to have a higher performance relative to the liquid phase reactor. The water and conventional media (MS, ½MS, and B5) were tested in a mist bioreactor to evaluate the performance of the misting system. The physicochemical properties of liquids were measured at 0 (Fresh state) and when treated by ultrasonic system for 1 and 2 h. At a given airflow rate, the misting rate for different heights of various media was determined. The results demonstrated that the electrical conductivity of all media increased with the duration of sonication. The ultrasonic effect caused an increase in surface tension of MS and ½MS, while the pH of MS and ½MS reduced with ultrasonic treatment. The inverse trends were observed for water and B5 for these properties. The misting rate increased by increasing liquid height to a maximum value, then decreased to a minimum value at the highest height. For all liquids studied in this experiment, this maximum value of misting performed at height ranged between 3 and 4 cm. The maximum and minimum values of the ultrasonic misting rate belonged to MS ([Formula: see text]) at height 3 cm and B5 ([Formula: see text]) at height 1 cm, respectively.

Optimized sonoreactor for accelerative amyloid-fibril assays through enhancement of primary nucleation and fragmentation.

Ultrasonication to supersaturated protein solutions forcibly forms amyloid fibrils, thereby allowing the early-stage diagnosis for amyloidoses. Previously, we constructed a high-throughput sonoreactor to investigate features of the amyloid-fibril nucleation. Although the instrument substantiated the ultrasonication efficacy, several challenges remain; the key is the precise control of the acoustic field in the reactor, which directly affects the fibril-formation reaction. In the present study, we develop the optimized sonoreactor for the amyloid-fibril assay, which improves the reproducibility and controllability of the fibril formation. Using ß2-microglobulin, we experimentally demonstrate that achieving identical acoustic conditions by controlling oscillation amplitude and frequency of each transducer results in identical fibril-formation behavior across 36 solutions. Moreover, we succeed in detecting the 100-fM seeds using the developed sonoreactor at an accelerated rate. Finally, we reveal that the acceleration of the fibril-formation reaction with the seeds is achieved by enhancing the primary nucleation and the fibril fragmentation through the analysis of the fibril-formation kinetics. These results demonstrate the efficacy of the developed sonoreactor for the diagnosis of amyloidoses owing to the accelerative seed detection and the possibility for further early-stage diagnosis even without seeds through the accelerated primary nucleation.

Development of ultrasound-assisted mixture extraction and online extraction solution concentration coupled with countercurrent chromatography for the preparation of pure phytochemicals from Phellinus vaninii.

Herein, ultrasound-assisted mixture extraction (UAME) and online extraction solution concentration (OESC) were conducted to extract products from crops and plants. These techniques were coupled with parallel countercurrent chromatography (PCCC) and applied for continuous extraction and online isolation of chemical constituents from Phellinus vaninii. The UAME instrument comprises extraction and solution separation chambers. It provides higher extraction efficiency and fewer impurities and is suitable for processing various sample matrices. The OESC device comprises a spray nozzle, concentrating cylinder, and hot-blast air nozzle. The mechanical parameters for UAME and OESC were optimized, and the operation of online UAME and OESC coupled with PCCC was described. Raw plant materials were extracted using a two-phase extractant comprising petroleum-ethyl acetate-ethanol-water (0.5:2.0:0.5:2.0, v/v/v/v). The aqueous and organic phases were then concentrated using the OESC technique. Two CCC runs were conducted for preparatory work. After extraction and online concentration, the concentrate was pumped into the CCC for separation. During PCCC separation, continuous automated extraction and concentration were still conducted. When the first cycle of the UAME/OESC/PCCC was completed, followed by the initiation of the second cycle, and the process was continued. Six target compounds with purities exceeding 97.22% were successfully separated using the CCC solvent systems comprising n-hexane-ethyl acetate-acetonitrile-water (5.5:2.5:5.0:0.4, v/v/v/v) and n-butanol-ethanol-water (4.5:1.3:6.5, v/v/v). Compared with conventional extraction methods, the proposed UAME/OESC/PCCC method has higher efficiency, facilitates high-purity separation of analytes, and offers opportunity for automation and systematic preparation of natural products.

Assessment of the olive oil extraction plant layout implementing a high-power ultrasound machine.

The objective of this study is to assess the effects of installation and operation of a high-power ultrasound machine (HPU) for the treatment of olive paste by using ultrasound technology in order to evaluate the best way installation and the best definition of the operating conditions of the machine. The study was conducted installing in an industrial olive oil mill a continuous processing ultrasound machine, which used a frequency of 20 kHz able to work at 3200 kg h-1 as feed capacity. Checking of performance has been carried out by the assessment of the different operating and process conditions, assessing in particular the impact of the ultrasound treatment before and after the malaxation phase on performance indicators of the continuous olive oil plant (plant extractability, olive paste rheological characteristic) and on selected chemical properties of the olive oil extracted (quality parameters, antioxidant content, and volatile profiles). In the tested conditions, high-power ultrasound treatment did not produce significant effect on the legal parameters (free acidity, peroxide index and spectrophotometric indexes), while a significant increase in the content of phenolic compounds was generally observed; higher enhancements were more evident when the high-power ultrasound treatment was carried out before the malaxation phase.

Preparation of Cell Extracts for Purification of Soluble Proteins Expressed in E. coli.

Recovery of intracellular proteins requires disruption of the host cell before the target protein is extracted and isolated. For cells enveloped in cell walls (such as Escherichia coli), vigorous methods are often required. This protocol focuses on E. coli lysis by sonication. Also included are methods for lysis by freeze-thaw and enzymatic treatments.

Mechanical/Physical Methods of Cell Disruption and Tissue Homogenization.

This chapter covers the various methods of mechanical cell disruption and tissue homogenization that are currently commercially available for processing small samples s < 1 mL) to larger multikilogram production quantities. These mechanical methods of lysing do not introduce chemicals or enzymes to the system. However, the energies required when using these "harsh," high mechanical energy methods can be enough to damage the very components being sought.The destruction of cell membranes and walls is effected by subjecting the cells (a) to shearing by liquid flow, (b) to exploding by pressure differences between inside and outside of cell, (c) to collision forces by impact of beads or paddles, or (d) a combination of these forces.Practical suggestions to optimize each method, where to acquire such equipment, and links to reference sources are included. Several novel technologies are presented.

Enhanced Radiosensitization for Cancer Treatment with Gold Nanoparticles through Sonoporation.

We demonstrate the megavoltage (MV) radiosensitization of a human liver cancer line by combining gold-nanoparticle-encapsulated microbubbles (AuMBs) with ultrasound. Microbubbles-mediated sonoporation was administered for 5 min, at 2 h prior to applying radiotherapy. The intracellular concentration of gold nanoparticles (AuNPs) increased with the inertial cavitation of AuMBs in a dose-dependent manner. A higher inertial cavitation dose was also associated with more DNA damage, higher levels of apoptosis markers, and inferior cell surviving fractions after MV X-ray irradiation. The dose-modifying ratio in a clonogenic assay was 1.56 ± 0.45 for a 10% surviving fraction. In a xenograft mouse model, combining vascular endothelial growth factor receptor 2 (VEGFR2)-targeted AuMBs with sonoporation significantly delayed tumor regrowth. A strategy involving the spatially and temporally controlled release of AuNPs followed by clinically utilized MV irradiation shows promising results that make it worthy of further translational investigations.

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis.

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

Blood-brain barrier disruption and delivery of irinotecan in a rat model using a clinical transcranial MRI-guided focused ultrasound system.

We investigated controlled blood-brain barrier (BBB) disruption using a low-frequency clinical transcranial MRI-guided focused ultrasound (TcMRgFUS) device and evaluated enhanced delivery of irinotecan chemotherapy to the brain and a rat glioma model. Animals received three weekly sessions of FUS, FUS and 10 mg/kg irinotecan, or irinotecan alone. In each session, four volumetric sonications targeted 36 locations in one hemisphere. With feedback control based on recordings of acoustic emissions, 98% of the sonication targets (1045/1071) reached a pre-defined level of acoustic emission, while the probability of wideband emission (a signature for inertial cavitation) was than 1%. BBB disruption, evaluated by mapping the R1 relaxation rate after administration of an MRI contrast agent, was significantly higher in the sonicated hemisphere (P < 0.01). Histological evaluation found minimal tissue effects. Irinotecan concentrations in the brain were significantly higher (P < 0.001) with BBB disruption, but SN-38 was only detected in <50% of the samples and only with an excessive irinotecan dose. Irinotecan with BBB disruption did not impede tumor growth or increase survival. Overall these results demonstrate safe and controlled BBB disruption with a low-frequency clinical TcMRgFUS device. While irinotecan delivery to the brain was not neurotoxic, it did not improve outcomes in the F98 glioma model.

The current binomial Sonochemistry-Analytical Chemistry.

Although around 90 years have passed since Richards and Loomis developed the first experiments in sonochemistry, ultrasound is still awakening interest in the scientific community, in particular in the Analytical Chemistry field, as a result of the high number of benefits achieved, which are also in accordance with Green Analytical Chemistry principles. In the last years, and among the different reported applications, an important number of works have arisen devoted to the synthesis of new materials (specially nanomaterials), to the development of sonoelectroanalytical sensors or to new spectroscopic approaches, among others. Efforts are also being made to try to understand the real mechanism of such applications. This review article is aimed at providing a general overview of the different applications of ultrasound in Analytical Chemistry, also briefly highlighting its fundamentals and traditional applications, with a special emphasis on the most recent and challenging works that are still in the horizon of its near future.

Microfluidic Sonication To Assemble Exosome Membrane-Coated Nanoparticles for Immune Evasion-Mediated Targeting.

Using natural membranes to coat nanoparticles (NPs) provides an efficient means to reduce the immune clearance of NPs and improve their tumor-specific targeting. However, fabrication of these drug-loaded biomimetic NPs, such as exosome membrane (EM)- or cancer cell membrane (CCM)-coated poly(lactic-co-glycolic acid) (PLGA) NPs, remains a challenging task owing to the heterogeneous nature of biomembranes and labor-intensive procedures. Herein, we report a microfluidic sonication approach to produce EM-, CCM-, and lipid-coated PLGA NPs encapsulated with imaging agents in a one-step and straightforward manner. Tumor cell-derived EM-coated PLGA NPs consisting of both endosomal and plasma membrane proteins show superior homotypic targeting as compared to CCM-PLGA NPs of similar sizes and core-shell structures in both in vitro and in vivo models. The underlying mechanism is associated with a significantly reduced uptake of EM-PLGA NPs by macrophages and peripheral blood monocytes, revealing an immune evasion-mediated targeting of EM-PLGA NPs to homologous tumors. Overall, this work illustrates the promise of using microfluidic sonication approach to fabricate biomimetic NPs for better biocompatibility and targeting efficacy.

Cellular Bioeffect Investigations on Low-Intensity Pulsed Ultrasound and Sonoporation: Platform Design and Flow Cytometry Protocol.

At low-intensity levels, ultrasound can potentially generate therapeutic effects on living cells, and it can trigger sonoporation when microbubbles (MBs) are present to facilitate drug delivery. Yet, our foundational knowledge of low-intensity pulsed ultrasound (LIPUS) and sonoporation remains to be critically weak because the pertinent cellular bioeffects have not been rigorously studied. In this article, we present a population-based experimental protocol that can effectively foster investigations on the mechanistic bioeffects of LIPUS and sonoporation over a cell population. Walkthroughs of different methodological details are presented, including the fabrication of the ultrasound exposure platform and its calibration, as well as the design of a bioassay procedure that uses fluorescent tracers and flow cytometry to isolate sonicated cells with similar characteristics. An application example is also presented to illustrate how our protocol can be used to investigate the downstream cellular bioeffects of leukemia cells. We show that, with 1-MHz LIPUS exposure (with 29.1 J/cm2 delivered acoustic energy density), variations in viability and morphology would be found among different types of sonicated leukemia cells (HL-60, Molt-4) in the absence and presence of MBs. Taken altogether, this article provides a reference on how cellular bioeffect experiments on LIPUS and sonoporation can be planned meticulously to acquire strong observations that are critical to establish the biological foundations for therapeutic applications.

Influence of Manothermosonication on the Physicochemical and Functional Properties of Ferritin as a Nanocarrier of Iron or Bioactive Compounds.

Ferritin is a multisubunit protein with a hollow interior interface and modifiable surfaces. In this study, the manothermosonication (MTS) technology was applied to apo-red bean seed ferritin (apoRBF) to produce the MTS-treated apoRBF (MTFS). MTS treatment (200 kPa, 50 °C, and 40 s) maintained the spherical morphology of apoRBF (12 nm), but reduced the content of α-helix structure and increased the content of random coil structure, and correspondingly decreased the ferritin stability. The MTS treatment also affected the ferritin's iron storage function by decreasing its iron oxidative deposition activity and increasing the iron release activity. Importantly, the disassembly and reassembly properties of the MTFS induced by pH changes were retained, which facilitated its usage in encapsulation of tea polyphenol-epigallocatechin gallate (EGCG) into the ferritin by a relatively benign pH conversion routine (pH 3.0/6.8). In addition, the water solubility of the MTFS was increased, leading to the improved encapsulation efficiency of the EGCG molecules. This study will facilitate the ferritin modification and functionalization by MTS to design a protein variant to be used as new scaffold for iron and bioactive compounds.

The Influence of an Ultrasonic Cleaning Protocol for CAD/CAM Abutment Surfaces on Cell Viability and Inflammatory Response In Vitro.

BACKGROUND/AIM: To evaluate the effect of an ultrasonic cleaning and disinfection method for CAD/CAM abutment surfaces on cell viability and inflammatory response in vitro. MATERIALS AND METHODS: Untreated and manually polished surfaces of CAD/CAM generated titanium and zirconia disks were randomly assigned, either to a 3-step ultrasonic cleaning and disinfection process (test: TiUF, TiPF, ZrUF, ZrPF) or to 30 sec steam cleaning (control: TiUS, TiPS, ZrUS, ZrPS). Pre-cleaning surface analyses using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and surface profilometry were performed. Human gingival fibroblasts (HGFs) were cultured on test and control specimens and subsequently examined for cell viability and inflammatory response. Expression of acute inflammatory cytokine interleukin (IL)-6 and vascular endothelial growth factor A (VEGFA) were assessed by means of RT-qPCR. RESULTS: Cells on all specimens exhibited a satisfactory viability, indicating firm attachment. Cells on polished zirconia samples, cleaned by means of sonication (ZrPF), exhibited significantly higher viability than cells on the same material cleaned by steam (ZrPS), p=0.019. For all other three material/ surface treatment combinations (TiU, TiP, ZrU), no such difference was observed between the cleaning methods. The messenger ribonucleic acid (mRNA) levels of IL-6 and VEGFA were between 50 and 105% of that of the control cells on the non-toxic control surface. mRNA levels of IL-6 and VEGFA correlated well with each other. CONCLUSION: Except for higher viability of cells cultured on polished zirconia specimens, no universally applicable advantage could be found for the ultrasonic cleaning procedure for zirconia and titanium abutment surfaces regarding cell viability, IL-6 expression or VEGFA expression. The cleaning procedures did not have any negative effect either.

Preparation, Characterization, and In Vitro Evaluation of Lipidoid-Polymer Hybrid Nanoparticles for siRNA Delivery to the Cytosol.

RNA interference (RNAi) therapeutics are one of the most promising biological interventions in the efficient management of difficult-to-treat diseases. RNAi is mediated by small interfering RNA (siRNA), which induces specific and highly potent gene silencing. However, intracellular delivery of exogenous, chemically synthesized siRNA to the RNAi pathway in the cytosol remains a challenge, and is fully dependent on technologies that can facilitate cytosolic delivery without undesired side effects. One example is a novel delivery system referred to as lipidoid-polymer hybrid nanoparticles (LPNs), which we recently showed mediates highly efficient and safe gene silencing. Here we describe a double emulsion solvent evaporation method for the preparation of siRNA-loaded LPNs and methodologies employed for their physicochemical characterization and biological performance. A solution of siRNA in aqueous buffer is emulsified by sonication with an organic phase containing lipid and polymer into a primary emulsion. Subsequently, the primary emulsion is emulsified with a secondary water phase containing polyvinyl alcohol by sonication, and the organic phase is evaporated, eventually resulting in LPNs. The physicochemical characterization includes determination of (1) hydrodynamic particle size distribution, (2) zeta potential, (3) siRNA encapsulation efficiency, and (4) practical siRNA loading. The transfection experiments are conducted in a cell-based model system using enhanced green fluorescence protein as reporter. The gene silencing effect is also confirmed at the mRNA level by reverse transcription polymerase chain reaction (RT-PCR). The effect of the siRNA-loaded LPNs on cell viability is measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Low-energy shock waves evoke intracellular Ca2+ increases independently of sonoporation.

Low-energy shock waves (LESWs) accelerate the healing of a broad range of tissue injuries, including angiogenesis and bone fractures. In cells, LESW irradiations enhance gene expression and protein synthesis. One probable mechanism underlying the enhancements is mechanosensing. Shock waves also can induce sonoporation. Thus, sonoporation is another probable mechanism underlying the enhancements. It remains elusive whether LESWs require sonoporation to evoke cellular responses. An intracellular Ca2+ increase was evoked with LESW irradiations in endothelial cells. The minimum acoustic energy required for sufficient evocation was 1.7 µJ/mm2. With the same acoustic energy, sonoporation, by which calcein and propidium iodide would become permeated, was not observed. It was found that intracellular Ca2+ increases evoked by LESW irradiations do not require sonoporation. In the intracellular Ca2+ increase, actin cytoskeletons and stretch-activated Ca2+ channels were involved; however, microtubules were not. In addition, with Ca2+ influx through the Ca2+ channels, the Ca2+ release through the PLC-IP3-IP3R cascade contributed to the intracellular Ca2+ increase. These results demonstrate that LESW irradiations can evoke cellular responses independently of sonoporation. Rather, LESW irradiations evoke cellular responses through mechanosensing.

Rapid Short-pulse Ultrasound Delivers Drugs Uniformly across the Murine Blood-Brain Barrier with Negligible Disruption.

Background Previous work has demonstrated that drugs can be delivered across the blood-brain barrier by exposing circulating microbubbles to a sequence of long ultrasound pulses. Although this sequence has successfully delivered drugs to the brain, concerns remain regarding potentially harmful effects from disrupting the brain vasculature. Purpose To determine whether a low-energy, rapid, short-pulse ultrasound sequence can efficiently and safely deliver drugs to the murine brain. Materials and Methods Twenty-eight female wild-type mice underwent focused ultrasound treatment after injections of microbubbles and a labeled model drug, while three control mice were not treated (May-November 2017). The left hippocampus of 14 mice was exposed to low-energy short pulses (1 MHz; five cycles; peak negative pressure, 0.35 MPa) of ultrasound emitted at a rapid rate (1.25 kHz) in bursts (0.5 Hz), and another 14 mice were exposed to standard long pulses (10 msec, 0.5 Hz) containing 150 times more acoustic energy. Mice were humanely killed at 0 (n = 5), 10 (n = 3), or 20 minutes (n = 3) after ultrasound treatment. Hematoxylin-eosin (H-E) staining was performed on three mice. The delivered drug dose and distribution were quantified with the normalized optical density and coefficient of variation. Safety was assessed by H-E staining, the amount of albumin released, and the duration of permeability change in the blood-brain barrier. Statistical analysis was performed by using the Student t test. Results The rapid short-pulse sequence delivered drugs uniformly throughout the parenchyma. The acoustic energy emitted from the microbubbles also predicted the delivered dose (r = 0.97). Disruption in the blood-brain barrier lasted less than 10 minutes and 3.4-fold less albumin was released into the brain than with long pulses. No vascular or tissue damage from rapid short-pulse exposure was observable using H-E staining. Conclusion The rapid short-pulse ultrasound sequence is a minimally disruptive and efficient drug delivery method that could improve the treatment, diagnosis, and study of neurologic diseases. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Klibanov and McDannold in this issue.

Assessment of a low-frequency ultrasound device on prevention of biofilm formation and carbonate deposition in drinking water systems.

A device generating low-frequency and low-intensity ultrasound waves was used for mitigating biofilm accumulation and scaling. Two systems were tested: a lab-scale plate heat exchanger operated with continuously recycled water and a continually fed flow-through drinking water pilot used for mimicking water circulation in pipes. Initial deposition of bacterial cells was not prevented by ultrasound wave treatment. However, whatever the tested system, both further calcium carbonate deposition and biofilm growth were markedly inhibited. Biofilms formed in reactors subjected to low-frequency and low-intensity ultrasound waves were weakly attached to the material. Even though the activity of bacteria was affected as shown by their lower cultivability, membrane permeability did not appear compromised. Ultrasound technology sounds very promising in both the mitigation of drinking water biofilm and carbonate accumulation.

Efficacy of sonic and ultrasonic irrigation devices in the removal of debris from canal irregularities in artificial root canals.

OBJECTIVE: To evaluate the efficacy of different sonic and ultrasonic devices in the elimination of debris from canal irregularities in artificial root canals. MATERIALS AND METHODS: A resin model of a transparent radicular canal filled with dentin debris was used. Five groups were tested, namely: Group 1 - ultrasonic insert 15.02; Group 2 - ultrasonic insert 25/25 IRRI K; Group 3 - ultrasonic insert 25/25 IRRI S; Group 4 - sonic insert 20/28 Eddy on a vibrating sonic air-scaler handpiece; Group 5 - 20.02 K-file inserted on a Safety M4 handpiece. Two different irrigants (5% sodium hypochlorite and 17% EDTA) and 3 different times of activation (20, 40, and 60 seconds) were tested. Means and standard deviations were calculated and statistically analyzed with the Kruskal-Wallis and Wilcoxon tests (p<0.05). RESULTS: No statistically significant differences were found between the two irrigants used. Group 4 removed more debris than the other groups (p<0.05). Groups 1, 2, and 3 removed more debris than group 5 (p<0.05). A statistically significant difference (p<0.05) was found for the time of activation in all groups and at all canal levels, except between 40 and 60 seconds in group 4 at coronal and middle third level (p>0.05). CONCLUSIONS: No significant differences were found between 5% sodium hypochlorite and 17% EDTA. When the time of activation rises, the dentin debris removal increases in all groups. Both sonic and ultrasonic activation demonstrate high capacity for dentin debris removal.

A green approach for alginate extraction from Sargassum muticum brown seaweed using ultrasound-assisted technique.

Ultrasound assisted aqueous extraction of alginate from Sargassum muticum was proposed to minimize the use of chemicals, high temperatures and prolonged times, with comparable extraction yields to conventional acid/alkali procedures. The alginate, precipitated from the liquors obtained after ultrasound assisted extraction of fucoidan and phlorotannin fractions, and converted to alginic acid sodium salt by a green treatment was characterized by FTIR-ATR, 1H NMR, HPSEC, MALDI-TOF, rheology and citotoxitity. A clear influence of the sonication time was observed on the alginate molar mass, block structure, thermo-rheological and tumoral cell growth inhibition features. All tested hydrogels featured stable and thermo-reversible characteristics.