Membrane reorganization after photochemical internalization to release transferrin-biofunctionalized polystyrene microparticles.

Therapeutic drug carriers can drive their cargo to their target cells. However, an obstacle is usually the entrapment of the drug inside the endolysosomal compartment, which physically impedes its actuation by the impossibility of reaching its molecular site of action. To overcome this hurdle, photochemical internalization (PCI) has been proposed, but the extent of PCI-induced membrane disruption and its capability to allow the release of microparticles is unknown. The aim of the present study was to determine if PCI allows the release of microparticles from the endolysosomal compartment to the cytosol and to analyze at the ultrastructural level the effect of PCI on the membrane surrounding the particles. Confocal microscope allowed us to detect that endolysosomal membranes suffered some disruption after PCI, evidenced by the diffusion of soluble transferrin from the endolysosomes to the cytosol and by a decrease of LAMP1-microparticles co-localization. Transmission electron microscopy (TEM) showed a decrease in the number of well-defined membranes around microparticles after PCI, and scanning TEM combined with energy dispersive x-ray revealed an increase in the width of endolysosomal membranes after treatment. These results suggest that endolysosomal membranes suffered an ultrastructure alteration after PCI, enough to liberate soluble transferrin but not the entire microparticles.

Electromechanical Nanogenerator-Cell Interaction Modulates Cell Activity.

Noninvasive methods for in situ electrical stimulation of human cells open new frontiers to future bioelectronic therapies, where controlled electrical impulses could replace the use of chemical drugs for disease treatment. Here, this study demonstrates that the interaction of living cells with piezoelectric nanogenerators (NGs) induces a local electric field that self-stimulates and modulates their cell activity, without applying an additional chemical or physical external stimulation. When cells are cultured on top of the NGs, based on 2D ZnO nanosheets, the electromechanical NG-cell interactions stimulate the motility of macrophages and trigger the opening of ion channels present in the plasma membrane of osteoblast-like cells (Saos-2) inducing intracellular calcium transients. In addition, excellent cell viability, proliferation, and differentiation are validated. This in situ cell-scale electrical stimulation of osteoblast-like cells can be extrapolated to other excitable cells such as neurons or muscle cells, paving the way for future bioelectronic medicines based on cell-targeted electrical impulses.

Investigation of the influence of calibration practices on cytogenetic laboratory performance for dose estimation.

PURPOSE: In the frame of the QA program of RENEB, an inter-laboratory comparison (ILC) of calibration sources used in biological dosimetry was achieved to investigate the influence of calibration practices and protocols on the results of the dose estimation performance as a first step to harmonization and standardization of dosimetry and irradiation practices in the European biological dosimetry network. MATERIALS AND METHODS: Delivered doses by irradiation facilities used by RENEB partners were determined with EPR/alanine dosimetry system. Dosimeters were irradiated in the same conditions as blood samples. A short survey was also performed to collect the information needed for the data analysis and evaluate the diversity of practices. RESULTS: For most of partners the deviation of delivered dose from the targeted dose remains below 10%. Deviations larger than 10% were observed for five facilities out of 21. Origins of the largest discrepancies were identified. Correction actions were evaluated as satisfactory. The re-evaluation of some ILC results for the fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC) assays has been performed leading to an improvement of the overall performances. CONCLUSIONS: This work has shown the importance of dosimetry in radiobiology studies and the needs of harmonization, standardization in irradiation and dosimetry practices and educational training for biologists using ionizing radiation.

Uncertainty of fast biological radiation dose assessment for emergency response scenarios.

PURPOSE: Reliable dose estimation is an important factor in appropriate dosimetric triage categorization of exposed individuals to support radiation emergency response. MATERIALS AND METHODS: Following work done under the EU FP7 MULTIBIODOSE and RENEB projects, formal methods for defining uncertainties on biological dose estimates are compared using simulated and real data from recent exercises. RESULTS: The results demonstrate that a Bayesian method of uncertainty assessment is the most appropriate, even in the absence of detailed prior information. The relative accuracy and relevance of techniques for calculating uncertainty and combining assay results to produce single dose and uncertainty estimates is further discussed. CONCLUSIONS: Finally, it is demonstrated that whatever uncertainty estimation method is employed, ignoring the uncertainty on fast dose assessments can have an important impact on rapid biodosimetric categorization.

The RENEB operational basis: complement of established biodosimetric assays.

PURPOSE: To set up an operational basis of the Realizing the European Network of Biodosimetry (RENEB) network within which the application of seven established biodosimetric tools (the dicentric assay, the FISH assay, the micronucleus assay, the PCC assay, the gamma-H2AX assay, electron paramagnetic resonance and optically stimulated luminescence) will be compared and standardized among the participating laboratories. METHODOLOGY: Two intercomparisons were organized where blood samples and smartphone components were irradiated, coded and sent out to participating laboratories for dosimetric analysis. Moreover, an accident exercise was organized during which each RENEB partner had the chance to practice the procedure of activating the network and to handle large amounts of dosimetric results. RESULTS: All activities were carried out as planned. Overall, the precision of dose estimates improved between intercomparisons 1 and 2, clearly showing the value of running such regular activities. CONCLUSIONS: The RENEB network is fully operational and ready to act in case of a major radiation emergency. Moreover, the high capacity for analyzing radiation-induced damage in cells and personal electronic devices makes the network suitable for large-scale analyses of low doses effects, where high numbers of samples must be scored in order to detect weak effects.

RENEB - Running the European Network of biological dosimetry and physical retrospective dosimetry.

PURPOSE: A European network was initiated in 2012 by 23 partners from 16 European countries with the aim to significantly increase individualized dose reconstruction in case of large-scale radiological emergency scenarios. RESULTS: The network was built on three complementary pillars: (1) an operational basis with seven biological and physical dosimetric assays in ready-to-use mode, (2) a basis for education, training and quality assurance, and (3) a basis for further network development regarding new techniques and members. Techniques for individual dose estimation based on biological samples and/or inert personalized devices as mobile phones or smart phones were optimized to support rapid categorization of many potential victims according to the received dose to the blood or personal devices. Communication and cross-border collaboration were also standardized. To assure long-term sustainability of the network, cooperation with national and international emergency preparedness organizations was initiated and links to radiation protection and research platforms have been developed. A legal framework, based on a Memorandum of Understanding, was established and signed by 27 organizations by the end of 2015. CONCLUSIONS: RENEB is a European Network of biological and physical-retrospective dosimetry, with the capacity and capability to perform large-scale rapid individualized dose estimation. Specialized to handle large numbers of samples, RENEB is able to contribute to radiological emergency preparedness and wider large-scale research projects.

Integration of new biological and physical retrospective dosimetry methods into EU emergency response plans - joint RENEB and EURADOS inter-laboratory comparisons.

PURPOSE: RENEB, 'Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,' is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation. MATERIALS AND METHODS: The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation-induced thermoluminescent signals in glass screens taken from mobile phones. RESULTS: In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques. CONCLUSIONS: Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios.

Novel Fe-Mn-Si-Pd alloys: insights into mechanical, magnetic, corrosion resistance and biocompatibility performances.

Two new Fe-based alloys, Fe-10Mn6Si1Pd and Fe-30Mn6Si1Pd, have been fabricated by arc-melting followed by copper mold suction casting. The Fe-30Mn6Si1Pd alloy mainly consists of ε-martensite and γ-austenite Fe-rich phases whereas the Fe-10Mn6Si1Pd alloy primarily contains the α-Fe(Mn)-ferrite phase. Additionally, Pd-rich precipitates were detected in both alloys. Good mechanical response was observed by nanoindentation: hardness values around 5.6 GPa and 4.2 GPa and reduced Young's moduli of 125 GPa and 93 GPa were measured for the as-prepared Fe-10Mn6Si1Pd and Fe-30Mn6Si1Pd alloys, respectively. Both alloys are thus harder and exhibit lower Young's modulus than 316L stainless steel, which is one of the most common Fe-based reference materials used for biomedical applications. Compared with the ferromagnetic Fe-10Mn6Si1Pd alloy, the paramagnetic Fe-30Mn6Si1Pd alloy is more appropriate to be used as an implant since it would be compatible for nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) analyses. Concerning biocompatibility, the more hydrophilic Fe-10Mn6Si1Pd alloy shows improved cell adhesion but its pronounced ion leaching has a negative effect on the proliferation of cells. The influence of immersion in a simulated body fluid on the composition, microstructure, mechanical and magnetic properties of both alloys is assessed, and the correlation between microstructure evolution and physical properties is discussed.

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells.

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 µm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

A new porphyrin for the preparation of functionalized water-soluble gold nanoparticles with low intrinsic toxicity.

A potential new photosensitizer based on a dissymmetric porphyrin derivative bearing a thiol group was synthesized. 5-[4-(11-Mercaptoundecyloxy)-phenyl-10,15,20-triphenylporphyrin (PR-SH) was used to functionalize gold nanoparticles in order to obtain a potential drug delivery system. Water-soluble multifunctional gold nanoparticles GNP-PR/PEG were prepared using the Brust-Schiffrin methodology, by immobilization of both a thiolated polyethylene glycol (PEG) and the porphyrin thiol compound (PR-SH). The nanoparticles were fully characterized by transmission electron microscopy and (1)H nuclear magnetic resonance spectroscopy, UV/Vis absorption spectroscopy, and X-ray photoelectron spectroscopy. Furthermore, the ability of GNP-PR/PEGs to induce singlet oxygen production was analyzed to demonstrate the activity of the photosensitizer. Cytotoxicity experiments showed the nanoparticles are nontoxic. Finally, cellular uptake experiments demonstrated that the functionalized gold nanoparticles are internalized. Therefore, this colloid can be considered to be a novel nanosystem that could potentially be suitable as an intracellular drug delivery system of photosensitizers for photodynamic therapy.

Multibiodose radiation emergency triage categorization software.

In this note, the authors describe the MULTIBIODOSE software, which has been created as part of the MULTIBIODOSE project. The software enables doses estimated by networks of laboratories, using up to five retrospective (biological and physical) assays, to be combined to give a single estimate of triage category for each individual potentially exposed to ionizing radiation in a large scale radiation accident or incident. The MULTIBIODOSE software has been created in Java. The usage of the software is based on the MULTIBIODOSE Guidance: the program creates a link to a single SQLite database for each incident, and the database is administered by the lead laboratory. The software has been tested with Java runtime environment 6 and 7 on a number of different Windows, Mac, and Linux systems, using data from a recent intercomparison exercise. The Java program MULTIBIODOSE_1.0.jar is freely available to download from http://www.multibiodose.eu/software or by contacting the software administrator: MULTIBIODOSE-software@gmx.com.

Optimized immobilization of lectins using self-assembled monolayers on polysilicon encoded materials for cell tagging.

Self-assembled monolayers (SAMs) have been used for the preparation of functional microtools consisting of encoded polysilicon barcodes biofunctionalized with proteins of the lectin family. These hybrid microtools exploit the lectins ability for recognizing specific carbohydrates of the cell membrane to give an efficient system for cell tagging. This work describes how the control of the methodology for SAM formation on polysilicon surfaces followed by lectin immobilization has a crucial influence on the microtool biofunction. Several parameters (silanization time, silane molar concentration, type of solvent or deposition methodology) have been studied to establish optimal function. Furthermore, silanes incorporating different terminal groups, such as aldehyde, activated ester or epoxide groups were tested in order to analyze their chemical coupling with the biomolecules, as well as their influence on the biofunctionality of the immobilized protein. Two different lectins - wheat germ agglutinin (WGA) and phytohemagglutinin (PHA-L) - were immobilized, because they have different and specific cell recognition behaviour and exhibit different cell toxicity. In this way we can assess the effect of intrinsic bulk toxicity with that of the cell compatibility once immobilized as well as the importance of cell affinity. A variety of nanometrical techniques were used to characterize the active surfaces, and lectin immobilization was quantified using ultraviolet-visible absorption spectroscopy (UV-vis) and optical waveguide light mode spectroscopy (OWLS). Once the best protocol was found, WGA and PHA were immobilized on polysilicon coded barcodes, and these microtools showed excellent cell tagging on living mouse embryos when WGA was used.

Novel Ti-Zr-Hf-Fe Nanostructured Alloy for Biomedical Applications.

The synthesis and characterization of Ti40Zr20Hf20Fe20 (atom %) alloy, in the form of rods (f = 2 mm), prepared by arc-melting, and subsequent Cu mold suction casting, is presented. The microstructure, mechanical and corrosion properties, as well as in vitro biocompatibility of this alloy, are investigated. This material consists of a mixture of several nanocrystalline phases. It exhibits excellent mechanical behavior, dominated by high strength and relatively low Young's modulus, and also good corrosion resistance, as evidenced by the passive behavior in a wide potential window and the low corrosion current densities values. In terms of biocompatibility, this alloy is not cytotoxic and preosteoblast cells can easily adhere onto its surface and differentiate into osteoblasts.

Detection of circulating cancer cells using electrocatalytic gold nanoparticles.

A rapid cancer cell detection and quantification assay, based on the electrocatalytic properties of gold nanoparticles towards the hydrogen evolution reaction, is described. The selective labeling of cancer cells is performed in suspension, allowing a fast interaction between the gold nanoparticle labels and the target proteins expressed at the cell membrane. The subsequent electrochemical detection is accomplished with small volumes of sample and user-friendly equipment through a simple electrochemical method that generates a fast electrochemical response used for the quantification of nanoparticle-labeled cancer cells. The system establishes a selective cell-detection assay capable of detecting 4 × 10(3) cancer cells in suspension that can be extended to several other cells detection scenarios.

Simple monitoring of cancer cells using nanoparticles.

Here we present a new strategy for a simple and fast detection of cancer circulating cells (CTCs) using nanoparticles. The human colon adenocarcinoma cell line (Caco2) was chosen as a model CTC. Similarly to other adenocarcinomas, colon adenocarcinoma cells have a strong expression of EpCAM, and for this reason this glycoprotein was used as the capture target. We combine the capturing capability of anti-EpCAM functionalized magnetic beads (MBs) and the specific labeling through antibody-modified gold nanoparticles (AuNPs), with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction (HER) detection technique. The fully optimized process was used for the electrochemical detection of Caco2 cells in the presence of monocytes (THP-1), other circulating cells that could interfere in real blood samples. Therefore we obtained a novel and simple in situ-like sensing format that we applied for the rapid quantification of AuNPs-labeled CTCs in the presence of other human cells.

Internalization and cytotoxicity analysis of silicon-based microparticles in macrophages and embryos.

Microchips can be fabricated, using semiconductor technologies, at microscopic level to be introduced into living cells for monitoring of intracellular parameters at a single cell level. As a first step towards intracellular chips development, silicon and polysilicon microparticles of controlled shape and dimensions were fabricated and introduced into human macrophages and mouse embryos by phagocytosis and microinjection, respectively. Microparticles showed to be non-cytotoxic for macrophages and were found to be localized mainly inside early endosomes, in tight association with endosomal membrane, and more rarely in acidic compartments. Embryos with microinjected microparticles developed normally to the blastocyst stage, confirming the non-cytotoxic effect of the particles. In view of these results silicon and polysilicon microparticles can serve as the frame for future intracellular chips development and this technology opens the possibility of real complex devices to be used as sensors or actuators inside living cells.

Radiation effects in interventional radiology using biological and physical dosimetry methods: a case-control study.

Interventional radiologists and staff members are frequently exposed to protracted and fractionated low doses of ionizing radiation, which extend during all their professional activities. These exposures can derive, due to the irradiation of skin tissues and peripheral blood, in deterministic effects (radiodermitis, aged skin, hands depilation) or stochastic ones (skin and non-solid cancers incidence). Epidemiological studies of population exposed to ionizing radiation provide information of radio-induced effects. The radiation risk or radiological detriment has been estimated from a group of six exposed interventionist radiologists of the Hospital La Fe (Valencia, Spain). Dosimetry has been periodically registered from TLDs and wrist dosimeters (physical methods) and estimated through translocations in lymphocytes of peripheral blood (biological methods), by extrapolating the yield of translocations to their respective dose-effect curves. The probability of non-melanoma skin cancer and leukaemia (acute myelogenous, acute lymphocytic and chronic myelogenous leukaemia) incidence has been estimated through the software RADRISK. This software is based on a transport model from epidemiological studies of population exposed to external low-LET ionizing radiation [1]. Other non-solid carcinomas have not been considered due to their low statistical power, such as myeloid and non-Hodgkin lymphomas. The discrepancies observed between the physically recorded doses and biological estimated doses could indicate that exposed workers did not always wear their dosimeters or these dosimeters were not always exposed to the radiation field.