Low-intensity Ultrasound Exposure Research Articles

Biomechanical Response of Cancer Stem Cells to Low-intensity Ultrasound.

The presence of stem cells in cancer may increase the chances of drug resistance and invasiveness. Low-intensity ultrasound (LIUS) can regulate the biological and mechanical properties of cells and participate in cellular migration and differentiation. Although LIUS has shown significant potential in cancer treatment, the effects of LIUS on migration and drug resistance of cancer stem cells (CSCs) have remained less understanding on an angle of biomechanical. Hence, the objective of this work is to analyze the biomechanical response of LIUS to CSCs. In this study, we selected human ovarian cancer cell line A2780 and ovarian cancer stem cells (OCSCs) were enriched from A2780 cells, and observed that OCSCs had higher drug sensitivity and lower invasiveness than A2780 cells after LIUS exposure. Furthermore, we further analyzed the changes in cell morphology, cytoskeleton, and membrane stiffness of A2780 cells and OCSCs at various intensities of LIUS, these results showed that LIUS could induce morphological changes, F-actin formation and increase membrane stiffness, which could help to suppress migration and reduce the drug resistance of OCSCs. Our findings will help establish a better understanding of the biomechanical response to LIUS in CSCs, and future studies on cancer will benefit from the careful consideration of the cellular response of CSCs to LIUS stimulation, ultimately allowing for the development of more effective therapies.

Dynamics of cellular factors of innate immunity and antioxidant protection in the patients with chronic recurrent vulvovaginal candidosis upon exposure to cavitated physiological solution

Dysfunction of colonization resistance factors at the mucous membranes of genitourinary system during adhesion of Candida fungi is a common pathogenic situation leading to impairment of the mucous membrane regeneration, unsuccessful attempts at in vitro fertilization (IVF), increased risk of complications during gestation. The aim of present study was to substantiate the opportunity of complex therapy for chronic vulvovaginal candidiasis using cavitated saline and usage suppositories with alpha-2b human recombinant interferon a (Viferon LLC, Russia). The study involved 90 women at the age of25.45±7.16 years, with clinically and laboratory confirmed recurrencies of chronic vulvovaginal candidiasis observed, on average, 5.55±0.45 times. Group 1 consisted of 30 women with C. albicans infection of urogenital tract, at the mean age of 27.11±1.52 years, who received therapy with fluconazole (150 mg once every 7 days). Group 2 consisted of 30 women with C. albicans infection of mucous membranes, mean age 27.31±1.99 years, treated with Fluconazole (150 mg 1 time in 7 days, and 5-min. irrigation of vagina with ultrasonicated saline solution, 7 days; Low-intensity ultrasound exposure was performed using the Fotek AK100-25 hardware complex, ultrasonic vibration frequency of 25 kHz (Yekaterinburg, Russia). Group 3 consisted of 30 women whose treatment included Fluconazole (150 mg once every 6 days), irrigation of mucous membranes with ultrasonicated cavitated solution and subsequent administration of suppositories containing Viferon® 500,000 IU (1 suppository daily for 7 days). Biochemical studies included determination of isopropanol- and heptane-soluble primary, secondary end products of lipid peroxidation. Activity of the antioxidant system was studied by determination of superoxide dismutase, glutathione peroxidase, ceruloplasmin, vitamin C. Qualitative and quantitative characteristics of neutrophilic granulocytes from vaginal secretions were evaluated by testing phagocytic activity, reduction of nitroblue tetrazolium compound to diformazan. The studies of IL-8, TNFα, IL-2, and IL-10 cytokines were carried out in cell-free supernates of vaginal secretions by ELISA test systems (LLC “Vector Best”, Novosibirsk). Results: upon therapeutic exposure to low-frequency ultrasonic cavitation combined with antioxidant drug, we have observed a decreased local concentration of pro-inflammatory cytokines, the balance of lipid peroxidationantioxidant defense system was restored, and functional metabolic status of the phagocytes in vaginal secretions was normalized.

Lipid bubbles combined with low-intensity ultrasound enhance the intratumoral accumulation and antitumor effect of pegylated liposomal doxorubicin in vivo

Pegylated liposomal doxorubicin (PLD) is a representative nanomedicine that has improved tumor selectivity and safety profile. However, the therapeutic superiority of PLD over conventional doxorubicin has been reported to be insignificant in clinical medicine. Combination treatment with microbubbles and ultrasound (US) is a promising strategy for enhancing the antitumor effects of chemotherapeutics by improving drug delivery. Recently, several preclinical studies have shown the drug delivery potential of lipid bubbles (LBs), newly developed monolayer microbubbles, in combination with low-intensity US (LIUS). This study aimed to elucidate whether the combined use of LBs and LIUS enhanced the intratumoral accumulation and antitumor effect of PLD in syngeneic mouse tumor models. Contrast-enhanced US imaging using LBs showed a significant decrease in contrast enhancement after LIUS, indicating that LIUS exposure induced the destruction of LBs in the tumor tissue. A quantitative evaluation revealed that the combined use of LBs and LIUS improved the intratumoral accumulation of PLD. Furthermore, tumor growth was inhibited by combined treatment with PLD, LBs, and LIUS. Therefore, the combined use of LBs and LIUS enhanced the antitumor effect of PLD by increasing its accumulation in the tumor tissue. In conclusion, the present study provides important evidence that the combination of LBs and LIUS is an effective method for enhancing the intratumoral delivery and antitumor effect of PLD in vivo.

The enhancing antifungal effect of AD1 aptamer-functionalized amphotericin B-loaded PLGA-PEG nanoparticles with a low-frequency and low-intensity ultrasound exposure on C.albicans biofilm through targeted effect

The prevalence and fatality rates with fungal biofilm-associated infections urgently need to develop targeted therapeutic approaches to augment the action of antifungal drugs. This study developed amphotericin B-loaded PLGA-PEG nanoparticles (AmB-NPs) with AD1 aptamer conjugation on its surface via an EDC/NHS technique. Their high nuclease resistance of the conjugation was confirmed by PAGE gel electrophoresis. The targeting and toxicity of AD1-AmB-NPs in the subcutaneous C. albicans infection model were evaluated. AD1-AmB-NPs can bind to different morphological forms(including yeast cells, germ tubes, hyphae) of C. albicans biofilms and extracellular matrix material. Low-frequency and low-intensity ultrasound (LFU, with a fixed frequency of 42 kHz, at the intensity of 0.30 W/cm2 for 15 min) significantly promoted permeability of the biofilm and allowed AD1-AmB-NPs into the deepest layers of the biofilm. After 7 days of treatment, the combination treatment of AD1-AmB-NPs and LFU, kills at least 99% of the biofilm fungal population in vivo comparison with ultrasound alone or AD1-AmB-NPs alone, and returned to normal subcutaneously. Our data suggest that the combined strategy of AD1-AmB-NPs and ultrasound treatment selective delivered of therapeutic drugs to the infection site and exhibited significant synergistic antifungal effects.

Experimental Data-Mining Analyses Reveal New Roles of Low-Intensity Ultrasound in Differentiating Cell Death Regulatome in Cancer and Non-cancer Cells via Potential Modulation of Chromatin Long-Range Interactions.

Background: The mechanisms underlying low intensity ultrasound (LIUS) mediated suppression of inflammation and tumorigenesis remain poorly determined.Methods: We used microarray datasets from NCBI GEO Dataset databases and conducted a comprehensive data mining analyses, where we studied the gene expression of 299 cell death regulators that regulate 13 different cell death types (cell death regulatome) in cells treated with LIUS.Results: We made the following findings: (1) LIUS exerts a profound effect on the expression of cell death regulatome in cancer cells and non-cancer cells. Of note, LIUS has the tendency to downregulate the gene expression of cell death regulators in non-cancer cells. Most of the cell death regulator genes downregulated by LIUS in non-cancer cells are responsible for mediating inflammatory signaling pathways; (2) LIUS activates different cell death transcription factors in cancer and non-cancer cells. Transcription factors TP-53 and SRF- were induced by LIUS exposure in cancer cells and non-cancer cells, respectively; (3) As two well-accepted mechanisms of LIUS, mild hyperthermia and oscillatory shear stress induce changes in the expression of cell death regulators, therefore, may be responsible for inducing LIUS mediated changes in gene expression patterns of cell death regulators in cells; (4) LIUS exposure may change the redox status of the cells. LIUS may induce more of antioxidant effects in non-cancer cells compared to cancer cells; and (5) The genes modulated by LIUS in cancer cells have distinct chromatin long range interaction (CLRI) patterns to that of non-cancer cells.Conclusions: Our analysis suggests novel molecular mechanisms that may be utilized by LIUS to induce tumor suppression and inflammation inhibition. Our findings may lead to development of new treatment protocols for cancers and chronic inflammation.

Dual-Frequency Ultrasound Induces Neural Stem/Progenitor Cell Differentiation and Growth Factor Utilization by Enhancing Stable Cavitation.

Neural stem/progenitor cells (NSPCs) have the potential to serve as the basic materials for treating severe neural diseases and injuries. Ultrasound exposure is an effective therapy for nonunion fractures and healing fresh wounds through an easy and noninvasive application. According to the results of our preliminary study, low-intensity ultrasound (LIUS) promotes the attachment and differentiation of NSPCs. However, the parameters of and mechanisms by which LIUS induces NSPC differentiation remain unclear. To the best of our knowledge, no published studies have reported and compared the biological effects of dual-frequency and single-frequency LIUS on NSPCs. The purpose of this study is to systematically compare several LIUS parameters, including single-frequency, single-transducer dual-frequency ultrasound, burst, and continuous cycling stimulation at several intensities. Furthermore, synergistic effects of single-/dual-frequency LIUS combined with neural growth factor addition on NSPCs were also evaluated. Based on the results of the cytotoxicity assay, low-intensity (40 kPa) ultrasound does not damage NSPCs compared with that observed in the control group. The morphology and immunostaining results show that all experimental groups exposed to ultrasound exhibit neurite outgrowth and NSPC differentiation. In particular, dual-frequency ultrasound promotes NSPCs differentiation to a greater extent than single-frequency ultrasound. In addition, more complicated and denser neural networks are observed in the dual-frequency group. Neural growth factor addition increased the percentage of neurons formed, particularly in the groups stimulated with ultrasound. Among these groups, the dual-frequency group exhibited significant differences in the percentage of differentiated neurons compared with the single-frequency group. This study may the first to prove that dual-frequency LIUS exposure further enhances NSPC differentiation and the utilization of growth factors than single-frequency LIUS. Moreover, the result also revealed that dual-frequency ultrasound generated higher calcium ion influx and extended the channel opening time. A potential explanation is that dual-frequency ultrasound generates more stable cavitation than single-frequency LIUS, which may stimulate cell membrane mechanochannels and enhance calcium ion influx but does not damage them. This in vitro study may serve as a useful alternative for ultrasound therapy.

The effect of low-intensity ultrasound and met signaling on cellular motility and morphology

During the past decade, the potential of low-intensity ultrasound (LIUS) was demonstrated in medical research including microvascular remodeling, wound repair, blood flow restoration, angiogenesis and activation of mechanosensitive signaling pathways. However, the current clinical application of LIUS hasn’t been extended beyond physiotherapy. In cancer therapy, LIUS related research is focused on ultrasound-mediated techniques for chemotherapeutic drug activation, drug delivery enhancement and gene transfection improvement. While clinical trials show highly promising results, the ambiguity regarding the mechanism of ultrasound-cell interaction is a major limitation in the road to clinical applications. In 2011, the bilayer sonophore (BLS) model suggested that cell intramembrane cavitation is the underlying mechanism for ultrasound-induced bio-effects. According to the BLS model, ultrasound affects cell shape and functionality by inducing intramembrane gas bubbles that deform the cell membrane. This mechanical effect might involve alterations in the bio-chemical signaling pathways. Many biological processes such as cell motility, proliferation and angiogenesis are triggered by MET tyrosine kinase growth factor receptor and its ligand HGF/SF (Hepatocyte growth factor/Scatter Factor). The activation of MET receptor increases cancer cell motility via membrane alterations, leading to epithelial to mesenchymal transformation (EMT), invasion and metastasis. The aim of this work is to examine the crosstalk between MET-activation and LIUS under the assumption that both induce cell membrane alterations whereby affecting cell morphology and motility. In this paper, we present preliminary observations regarding the effect of MET-activation, LIUS exposure and their combined effect on living cells. Measurements of morphokinetic parameters using single cell time-lapse analysis are presented as well. The analysis demonstrates that LIUS inhibits the motility and modulates the morphology of MET-activated cells. These findings demonstrate the balance between the inner cellular processes (MET signaling pathways) and the external forces (LIUS exposure). Understanding the mechanism of LIUS on motile cells might improve the performance of existing LIUS-based cancer treatment modalities and help develop new ones.

The frequency-dependent effects of low-intensity ultrasound exposure on human colon carcinoma cells

Previous studies have established a correlative relationship between the acoustic properties of normal and malignant epithelial cells in response to high-frequency ultrasound exposure, indicating ultrasound’s value as a tool in modern non-invasive cancer detection. More recently, ultrasound exposure has been extended into therapeutic fields, manipulated in frequency and power to stress and destroy specific cancer cells based on their determined acoustic properties. The present study seeks to determine the frequency-dependence of low intensity ultrasound exposure on varying cell types, beginning with colon carcinoma cells. The cells were exposed to a single element unfocused piezoelectric transducer at energies of less than 3 W/cm2 and frequencies of 0, 5, 10, 15, 20, and 25 MHz, and evaluated for cytotoxicity indicated by lack of cell adhesion before and after treatment. Comparison is also made to non-cancerous human colon epithelial cells. This project compares the frequency-dependent effects of low intensity ultrasound on cells of varying cancerous and non- cancerous lineages, especially highlighting the low-point threshold.

Differentiation of Neural Stem/Progenitor Cells Using Low-Intensity Ultrasound

Herein, we report the evaluation of apoptosis, cell differentiation, neurite outgrowth and differentiation of neural stem/progenitor cells (NSPCs) in response to low-intensity ultrasound (LIUS) exposure. NSPCs were cultured under different conditions, with and without LIUS exposure, to evaluate the single and complex effects of LIUS. A lactic dehydrogenase assay revealed that the cell viability of NSPCs was maintained with LIUS exposure at an intensity range from 100 to 500 mW/cm2. Additionally, in comparison with no LIUS exposure, the cell survival rate was improved with the combination of medium supplemented with nerve growth factor and LIUS exposure. Our results indicate that LIUS exposure promoted NSPC attachment and differentiation on a glass substrate. Neurite outgrowth assays revealed the generation of longer, thicker neurites after LIUS exposure. Furthermore, LIUS stimulation substantially increased the percentage of differentiating neural cells in NSPCs treated with nerve growth factor in comparison with the unstimulated group. The high percentage of differentiated neural cells indicated that LIUS induced neuronal networks denser than those observed in the unstimulated groups. Furthermore, the release of nitric oxide, an important small-molecule neurotransmitter, was significantly upregulated after LIUS exposure. It is therefore reasonable to suggest that LIUS promotes the differentiation of NSPCs into neural cells, induces neurite outgrowth and regulates nitric oxide production; thus, LIUS may be a potential candidate for NSPC induction and neural cell therapy.

Novel Dianostics and Therapeutics with Ultrasound Technologies and Nanotechnologies

Ultrasound is a good tool for theranostics due to have multi-potency both of diagnostics with sonography and therapeutics with high intensity focused ultrasound (HIFU). In addition, microbubbles and nanobubbles are utilized as not only contrast imaging agent but also enhancer of drug and gene delivery by combination of ultrasound. Recently, we developed novel liposomal nanobubbles (Bubble liposomes) which were containing perfluoropropane. Bubble liposomes induced jet stream by low intensity ultrasound exposure and resulted in enhancing permeability of cell membrane. This phenomenon has been utilized as driving force for drug and gene delivery. On the other hand, the combination of Bubble liposomes and high intensity ultrasound induces strong jet stream and increase temperature. This condition can directly damage to tumor cells, we are applying this for cancer therapy. Therefore, their combination has potency for various cancer therapies such as gene therapy, immunotherapy and hyperthermia. In this review, we discuss about cancer therapy by the combination of Bubble liposomes and ultrasound.

Outcome Measures Following Sonodynamic Photodynamic Therapy – A Case Series

Sonodynamic Photodynamic Therapy (SPDT) is a novel cancer treatment approach using a photosensitive agent (Sonnelux-1) with reported ultrasound-activated properties. The sensitiser is administered prior to a cycle of light and low-intensity ultrasound exposure. Ultrasound has the advantage of significantly greater tissue penetrance compared to light, which potentially allows non-invasive activation of the sensitiser within deep-sited tumours. Sonnelux-1 has previously demonstrated significant tumour cell inhibition following ultrasound administration in animal studies, and several case reviews have been published reporting clinical benefits in metastatic cancer patients. This current case series presents outcome measures of five patients with a variety of cancer diagnoses following SPDT, providing further evidence of beneficial treatment outcomes.

Upgraded Nerve Growth Factor Expression Induced by Low-Intensity Continuous-Wave Ultrasound Accelerates Regeneration of Neurotometicly Injured Sciatic Nerve in Rats

Low-intensity ultrasound (LIU) can stimulate injured nerve regeneration but the mechanism is still unclear. We investigated the stimulating effect and its mechanism of continuous-wave LIU on neurotometic injury of sciatic nerve. The right sciatic nerves of 64 adult Wistar rats were first crushed and then exposed (32 rats) or sham-exposed (32 rats) to LIU at the crush site. The LIU had a spatial averaged and temporal averaged intensity of 0.25W/cm 2 operated at 1.0 MHz for 1 min every other day. At various stages (the second, fourth, sixth and eighth weeks) after LIU exposure, the sciatic nerve function index (SFI), the sensory nerve conduction velocity (SNCV), the expression of nerve growth factor (NGF) and sample histology were studied. It was found that the density of nerve fibers with myelin sheath, SFI, SNCV and NGF expression of the treatment group were higher than that of control group ( p < 0.05). It has been determined that LIU treatment can accelerate the regeneration and functional recovery of neurotometic injured sciatic nerve at earlier stages after injury, the upgraded expression of NGF induced by LIU may be the primary mechanism of the acceleration effects. (E-mail: wangzhibiao@haifu.com.cn)

Activated Cancer Therapy Using Light and Ultrasound - A Case Series of Sonodynamic Photodynamic Therapy in 115 Patients Over a 4 Year Period

Activated Ca ncer Ther apy ( ACT), al so know n a s Sonodyna mic Phot odynamic Ther apy ( SPDT) i s a nove l therapeutic modality that utilises a non-toxic photosensitive agent with reported ultrasound-activated properties. SPDT has previously demonstrated significant tumour cell inhibition in animal studies. There has been much research into the effi- cacy of phot odynamic therapy and development in understanding of the underlying mechanism of tumour cytotoxicity. Synergistic ultrasound activation represents a promising development to activated sensitiser therapy, as photo-activation is limited by access and penetrance issues. Ultrasound has been demonstrated to activate a number of sono-sensitive agents allowing the possibility of non-invasive targeted treatment o f deeper tumour sites than is currently achievable with pho- todynamic therapy. Thi s case se ries of 115 pa tients w ith a v ariety of ca ncer di agnoses reports on experiences of this treatment over a 4 year period using sublingual administration of a new dual activation agent, Sonnelux-1, followed by a protocol of LED light and low-intensity ultrasound exposure. Initial clinical observation suggests SPDT is worthy of fur- ther investigation a s an ef fective and w ell to lerated t reatment fo r a w ide v ariety o f p rimary an d m etastatic tumours, in- cluding those refractory to chemotherapy. Key Wo rds: Sonodynamic therapy, photodynamic therapy, activated cancer therapy, u ltrasound activated therapy, m etastatic cancer, sonnelux-1, dove clinic, sonnemed.

Experimental study on low intensity ultrasound and tissue engineering to repair segmental bone defects

In order to evaluate the efficacy of low intensity ultrasound and tissue engineering technique to repair segmental bone defects, the rabbit models of 1.5-cm long rabbit radial segmental osteoperiosteum defects were established and randomly divided into 2 groups. All defects were implanted with the composite of calcium phosphate cement and bone mesenchymal stem cells, and additionally those in experimental group were subjected to low intensity ultrasound exposure, while those in control group to sham exposure. The animals were killed on the postoperative week 4, 8 and 12 respectively, and specimens were harvested. By using radiography and the methods of biomechanics, histomorphology and bone density detection, new bone formation and material degradation were observed. The results showed that with the prolongation of time after operation, serum alkaline phosphatase (AKP) levels in both groups were gradually increased, especially in experimental group, reached the peak at 6th week (experimental group: 1.26 mmol/L; control group: 0.58 mmol/L), suggesting the new bone formation in both two group, but the amount of new bone formation was greater and bone repairing capacity stronger in experimental group than in control group. On the 4th week in experimental group, chondrocytes differentiated into woven bone, and on the 12th week, remodeling of new lamellar bone and absorption of the composite material were observed. The mechanical strength of composite material and new born density in experimental group were significantly higher than in control group, indicating that low intensity ultrasound could not only effectively increase the formation of new bone, but also accelerate the calcification of new bone. It was concluded that low intensity ultrasound could evidently accelerate the healing of bone defects repaired by bone tissue engineering.

The use of low-intensity ultrasound to accelerate the healing of fractures.

Double-blind, prospective, placebo-controlled clinical trials demonstrate that healing times of fresh fractures of the radius and tibia are reduced by up to 40% with the use of low-intensity ultrasound. Animal studies indicate that low-intensity ultrasound exposure results in stronger and stiffer callus formation and in acceleration of the endochondral ossification process. Extensive clinical evidence demonstrates that ultrasound represents a safe, noninvasive method of accelerating the healing of fresh fractures of the tibia, the distal aspect of the radius, the scaphoid, and the metatarsals. Clinical studies indicate that ultrasound reduces the confounding effect of smoking and patient age on the fracture-healing process. Ultrasound requires a brief, twenty-minute, daily at-home treatment regimen and has no known contraindications. The effectiveness of low-intensity ultrasound has also been demonstrated in the clinical treatment of delayed unions and nonunions. Fracture-healing is a complex biological process that involves the spatial and temporal orchestration of numerous cell types, hundreds if not thousands of genes, and the intricate organization of an extracellular matrix, all working toward restoring the bone's mechanical strength and rapid return to full function. It has often been argued that nature has optimized this process and thus it would be difficult to interventionally accelerate or augment fracture-healing. How can science conceivably improve upon 600 million years of vertebrate evolution? Nevertheless, it is just this goal that has inspired an intense effort among basic-science and clinical investigators from a vast array of biotechnology and bioengineering disciplines at academic as well as industrial laboratories, to seek a means of accelerating the healing of fractured bones. In this article, the basic-science and clinical evaluation of the use of low-intensity ultrasound is reviewed and the case is made that nature's process of fracture-healing, while elegant, can be accelerated with respect to achieving the ability to support clinically relevant loads. The …

Ultrasound stimulation of rat testes damaged by busulfan

The purpose of the present study was to investigate whether or not low-intensity ultrasound exposure (20 mW/cm 2 average intensity) accelerates the repair of rat germinal epithelium damaged by an antispermatogenic agent. The results from analysis of testicular weight and DNA content, sperm production and epididymal sperm concentration showed that the time needed for the reestablishment of the spermatogenic process following busulfan treatment was not reduced by ultrasound energy suggesting that, in contrast to many other mammalian tissues, the seminiferous epithelium is refractory to ultrasound stimulation.