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.

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.

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.

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.

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.

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.

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.

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

Abstract 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.

Enhanced drug delivery using sonoactivatable liposomes with membrane-embedded porphyrins.

Small molecules that interfere with nucleic acid are widely used in chemotherapy, however, improved delivery approaches are required to improve anti-tumor outcomes. Here, we present the development of an ultrasound-activatable porphyrin-phospholipid-liposome (pp-lipo) that responds to low intensity focused ultrasound (LIFU) for sonodynamic therapy (SDT). The pp-lipo is constructed by incorporating a small proportion of porphyrin (pyropheophorbide) conjugated lipid into a liposome formulation. This enables sonosensitization-induced lipid oxidation and efficient disruption of liposomes to release loaded doxorubicin (Dox). This results in increased Dox nuclear subcellular location and cytotoxicity in cancer cells in vitro upon pp-lipo exposure to LIFU. Following intravenous administration, LIFU enhanced deposition of Dox within tumor tissue, suppressed tumor growth, and also increased porphyrin near infrared tumor fluorescence. Thus, pp-lipo is a versatile carrier that can be extended to many ultrasound-controllable drug delivery applications.

Towards enhanced automated elution systems for waterborne protozoa using megasonic energy.

Continuous and reliable monitoring of water sources for human consumption is imperative for public health. For protozoa, which cannot be multiplied efficiently in laboratory settings, concentration and recovery steps are key to a successful detection procedure. Recently, the use of megasonic energy was demonstrated to recover Cryptosporidium from commonly used water industry filtration procedures, forming thereby a basis for a simplified and cost effective method of elution of pathogens. In this article, we report the benefits of incorporating megasonic sonication into the current methodologies of Giardia duodenalis elution from an internationally approved filtration and elution system used within the water industry, the Filta-Max®. Megasonic energy assisted elution has many benefits over current methods since a smaller final volume of eluent allows removal of time-consuming centrifugation steps and reduces manual involvement resulting in a potentially more consistent and more cost-effective method. We also show that megasonic sonication of G. duodenalis cysts provides the option of a less damaging elution method compared to the standard Filta-Max® operation, although the elution from filter matrices is not currently fully optimised. A notable decrease in recovery of damaged cysts was observed in megasonic processed samples, potentially increasing the abilities of further genetic identification options upon isolation of the parasite from a filter sample. This work paves the way for the development of a fully automated and more cost-effective elution method of Giardia from water samples.

Antioxidant Potential of Fruit Juice with Added Chokeberry Powder (Aronia melanocarpa).

The purpose of this study was to determine the possibility of using chokeberry powder as a supplement in apple juice to increase the nutritional value of the final product with the aim of developing a new functional food product. Also, to determine the influence of ultrasound assisted extraction on the bioactive compounds content, nutritional composition and antioxidant potential of apple juice with added chokeberry powder. The juice samples with added chokeberry powder had higher antioxidant capacity, irrespective of the extraction technique used. Apple juice samples with added chokeberry powder treated with high intensity ultrasound had significantly higher content of all analyzed bioactive compounds. The application of high intensity ultrasound significantly reduced the extraction time of the plant material. A positive correlation between vitamin C content, total phenols, flavonoids and anthocyanins content and antioxidant capacity was determined in juice samples with added chokeberry powder treated with high intensity ultrasound.

Rapid cell separation with minimal manipulation for autologous cell therapies.

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of >98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities.

Direct brain infusion can be enhanced with focused ultrasound and microbubbles.

The delivery of most therapeutic agents is rendered ineffective for the treatment of brain diseases due to the presence of the blood-brain barrier (BBB). The goal of this study was to investigate the effect of pre-infusion focused ultrasound (FUS) and microbubbles on the distribution of direct brain infusion in vivo. A single-element FUS transducer was used in all sonications, which were carried out immediately prior to direct infusion procedures. Mice received direct infusion of either Gadolinium-labeled albumin (Gd-albumin, 74 kDa) or adeno-associated virus (AAV, ∼4 MDa). The volumes of Gd-albumin at 30 min were deemed comparable ( P = 0.334) between the direct infusion (DI)-only group and the FUS + DI group. At 120 min, the FUS + DI group showed significantly higher contrast-enhanced volume (9.76 ± 0.74 mm3) than the DI-only group (7.14 ± 0.34 mm3). For mice infused with AAV, the total volume of transduction was estimated as GFP-positive regions and FUS + DI group demonstrated significantly higher ( P = 0.017) transduction efficiency in vivo. In conclusion, enhanced bio-distribution of directly infused agents was observed when the targeted region was pre-conditioned with FUS and microbubbles. Focused ultrasound has the potential, as an adjuvant technique, to significantly enhance direct brain infusion and achieve the desired therapeutic outcomes.

Sonochemical and sustainable synthesis of graphene-gold (G-Au) nanocomposites for enzymeless and selective electrochemical detection of nitric oxide.

In this study, a sonochemical approach was utilised for the development of graphene-gold (G-Au) nanocomposite. Through the sonochemical method, simultaneous exfoliation of graphite and the reduction of gold chloride occurs to produce highly crystalline G-Au nanocomposite. The in situ growth of gold nanoparticles (AuNPs) took place on the surface of exfoliated few-layer graphene sheets. The G-Au nanocomposite was characterised by UV-vis, XRD, FTIR, TEM, XPS and Raman spectroscopy techniques. This G-Au nanocomposite was used to modify glassy carbon electrode (GCE) to fabricate an electrochemical sensor for the selective detection of nitric oxide (NO), a critical cancer biomarker. G-Au modified GCE exhibited an enhanced electrocatalytic response towards the oxidation of NO as compared to other control electrodes. The electrochemical detection of NO was investigated by linear sweep voltammetry analysis, utilising the G-Au modified GCE in a linear range of 10-5000µM which exhibited a limit of detection of 0.04µM (S/N=3). Furthermore, this enzyme-free G-Au/GCE exhibited an excellent selectivity towards NO in the presence of interferences. The synergistic effect of graphene and AuNPs, which facilitated exceptional electron-transfer processes between the electrolyte and the GCE thereby improving the sensing performance of the fabricated G-Au modified electrode with stable and reproducible responses. This G-Au nanocomposite introduces a new electrode material in the sensitive and selective detection of NO, a prominent biomarker of cancer.

Controlled Drug Release and Chemotherapy Response in a Novel Acoustofluidic 3D Tumor Platform.

Overcoming transport barriers to delivery of therapeutic agents in tumors remains a major challenge. Focused ultrasound (FUS), in combination with modern nanomedicine drug formulations, offers the ability to maximize drug transport to tumor tissue while minimizing toxicity to normal tissue. This potential remains unfulfilled due to the limitations of current approaches in accurately assessing and quantifying how FUS modulates drug transport in solid tumors. A novel acoustofluidic platform is developed by integrating a physiologically relevant 3D microfluidic device and a FUS system with a closed-loop controller to study drug transport and assess the response of cancer cells to chemotherapy in real time using live cell microscopy. FUS-induced heating triggers local release of the chemotherapeutic agent doxorubicin from a liposomal carrier and results in higher cellular drug uptake in the FUS focal region. This differential drug uptake induces locally confined DNA damage and glioblastoma cell death in the 3D environment. The capabilities of acoustofluidics for accurate control of drug release and monitoring of localized cell response are demonstrated in a 3D in vitro tumor mode. This has important implications for developing novel strategies to deliver therapeutic agents directly to the tumor tissue while sparing healthy tissue.

Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells.

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In addition, this method can be used in combination with specialized agents known as sonosensitizers to increase the extent of preferential damage exerted by ultrasound against neoplastic cells, an approach referred to as sonodynamic therapy (SDT). The methodology for generating and applying low frequency ultrasound in a preclinical in vitro setting is presented to demonstrate that reproducible cell destruction can be attained in order to examine and compare the effects of sonication on neoplastic and normal cells. This offers a means by which to reliably sonicate neoplastic cells at a level of consistency required for preclinical therapeutic assessment. In addition, the effects of cholesterol-depleting and cytoskeletal-directed agents on potentiating ultrasonic sensitivity in neoplastic cells are discussed in order to elaborate on mechanisms of action conducive to sonochemotherapeutic approaches.