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Background The possibility to combine Low Intensity UltraSound (LIUS) and Nanoparticles (NP) could represent a promising strategy for drugs delivery in tumors difficult to treat overcoming resistance to therapies. On one side the NP can carry drugs that specifically target the tumors on the other the LIUS can facilitate and direct the delivery to the tumor cells. In this study, we investigated whether Very Low Intensity UltraSound (VLIUS), at intensities lower than 120 mW/cm2, might constitute a novel strategy to improve delivery to tumor cells. Thus, in order to verify the efficacy of this novel modality in terms of increase selective uptake in tumoral cells and translate speedily in clinical practice, we investigated VLIUS in three different in vitro experimental tumor models and normal cells adopting three different therapeutic strategies. Methods VLIUS at different intensities and exposure time were applied to tumor and normal cells to evaluate the efficiency in uptake of labeled human ferritin (HFt)-based NP, the delivery of NP complexed Firefly luciferase reported gene (lipoplex-LUC), and the tumor-killing of chemotherapeutic agent. Results Specifically, we found that specific VLIUS intensity (120 mW/cm2) increases tumor cell uptake of HFt-based NPs at specific concentration (0.5 mg/ml). Similarly, VLIUS treatments increase significantly tumor cells delivery of lipoplex-LUC cargos. Furthermore, of interest, VLIUS increases tumor killing of chemotherapy drug trabectedin in a time dependent fashion. Noteworthy, VLIUS treatments are well tolerated in normal cells with not significant effects on cell survival, NPs delivery and drug-induced toxicity, suggesting a tumor specific fashion. Conclusions Our data shed novel lights on the potential application of VLIUS for the design and development of novel therapeutic strategies aiming to efficiently deliver NP loaded cargos or anticancer drugs into more aggressive and unresponsive tumors niche. Electronic supplementary material The online version of this article (10.1186/s13046-018-1018-6) contains supplementary material, which is available to authorized users.

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.

Genotoxic effects of therapeutic ultrasound are poorly documented, when compared with the wide use of this physical agent. The aim of this work was to investigate the clastogenic and aneugenic potential of 1 MHz ultrasound, employing intensities (200 and 300 mW/cm2 ) above the cavitational threshold, but in the range of those normally used in therapeutics. Both normal fibroblasts (AG01522) and tumoral cells (MCF-7) were sonicated. While no effects on viability were noted, significant increases of CREST-negative micronuclei (indicative of clastogenesis) and CREST-positive micronuclei (indicative of aneuploidy) were detected. Clastogenesis was confirmed by increases of γ-H2AX foci, while increases of spindle anomalies confirmed the induction of aneuploidy. Our results confirm previous works that showed ultrasound-induced DNA breakage. Moreover, our experiments show that the known effect of ultrasound-induced damage to microtubules is also able to damage the mitotic spindle and induce aneuploidy. On the overall, this work highlights the importance to further investigate the potential risks related to therapeutics US. Environ. Mol. Mutagen. 59:60-68, 2018. © 2017 Wiley Periodicals, Inc.

Microbubbles are a well-established contrast agent which improves diagnostic ultrasound imaging. During the last decade research has focused on expanding their use to include molecular imaging, targeted therapy and imaging modalities other than ultrasound. However, bioadhesion of targeted microbubbles under physiological flow conditions is still difficult to achieve, the main challenge being connected to the poor stability of lipid microbubbles in the body's circulation system. In this article, we investigate the use of polymeric microbubbles based on a poly (vinyl alcohol) shell as an alternative to lipid microbubbles. In particular, we report on the development of microbubble shell modification, using mild reaction conditions, with the aim of designing a multifunctional platform to enable diagnosis and therapy. Superparamagnetic iron oxide nanoparticles and a near infrared fluorescent probe, indocyanine green, are coupled to the bubbles surface in order to support magnetic resonance and fluorescence imaging. Furthermore, anchoring cyclic arginyl-glycyl-aspartic acid (RGD) peptide, and cyclodextrin molecules, allows targeting and drug loading, respectively. Last but not least, shell topography is provided by atomic force microscopy. These applications and features, together with the high echogenicity of poly (vinyl alcohol) microbubbles, may offer a more stable alternative to lipid microbubbles for the development of a multimodal theranostic platform.

Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation Fourier Transform Infrared micro-spectroscopy (SR-microFTIR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR-microFTIR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. Our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies.

Hypothesis One of the main assets of crosslinked polymer-shelled microbubbles (MBs) as ultrasound-active theranostic agents is the robustness of the shells, combined with the chemical versatility in modifying the surface with ligands and/or drugs. Despite the long shelf-life, subtle modifications occur in the MB shells involving shifts in acoustic, mechanical and structural properties. Experiments We carried out a long-term morphological and acoustic evolution analysis on elastomeric polyvinyl-alcohol (PVA)-shelled MBs, a novel platform accomplishing good acoustic and surface performances in one agent. Confocal laser scanning microscopy, acoustic spectroscopy and AFM nanomechanics were integrated to understand the mechanism of PVA MBs ageing. The changes in the MB acoustic properties were framed in terms of shell thickness and viscoelasticity using a linearised oscillation theory, and compared to MB morphology and to nanomechanical analysis. Findings We enlightened a novel, intriguing ageing time evolution of the PVA MBs with double behaviour with respect to a crossover time of ∼50 days. Before, significant changes occur in MB stiffness and shell thickness, mainly due to a massive release of entangled PVA chains. Then, the MB resonance frequency increases together with shell thickening and softening. Our benchmark study is of general interest for emerging viscoelastomeric bubbles towards personalised medicine.

Genotoxic effects of therapeutic ultrasound are poorly documented, when compared with the wide use of this physical agent. The aim of this work was to investigate the clastogenic and aneugenic potential of 1 MHz ultrasound, employing intensities (200 and 300 mW/cm

Ultrasound (US) induced enhancement of plasma membrane permeability is a hugely promising tool for delivering exogenous vectors at the specific biological site in a safe and efficient way. In this respect, here we report effects of membrane permeability alteration on fibroblast-like cells undergoing very low-intensity of US. The change in permeability was pointed out in terms of high uptake efficiency of the fluoroprobe calcein, thus resembling internalization of small cell-impermeable model drugs, as measured by fluorescence microscopy and flow cytometry. Fluorescence evidences moreover suggests that the higher the time of exposure, the larger will be the size of molecules can be internalized. The uptake events were related to the cell viability and also with structural changes occurring at membrane level as revealed by infrared spectroscopy and preliminary membrane fluidity and atomic force microscopy (AFM) investigation. Thus, the question of whether the uptake of cell-impermeable molecules is consistent with the presence of disruptions on the cell membrane (sonopore formation) has been addressed. In this framework, our findings may constitute experimental evidence in support of sub-cavitation sonoporation models recently proposed, and they may also provide some hints towards the actual working condition of medical US dealing with the optimum risk to benefit therapeutic ratio.

Although medical ultrasound offers compelling opportunities to improve therapy in principle, progress in the field has been limited because of an insufficient understanding of the potential genotoxic and cytotoxic effects of ultrasound on biological systems. This paper is mainly focused on an in vitro study of effects with respect to genotoxicity and viability induced by 1- and 3-MHz medical ultrasound in murine fibroblasts (NIH-3T3) at low-intensity exposure (spatial peak temporal average intensity Ita

In this paper we report on the study of the interface of hybrid shell droplets encapsulating decafluoropentane (DFP), which exhibit interesting potentialities for ultrasound (US) imaging. The fabrication of the droplets is based on the deposition of a dextran methacrylate layer onto the surface of surfactants. The droplets have been stabilized against coalescence by UV curing, introducing crosslinks in the polymer layer and transforming the shell into an elastomeric membrane with a thickness of about 300 nm with viscoelastic behaviour. US irradiation induces the evaporation of the DFP core of the droplets transforming the particles into microbubbles (MBs). The presence of a robust crosslinked polymer shell introduces an unusual stability of the droplets also during the core phase transition and allows the recovery of the initial droplet state after a few minutes from switching off US. The interfacial tension of the droplets has been investigated by two approaches, the pendant drop method and an indirect method, based on the determination of the liquid ↔ gas transition point of DFP confined in the droplet core. The re-condensation process has been followed by capturing images of single MBs by confocal microscopy. The time evolution of MB relaxation to droplets was analysed in terms of a modified Church model to account for the structural complexity of the MB shell, i.e. a crosslinked polymer layer over a layer of surfactants. In this way the microrheology parameters of the shell were determined. In a previous paper (Chem. Commun., 2013, 49, 5763–5765) we showed that these systems could be used as ultrasound contrast agents (UCAs). In this work we substantiate this view assessing some key features offered by the viscoelastic nature of the droplet shell.

PurposeThe purpose of this paper is to apply a dedicated checklist for the evaluation of good practice information in the design of Italian internet sites on electromagnetic fields (emf) and health.Design/methodology/approachThe “Guidelines on the collection, evaluation and dissemination of good practice information on the Internet” by European Occupational Safety and Health Agency (EU‐OSHA) and the criteria of effective health communication, by the American National Institute of Health (NIH), have been reoriented to the issue “emf and health”. In total, 100 Italian internet sites, provided by the search engine GoogleItalia, have been analysed.FindingsThe results show how Italian official sources of information on “emf and health”, mainly affiliated to public institutions, apply good practice criteria in the design of internet sites, in terms of Identity card of the provider, quality of information, usability, interactivity and content of the sites.Practical implicationsIn recent years, the internet has become a powerful tool for information, especially concerning health issues. Internet users need to find a trusted source providing accurate, balanced, evidence‐based, reliable, understandable information. This research provides practical criteria for selecting good practice information in the internet sites dedicated to “emf and health”.Originality/valueThe authors' effort in collecting and evaluating main criteria for an ergonomic design of internet sites dedicated to “emf and health”, confirms the need to implement usability of the internet sites and enhance the quality of information provided in the web in terms of “good practice”.

The structural effect induced by therapeutic ultrasound on proteins in aqueous solution has been investigated with FTIR spectroscopy, UV-VIS spectroscopy, circular dichroism and light scattering. Six proteins (cytochrome, lysozyme, myoglobin, bovine serum albumin, trypsinogen, and alpha-chymotrypsinogen A) with different molecular weight and secondary structure have been studied. The experiment has been performed using an ultrasound source at resonant frequency of 1 MHz and sonication times of 10, 20, 30, 40, 50, and 60 min. A different behaviour of proteins under sonication depends on the dominant secondary structure type (alpha-helix or beta-sheets) and on the grade of the ordered structure. The results suggest that the free radicals, produced by water sonolysis, have an important role in the changes of structural order.

In this work, the theoretical evaluation of the electromagnetic fleld (emf ) produced by radio base stations (rbs) in the city of Monselice, sited in the northern part of Italy is presented. An area of 16km 2 , characterized by 11 rbs sites has been selected. A census of the rbs sites has been carried out, collecting: the location of the sites, the technical data of their antennas, the shape of the ground, the position of all the buildings (Gauss-Boaga coordinates) in the area and their heights. Two difierent evaluations of the emf in this area are presented. The flrst one, representing the buildings by their barycentre placed at the height of each building, calculates in these points the electric fleld levels using the classical far-fleld equation, assuming the rbs working at the maximum power declared. Another theoretical evaluation of the emf has been carried out by the application of a calculation code working with the ray tracing method. It provides an output where a map of the electric fleld produced by the rbs on the surfaces of the buildings in an area around a electromagnetic sites is shown, assuming the rbs working at the maximum power. In the last years, the number of the electromagnetic fleld (emf ) sources in the frequency range of the microwaves has increased, principally in the urban areas, due to the proliferation of radio-base stations (rbs) for mobile communications. It has produced concern among the public about the potential health risks of long time exposition to the emf emitted from the rbs, in the working and living environments. This leads to the necessity of continuous monitoring and prediction of the emf strength. At the international level, several organizations have developed guidelines to protect the general public from the emf emitted from the rbs antennas. In Italy, the issue of public exposure to sources with frequencies between 100kHz and 300GHz is regulated by one decree promulgated in 2003 (1), in terms of three levels: (i) limits, levels that haven't to be exceeded in any circumstances for the protection from short terms efiects; (ii) attention levels, that have not to be exceeded into gambling areas, schools and buildings where people stay for more than 4 hours, as a precaution from the potential risks on health due to long time exposition; (iii) quality targets, levels to achieve in a long period in order to minimize people exposure. In Table 1, the values related to the upper three levels, deflned in terms of efiective electric and magnetic flelds and power density, are presented. Moreover, the decree determines how the measurements have to be performed by averaging the efiective electric and magnetic fleld on 6 minutes and on an area equivalent to the vertical section of the human body. In this work, we present an example of the electromagnetic mapping of an urban area, performed in the territory of the town of Monselice, sited in the northern part of Italy. Here, the theoretical evaluation of the distribution of the emf generated by the rbs, working in the urban territory, has been carried out by calculation programs used in the Laboratory of Radiation and Ultrasounds Pollution of ISPESL.

This work is focused on the in vitro study of the effects induced by medical ultrasound (US) in murine fibroblast cells (NIH-3T3) at a low-intensity of exposure (spatial peak temporal average intensity Ita

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1 MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H(2), H(2)O(2), and HO(2). The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1 MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.

In this study we have used for the first time the infrared spectroscopy as a tool to reveal the effects induced on a cell line, Jurkat T-lymphocytes, by ultrasound (US) used in therapy at 1 MHz frequency. We carried out the experiments using two different setups allowing to change the acoustic field parameters by varying the position of the sample with respect to the ultrasonic transducer and the sonication time with different duty cycles. The parameters of acoustic field measured by a hydrophone were compatible with the cavitation phenomenon in both setups. Genotoxic damage in exposed cells was ascertained by the cytokinesis-block micronucleus (CBMN) assay whilst cell structural changes due to sonication, were revealed by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, a non-destructive technique that allows, by the acquisition of IR spectra within a few minutes, one to obtain simultaneous information on all cellular macromolecules in a cell population. Infrared spectroscopy results highlight changes of functional groups typical of proteins, nucleic acids and lipids at 118 J/cm 2 energy dose, while the micronuclei test shows no toxic damage to the cellular samples; higher energy doses are necessary to reveal significant toxic effects together with structural changes.

The experiment was performed to prove that exposure to a relatively weak extremely low frequency (ELF) magnetic field retards tadpoles' development.Two cohorts of Xenopus laevis laevis (Daudin) tadpoles were exposed during their immature period ( approximately 60 days) to a 50 Hz magnetic field of 63.9or = Bor = 76.4 microT rms (root mean square, average values) magnetic flux density in a solenoid. At the same time, as controls, two comparable cohorts were reared in two aquariums remote from the solenoid. Cohorts' degree of development was quantified by daily inspections of animal limbs and attributing them to a stage of the Nieuwkoop and Faber ( 1956 ) classification. The experiment was replicated three times.(a) Mean developmental rate of exposed cohorts was reduced with respect to controls (0.43 vs. 0.48 stages/day, p0.001) starting from early larval stages; (b) Exposure increased the mean metamorphosis period of tadpoles by 2.4 days compared with the controls (p0.001); (c) Maturation rates of exposed and control tadpoles changed during maturation period; and (d) Important mortality, malformations or teratogenic effects were not observed in exposed matured tadpoles.A long-term exposure of X. laevis tadpoles to a relatively weak 50 Hz magnetic field causes a sub-lethal effect that slows down their larval developmental rate and delays their metamorphosis.

Background The paper reports the electric, magnetic and electromagnetic fields (emf) measurements carried out in the Regina Elena National Cancer Institute (NCI). Several devices, used in diagnostics and in medical cures, can represent sources of emf for the workers and for the public subjected to the treatments. The aim is to evaluate their exposition, in order to assess the compliance with the law. Methods The investigations have been carried out in the departments of: intensive care, physiotherapy, MR presstherapy and in the surgical rooms. The measurements have been performed using broad band probes in the frequency ranges 5 Hz÷30 kHz and 100 kHz-3 GHz. Results The variability of the magnetic induction (B(μT)) levels is between 0,05 μT and 80 μT. The statistical distribution shows that most of the measurements are in the range 0,05 Conclusion The measurement of the emf levels in the NCI is recommended because of the presence of the oncological patients; their long stay near the equipments and their day-long exposure represent additional risk factors for which a prudent avoidance strategy have to de adopted.

Great effort is now devolved to the study of the biological effects induced by exposure to the electromagnetic fields. Using flat strip lines and slotted cables, we realised an emitting system to irradiate a spatial region with opportune sizes and shapes. The first system provides the use of a flat strip line 8 m long, supplied by a microwave signal at the frequency in the range 900-960 MHz to reproduce a typical frequency range used by the TACS-GSM radio mobile systems. The second system, provides the use of a slotted cable 6 m long, supplied by the same microwave signal. The effective electric field emitted has been measured in a box close to the emitting system. The analysis shows that: most of the values, 88.5%, measured close to the flat strip line in a total volume whose dimensions are 800times20times6 centimetres are distributed in the range of level 1,6divide3,0 V/m; also most of the values, 77.0%, measured close to the slotted cables in a total volume whose dimensions are 600times6times10 centimetres are distributed in the range of level 1,0divide3,0 V/m. Then, the e. e. f. values produced by the strip line are distributed more uniformly than that of the slotted cable in a wider volume.

It is presented the work performed by ISPESL, related to the analysis of the wide band electromagnetic field measurements, carried out in the urban area of Rome, in the period from 1998 to 2001. ISPESL carried out approximately 500 wide band measurements near the sites selected and proposed by the network companies for the installation of new radio base stations (SRB). The measurements have been carried out using a wide band probe equipped with an isotropic electric field probe, in areas where people stay for more than four hours. The results of the electromagnetic measurements have been compared with the electric field levels fixed by the Italian law to prevent hazardous due to long term people exposure to the electromagnetic field sources with frequency between 100 kHz-300 GHz, which is 6 V/m. The electric field levels have been shared in classes, to analyse the statistical distribution of the electric field levels measured and calculated in the urban environment of Rome.

The concern about health effects of extremely low frequency (ELF) magnetic fields has continued to grow, since Wertheimer and Leeper in 1979 indicated a possible relationship between electrical wiring and childhood cancer. This has led to a great amount of studies, including in vivo and in vitro models, but a causal relationship between ELF exposure and cancer has not been explicitly demonstrated. In this work, in order to detect a possible clastogenic and aneugenic damage, the micronucleus test with CREST staining was performed on erythrocytes from liver and peripheral blood sampled from newborn mice exposed to an ELF magnetic field during the whole intra-uterine life (21 days), and from bone marrow and peripheral blood sampled from adult mice exposed to the same magnetic field for the same period. Results obtained in newborn mice show a significant increase of the micronuclei frequencies. In absolute terms, most of the induced micronuclei were CREST-negative (i.e., formed by a chromosome fragment). However, in relative terms, ELF exposure caused a two-fold increase in CREST-negative micronuclei and a four-fold increase in CREST-positive micronuclei (i.e., formed by a whole chromosome). No significant effect was recorded on exposed adults. These findings suggest to investigate the aneugenic properties of ELF magnetic fields in order to establish a possible relationship between these and carcinogenesis

Presents the work carried out by the Laboratory of Radiations and Ultrasounds Pollution of the National Institute of Occupation Safety and Prevention (ISPESL) about the measurement of radiofrequency and microwave electromagnetic field in the urban area of Rome (Italy) from 1998 to 2001. The measurements have been carried out within the framework of the technical and scientific advice paid by ISPESL to the public authority and to the companies to implement the cellular networks in TACS, GSM and DCS technologies. Presents the result of approximately 220 wide band measurements of the electromagnetic field background in the urban area of Rome, carried out near the sites selected for the installation of new base stations.

The aim of this research is to investigate some bioeffects due to Therapeutic Ultrasound (1 MHz and 50