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1.
Sci Rep ; 11(1): 19033, 2021 09 24.
Article in English | MEDLINE | ID: mdl-34561481

ABSTRACT

Keratinocytes, the main cell type of the skin, are one of the most exposed cells to environmental factors, providing a first defence barrier for the host and actively participating in immune response. In fact, keratinocytes express pattern recognition receptors that interact with pathogen associated molecular patterns and damage associated molecular patterns, leading to the production of cytokines and chemokines, including interleukin (IL)-6. Herein, we investigated whether mechanical energy transported by low intensity ultrasound (US) could generate a mechanical stress able to induce the release of inflammatory cytokine such IL-6 in the human keratinocyte cell line, HaCaT. The extensive clinical application of US in both diagnosis and therapy suggests the need to better understand the related biological effects. Our results point out that US promotes the overexpression and secretion of IL-6, associated with the activation of nuclear factor-κB (NF-κB). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability, paving the way for further investigating the molecular mechanisms related to US exposure.


Subject(s)
Gene Expression/radiation effects , Inflammation Mediators/metabolism , Interleukin-6/metabolism , Keratinocytes/metabolism , Ultrasonic Waves/adverse effects , Cell Membrane Permeability/radiation effects , Cell Survival/radiation effects , Dose-Response Relationship, Radiation , HaCaT Cells , Humans , Interleukin-6/genetics , NF-kappa B/metabolism , Stress, Mechanical
2.
Environ Mol Mutagen ; 60(7): 568-575, 2019 08.
Article in English | MEDLINE | ID: mdl-30942920

ABSTRACT

The effects of ultrasound on the cytoskeleton, comprising microtubules, had been studied decades ago. Nonetheless, very little attention has been paid to the effects of ultrasound on the mitotic spindle, which is also formed by microtubules. In this study, we treated human fibroblasts and human cancer cells (HeLa and MCF-7) with 1-MHz ultrasound at low intensities (70, 140, and 300 mW/cm2 ). In all cell lines, 5 min after the end of sonication, we found an intensity-dependent increase of mitotic abnormalities (including multipolar spindles). Two hours after sonication, these abnormalities were present, but at much lower frequencies. Twenty-four hours after sonication, mitotic abnormalities were at the same level of untreated samples, suggesting a transient effect due to ultrasound. Beside abnormalities of the mitotic spindle, we also observed an increase of metaphases with nonaligned chromosomes. The mitotic index of fibroblasts and HeLa cells, two hours after sonication, showed an intensity-dependent decrease; this was not observed in MCF-7 cells. In agreement with this last result, ultrasound-induced growth inhibition (which was also intensity-dependent) was more marked in fibroblasts and HeLa cells compared to MCF-7 cells. This work indicates that therapeutic ultrasound, even at intensities below the cavitation threshold, can affect genome integrity, showing the need to increase the knowledge of the potential risks of ultrasound to human health. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.


Subject(s)
Spindle Apparatus/physiology , Cell Line , Cell Line, Tumor , Chromosomes/physiology , Fibroblasts/physiology , HeLa Cells , Humans , MCF-7 Cells , Mitosis/physiology , Sonication/methods
3.
J Exp Clin Cancer Res ; 38(1): 1, 2019 Jan 03.
Article in English | MEDLINE | ID: mdl-30606223

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/therapeutic use , Colonic Neoplasms/diagnostic imaging , Colonic Neoplasms/drug therapy , Drug Delivery Systems/methods , Nanoparticles/metabolism , Ultrasonography/methods , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Colonic Neoplasms/pathology , Humans
4.
Environ Mol Mutagen ; 59(1): 60-68, 2018 01.
Article in English | MEDLINE | ID: mdl-28833460

ABSTRACT

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.


Subject(s)
DNA Damage/genetics , Ultrasonic Waves/adverse effects , Aneugens/adverse effects , Aneuploidy , Cell Line, Tumor , Cell Survival/genetics , Cells, Cultured , Fibroblasts/physiology , Genomics/methods , Humans , MCF-7 Cells , Micronuclei, Chromosome-Defective , Micronucleus Tests/methods , Microtubules/genetics , Mutagens/adverse effects , Spindle Apparatus/genetics
5.
J Colloid Interface Sci ; 491: 151-160, 2017 Apr 01.
Article in English | MEDLINE | ID: mdl-28024192

ABSTRACT

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.


Subject(s)
Diagnostic Imaging , Polyvinyl Alcohol/chemistry , Ultrasonics , Drug Delivery Systems , Molecular Structure , Particle Size , Surface Properties , beta-Cyclodextrins/chemistry
6.
Phys Chem Chem Phys ; 18(12): 8378-88, 2016 Mar 28.
Article in English | MEDLINE | ID: mdl-26931337

ABSTRACT

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.


Subject(s)
Contrast Media/chemistry , Biocompatible Materials/chemistry , Calorimetry, Differential Scanning , Dextrans/chemistry , Dynamic Light Scattering , Microbubbles , Microscopy, Atomic Force , Microscopy, Confocal , Polymers/chemistry , Surface Tension , Surface-Active Agents/chemistry , Thermogravimetry , Ultrasonography
7.
Article in English | MEDLINE | ID: mdl-25308543

ABSTRACT

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<0.1 W/cm(2)). The NIH-3T3 cells constitute a well-characterized in vitro cell model in which a genotoxic effect can be predicted by means of a reliable and precise murine cytokinesis-block micronucleus assay. A statistically significant increase in the incidence of micronuclei was observed in sonicated 3T3 cells. In particular, the effects were more evident at 1 MHz. Moreover, for each frequency investigated, the occurrence of micronuclei was comparatively more frequent with increasing time of exposure. The possible toxicological implications of the medical ultrasound employed herein deal with the existence of a window of exposure parameters (set well below the intensity of ultrasound cavitation) in which some genotoxic effects may occur without significant cytotoxicity. In this respect, they provide new insight toward the correct risk to benefit balancing of ultrasound-based treatments and for designing innovative therapeutic strategies.


Subject(s)
Fibroblasts/metabolism , Micronuclei, Chromosome-Defective , Sound/adverse effects , 3T3 Cells , Animals , Fibroblasts/pathology , Mice
8.
Ultrasonics ; 54(4): 1020-8, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24370376

ABSTRACT

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<0.1Wcm(-2)). Conventional 1MHz and 3MHz US devices of therapeutic relevance were employed with varying intensity and exposure time parameters. In this framework, upon cells exposure to US, structural changes at the molecular level were evaluated by infrared spectroscopy; alterations in plasma membrane permeability were monitored in terms of uptake efficiency of small cell-impermeable model drug molecules, as measured by fluorescence microscopy and flow cytometry. The results were related to the cell viability and combined with the statistical PCA analysis, confirming that NIH-3T3 cells are sensitive to therapeutic US, mainly at 1MHz, with time-dependent increases in both efficiency of uptake, recovery of wild-type membrane permeability, and the size of molecules entering 3T3. On the contrary, the exposures from US equipment at 3MHz show uptakes comparable with untreated samples.


Subject(s)
Cell Membrane Permeability , Fibroblasts/cytology , Fluoresceins/metabolism , Ultrasonics/methods , Animals , Cells, Cultured , Flow Cytometry , In Vitro Techniques , Mice , Microscopy, Fluorescence , Spectrophotometry, Infrared
9.
Drug Deliv ; 20(7): 285-95, 2013.
Article in English | MEDLINE | ID: mdl-24044646

ABSTRACT

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.


Subject(s)
Cell Membrane Permeability , Fibroblasts/metabolism , Fluoresceins/pharmacokinetics , Ultrasonics/methods , Animals , Cell Survival , Flow Cytometry , Fluorescent Dyes/pharmacokinetics , Membrane Fluidity , Mice , Microscopy, Atomic Force , Microscopy, Fluorescence , NIH 3T3 Cells , Spectrophotometry, Infrared
10.
Spectrochim Acta A Mol Biomol Spectrosc ; 84(1): 74-85, 2011 Dec 15.
Article in English | MEDLINE | ID: mdl-21975044

ABSTRACT

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.


Subject(s)
Drug Delivery Systems , Fibroblasts/cytology , Gene Transfer Techniques , Spectroscopy, Fourier Transform Infrared/methods , T-Lymphocytes/cytology , Ultrasonics , Amides/chemistry , Animals , Cell Nucleus Division , Cytokinesis , Humans , Jurkat Cells , Lipids/chemistry , Mice , Micronucleus Tests , NIH 3T3 Cells , Nucleic Acids/chemistry , Principal Component Analysis , Protein Structure, Secondary , Sonication
11.
Int J Radiat Biol ; 86(1): 37-46, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20070214

ABSTRACT

PURPOSE: The experiment was performed to prove that exposure to a relatively weak extremely low frequency (ELF) magnetic field retards tadpoles' development. METHODS: Two cohorts of Xenopus laevis laevis (Daudin) tadpoles were exposed during their immature period ( approximately 60 days) to a 50 Hz magnetic field of 63.9 < or = B < or = 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. RESULTS: (a) Mean developmental rate of exposed cohorts was reduced with respect to controls (0.43 vs. 0.48 stages/day, p < 0.001) starting from early larval stages; (b) Exposure increased the mean metamorphosis period of tadpoles by 2.4 days compared with the controls (p < 0.001); (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. CONCLUSION: 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.


Subject(s)
Electromagnetic Fields , Larva/radiation effects , Magnetics , Metamorphosis, Biological/radiation effects , Xenopus laevis/growth & development , Animals , Larva/growth & development , Time Factors
12.
J Exp Clin Cancer Res ; 27: 16, 2008 Jul 03.
Article in English | MEDLINE | ID: mdl-18598357

ABSTRACT

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(microT)) levels is between 0,05 microT and 80 microT. The statistical distribution shows that most of the measurements are in the range 0,05

Subject(s)
Electromagnetic Fields/adverse effects , Environmental Exposure , Environmental Health , Environmental Monitoring , Humans , Italy , Occupational Exposure , Occupational Health , Radiation Monitoring , Risk Assessment
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