Cécile Sicard-Roselli

Parameters governing gold nanoparticle X-ray radiosensitization of DNA in solution.

Radiosensitization by gold nanoparticles (GNP) is a promising approach for improving radiotherapy. We report herein the results of an investigation of three key-parameters governing such radiosensitization in DNA, namely, DNA:GNP molar ratio, GNP diameter and incident X-ray energy. We performed irradiations with a clinical orthovoltage source and tested concentration ratios up to 1:1, five sizes of GNP from 8 to 92 nm and six effective X-ray energies from 14.8 to 70 keV. The most efficient parameters are found to be large-sized GNP, high molar concentration and 50-keV photons, which could potentially result in a dose enhancement factor of 6. The relevance of such parameters as regards the development of future therapeutic applications is discussed. To the best of our knowledge, this study constitutes the first report of systematic data on radiosensitization by GNP.

Damage induced to DNA by low-energy (0-30 eV) electrons under vacuum and atmospheric conditions.

In this study, we show that it is possible to obtain data on DNA damage induced by low-energy (0-30 eV) electrons under atmospheric conditions. Five monolayer films of plasmid DNA (3197 base pairs) deposited on glass and gold substrates are irradiated with 1.5 keV X-rays in ultrahigh vacuum and under atmospheric conditions. The total damage is analyzed by agarose gel electrophoresis. The damage produced on the glass substrate is attributed to energy absorption from X-rays, whereas that produced on the gold substrate arises from energy absorption from both the X-ray beam and secondary electrons emitted from the gold surface. By analysis of the energy of these secondary electrons, 96% are found to have energies below 30 eV with a distribution peaking at 1.4 eV. The differences in damage yields recorded with the gold and glass substrates is therefore essentially attributed to the interaction of low-energy electrons with DNA under vacuum and hydrated conditions. From these results, the G values for low-energy electrons are determined to be four and six strand breaks per 100 eV, respectively.

Radiation chemistry of proteins

Publisher Summary This chapter reviews most recent data on radiation chemistry of proteins and provides an overview of the unknown aspects in protein radiation chemistry as well as in some of the expected biological consequences of protein radiolytic modifications. Under irradiation, proteins are affected by direct and indirect effects of ionizing radiations. When these macromolecules are in liquid solution, the indirect effects are predominant and the direct effects can be neglected. On the contrary, in a solid state, proteins are ionized mainly by direct interaction. Proteins in a solid state can be found in different forms, lyophilized or in frozen aqueous solution. Under irradiation, lyophilized proteins mostly aggregate. On the contrary, irradiation of frozen protein solutions gives rise to fragmentation. In an aqueous solution, the first step is the reaction of free radicals. This reaction proceeds with a rate constant that varies with the nature of the free radical but very little with the protein. The chemical nature of the resulting odd-electron site(s) can be hypothesized, but prediction of its location is still difficult. The reactivity of residues depends on many factors, including accessibility, neighboring residues, and electrostatic guidance.

Impact of gold nanoparticles combined to X-Ray irradiation on bacteria

Recent increase of multi drug-resistant bacteria represents a crucial issue of public health. As novative approaches are required to face that problem, those emerging from nanotechnology are of great interest. In that context we propose the possibility to use gold nanoparticles combined with ionising radiation to destroy pathogenic bacteria. For that, we investigated the potential X-Rays enhanced reduction of bacterial cell viability, following nanoparticle exposure, on a bacterial model,Escherichia coli. Our first concern was to confirm the absence of toxicity of the colloidal solution used. Then, we developed an X-Ray irradiation system and showed that gold nanoparticles increased the efficiency of ionising radiation to induce bacteria cell death.

Gold nanoparticles functionalization notably decreases radiosensitization through hydroxyl radical production under ionizing radiation

The purpose of this work was to study the influence of gold nanoparticles (GNP) coating on hydroxyl radical (HO) production under ionizing radiation. Though radiosensitizing mechanisms are still unknown, radical oxygen species are likely to be involved, especially HO. We synthesized six different types of GNP, choosing relevant ligands such as polyethylene glycol or human serum albumin. A great attention was paid to characterize these GNP in terms of size, charge and number of atoms in the coating. Our results show that functionalization dramatically decreases HO production, which is correlated to reduced plasmidic DNA damages. These findings are of high importance as GNP translation from fundamental research to applied medicine requires their functionalization to increase blood circulation time and specific cancerous cells addressing. We suggest that to keep GNP efficient for radiotherapy, a wispy coating is required.

Electronic emission of radio-sensitizing gold nanoparticles under X-ray irradiation: experiment and simulations

In this paper we present new results on electronic emission of Gold Nanoparticles (GNPs) using X-ray photoelectron spectroscopy (XPS) and compare them to the gold bulk electron emission. This subject has undergone new interest within the perspective of using GNPs as a radiotherapy enhancer. The experimental results were simulated using various models (Livermore and PENELOPE) of the Geant 4 simulation toolkit dedicated to the calculation of the transportation of particles through the matter. Our results show that the GNPs coating is a key parameter to correctly construe the experimental GNPs electronic emission after X-ray irradiation and point out some limitations of the PENELOPE model. Using XPS spectra and Geant4 Livermore simulations,we propose a method to determine precisely the coating surface density of the GNPs. We also show that the expected intrinsic nanoscale electronic emission enhancement effect suspected to contribute to the GNPs radio-sensitizing properties participates at most for a few percent of the global electronic emission spectra of the GNPs compared to gold bulk. ∗Electronic address: romain.casta@irsamc.ups-tlse.frIn this paper, we present new results on electronic emission of Gold Nanoparticles (GNPs) using X-ray photoelectron spectroscopy (XPS) and compare them to the gold bulk electron emission. This subject has undergone new interest within the perspective of using GNPs as a radiotherapy enhancer. The experimental results were simulated using various models (Livermore and PENELOPE) of the Geant4 simulation toolkit dedicated to the calculation of the transportation of particles through the matter. Our results show that the GNPs coating is a key parameter to correctly construe the experimental GNPs electronic emission after X-ray irradiation and point out some limitations of the PENELOPE model. Using XPS spectra and Geant4 Livermore simulations, we propose a method to determine precisely the coating surface density of the GNPs. We also show that the expected intrinsic nano-scale electronic emission enhancement effect—suspected to contribute to the GNPs radio-sensitizing properties—participates at most for a few percent of the global electronic emission spectra of the GNPs compared to gold bulk.

Radiolysis of proteins in the solid state: an approach by EPR and product analysis.

Radio-induced modifications in proteins have been studied using several techniques. Electron paramagnetic resonance (EPR) was used to characterize free radicals, and analysis methods (high-performance liquid chromatography, capillary electrophoresis) were employed to visualize final degraded forms. Whereas EPR indicates that perthiyl radicals are formed, analysis does not detect any compound in which such bonds would be broken. Since EPR signals decay with time, it is concluded that rearrangements occur at subsequent steps, in which the solvent used during the analysis might play a role.

Oxidative stress induces mainly human centrin 2 polymerisation.

Purpose: To determine the human centrin 2 (Hscen 2) protein response to oxidising radicals in vitro and to evaluate the consequences on its biological functions. Materials and methods: Hscen 2 was submitted to hydroxyl and azide radicals produced by radiolysis in the absence of oxygen. The resulting products were characterised by biochemical, spectroscopic and mass spectrometry techniques. Their thermodynamics parameters of complexation with C-terminal fragment of Xeroderma pigmentosum C protein (C-XPC), one of the Hscen 2 cellular partners, were quantified by isothermal titration calorimetry (ITC). Results: Both hydroxyl and azide radicals induce centrin 2 polymerisation as we characterised several intermolecular cross-links generating dimers, trimers, tetramers and higher molecular mass species. These cross-links result from the formation of a covalent bond between the only tyrosine residue (Tyr 172) located in the C-terminal region of each monomer. Remarkably, dimerisation occurs for doses as low as a few grays. Moreover, this Hscen2 dimer has a lower affinity and stoechiometry binding to C-XPC. Conclusions: These results show that as oxidative radicals induce high proportions of irreversible damages (polymerisation) centrin 2 is highly sensitive to ionising radiation. This could have important consequences on its biological functions.

A novel cryo-reduction method to investigate the molecular mechanism of nitric oxide synthases.

Nitric oxide synthases (NOSs) are hemoproteins responsible for the biosynthesis of NO in mammals. They catalyze two successive oxidation reactions. The mechanism of oxygen activation is based on the transfer of two electrons and two protons. Despite structural analogies with cytochromes P450, the molecular mechanism of NOS remains yet to be elucidated. Because of extremely high reaction rates, conventional kinetics methods failed to trap and characterize the major reaction intermediates. Cryo-reduction methods offer a possibility to circumvent this technological lock, by triggering oxygen activation at cryogenic temperatures by using water radiolysis. However, this method is not adapted to the NOS mechanism because of the high instability of the initial Fe(II)O2 complex (extremely fast autoxidation and/or reaction with the cofactor H4B). This imposed a protocol with a stable Fe(II)O2 complex (observed only for one NOS-like protein) and that excludes any redox role for H4B. A relevant approach to the NOS mechanism would use H4B to provide the (second) electron involved in oxygen activation; water radiolysis would thus provide the first electron (heme reduction). In this context, we report here an investigation of the first electron transfer by this alternative approach, i.e., the reduction of native NOS by water radiolysis. We combined EPR and resonance Raman spectroscopies to analyze NOS reduction for a combination of different substrates, cofactor, and oxygen concentrations, and for different NOS isoforms. Our results show that cryo-reduction of native NOS is achieved for all conditions that are relevant to the investigation of the NOS mechanism.

A novel experimental approach to investigate radiolysis processes in liquid samples using collimated radiation sources

Here is detailed a novel and low-cost experimental method for high-throughput automated fluid sample irradiation. The sample is delivered via syringe pump to a nozzle, where it is expressed in the form of a hanging droplet into the path of a beam of ionising radiation. The dose delivery is controlled by an upstream lead shutter, which allows the beam to reach the droplet for a user defined period of time. The droplet is then further expressed after irradiation until it falls into one well of a standard microplate. The entire system is automated and can be operated remotely using software designed in-house, allowing for use in environments deemed unsafe for the user (synchrotron beamlines, for example). Depending on the number of wells in the microplate, several droplets can be irradiated before any human interaction is necessary, and the user may choose up to 10 samples per microplate using an array of identical syringe pumps, the design of which is described here. The nozzles consistently produce droplets of 25.1 ± 0.5 μl.