Quantifying hydroxyl radicals generated by a low-temperature plasma using coumarin: methodology and precautions.

The detection and quantification of hydroxyl radicals (HO˙) generated by low-temperature plasmas (LTPs) are crucial for understanding their role in diverse applications of plasma radiation. In this study, the formation of HO˙ in the irradiated aqueous phase is investigated at various plasma parameters, by probing them indirectly using the coumarin molecule. We propose a quantification methodology for these radicals, combining spectrophotometry to study the coumarin reaction with hydroxyl radicals and fluorimetry to evaluate the formation yield of the hydroxylated product, 7-hydroxycoumarin. Additionally, we thoroughly examine and discuss the impact of pH on this quantification process. This approach enhances our comprehension of HO˙ formation during LTP irradiation, adding valuable insights to plasma's biological applications.

In-depth theoretical understanding of the chemical interaction of aromatic compounds with a gold nanoparticle.

Gold Nanoparticles (GNPs), owing to their unique properties and versatile preparation strategy, have been demonstrated to exhibit promising applications in diverse fields, which include bio-sensors, catalysts, nanomedicines and radiotherapy. Yet, the nature of the interfacial interaction of GNPs with their chemical environment remains elusive. Experimental vibrational spectroscopy can reveal different interactions of aromatic biological molecules absorbed on GNPs, that may result from changes in the orientation of the molecule. However, the presence of multiple functional groups and the aqueous solvent introduces competition, and complexifies the spectral interpretations. Therefore, our objective is to theoretically investigate the adsorption of aromatic molecules containing various functional groups on the surface of GNPs to comparatively study their preferred adsorption modes. The interaction between Au32, as a model of GNPs, and a series of substituted aromatic compounds that includes benzene, aniline, phenol, toluene, benzoic acid, acetophenone, methyl benzoate, and thiophenol, is investigated. Our computed interaction energies highlight the preference of the aromatic ring to lie flat on the surface. The orientations of the molecules can be distinguished using infrared spectroscopy along with strong changes in intensity and significant shifts of some vibrational modes when the molecule interacts with the GNP. The interaction energy and the electron transfer between the nanoparticle and the aromatic molecule are not found to correlate, possibly because of significant back donation of electrons from GNPs to organic molecules as revealed by charge decomposition analysis. A thorough quantum topological analysis identifies multiple non-covalent interactions and assigns the nature of the interaction mostly to dative interactions between the aromatic ring and the GNP as well as dispersive interaction. Finally, energy decomposition analyses point out the role of the charge transfer energy contribution in the subtle balance of the different physical components.

Protein Dimerization via Tyr Residues: Highlight of a Slow Process with Co-Existence of Numerous Intermediates and Final Products.

Protein dimerization via tyrosine residues is a crucial process in response to an oxidative attack, which has been identified in many ageing-related pathologies. Recently, it has been found that for isolated tyrosine amino acid, dimerization occurs through three types of tyrosine-tyrosine crosslinks and leads to at least four final products. Herein, considering two protected tyrosine residues, tyrosine-containing peptides and finally proteins, we investigate the dimerization behavior of tyrosine when embedded in a peptidic sequence. After azide radical oxidation and by combining UPLC-MS and H/D exchange analyzes, we were able to evidence: (i) the slow kinetics of Michael Addition Dimers (MAD) formation, i.e., more than 48 h; (ii) the co-existence of intermediates and final cyclized dimer products; and (iii) the probable involvement of amide functions to achieve Michael additions even in proteins. This raises the question of the possible in vivo existence of both intermediates and final entities as well as their toxicity and the potential consequences on protein structure and/or function.

Advanced methodology combining UPLC-MS, isotopic labelling and H/D exchanges reveals three tyrosine-tyrosine cross-links induced by oxidative radicals evolving to at least four dimeric structures.

Di-tyrosine is one of the major protein cross-links involved in a large number of neurodegenerative or ageing-related diseases. Recently, no less than four different di-tyrosine bridge isomers have been highlighted while only two structures are characterized at the moment in the literature. In this study, the four dimers were produced by radiolytical-induced oxidation. Although the abundance of these additional dimers precluded the use of NMR or other structural characterization methods, we propose a new methodology combining UPLC-MS analysis, specific deuterium labelling and isotopic (H/D) exchanges with the solvent. Thus, we were able to identify three different covalent cross-links and propose different new original di-tyrosine structures based on double Michael additions, leading to tetracyclic products. Absorption and fluorescence characterizations of the four species were performed and consolidate our proposal.

New Insights into the Reactivity of Detonation Nanodiamonds during the First Stages of Graphitization.

The present study aims to compare the early stages of graphitization of the same DND source for two annealing atmospheres (primary vacuum, argon at atmospheric pressure) in an identical set-up. DND samples are finely characterized by a combination of complementary techniques (FTIR, Raman, XPS, HR-TEM) to highlight the induced modifications for temperature up to 1100 °C. The annealing atmosphere has a significant impact on the graphitization kinetics with a higher fraction of sp2-C formed under vacuum compared to argon for the same temperature. Whatever the annealing atmosphere, carbon hydrogen bonds are created at the DND surface during annealing according to FTIR. A "nano effect", specific to the <10 nm size of DND, exalts the extreme surface chemistry in XPS analysis. According to HR-TEM images, the graphitization is limited to the first outer shell even for DND annealed at 1100 °C under vacuum.

Probing the structural properties of the water solvation shell around gold nanoparticles: A computational study.

While subjected to radiation, gold nanoparticles (GNPs) have been shown to enhance the production of radicals when added to aqueous solutions. It has been proposed that the arrangement of water solvation layers near the water-gold interface plays a significant role. As such, the structural and electronic properties of the first water solvation layer surrounding GNPs of varying sizes were compared to bulk water using classical molecular dynamics and quantum and semi-empirical methods. Classical molecular dynamics was used to understand the change in macroscopic properties of bulk water in the presence of different sizes of GNP, as well as by including salt ions. The analysis of these macroscopic properties has led to the conclusion that larger GNPs induce the rearrangement of water molecules to form a 2D hydrogen-bond network at the interface. Quantum methods were employed to understand the electronic nature of the interaction between water molecules and GNPs along with the change in the water orientation and the vibrational density of states. The stretching region of vibrational density of states was found to extend into the higher wavenumber region, as the size of the GNP increases. This extension represents the dangling water molecules at the interface, as a result of reorientation of the water molecules in the first solvation shell. This multi-level study suggests that in the presence of GNP of increasing sizes, the first water solvation shell undergoes a rearrangement to maximize the water-water interactions as well as the water-GNP interactions.

Oxidative radicals (HO• or N3•) induce several di-tyrosine bridge isomers at the protein scale.

Among protein oxidative damages, di-tyrosine bridges formation has been evidenced in many neuropathological diseases. Combining oxidative radical production by gamma radiolysis with very performant chromatographic separation coupled to mass spectrometry detection, we brought into light new insights of tyrosine dimerization. Hydroxyl and azide radical tyrosine oxidation leading to di-tyrosine bridges formation was studied for different biological compounds: a full-length protein (Δ25-centrin 2), a five amino acid peptide (KTSLY) and free tyrosine. We highlighted that both radicals generate high proportion of dimers even for low doses. Surprisingly, no less than five different di-tyrosine isomers were evidenced for the protein and the peptide. For tyrosine alone, at least four distinct dimers were evidenced. These results raise some questions about their respective role in vivo and hence their relative toxicity. Also, as di-tyrosine is often used as a biomarker, a better knowledge of the type of dimer detected in vivo is now required.

Quality control of gold nanoparticles as pharmaceutical ingredients.

Nanoparticles are being developed for a wide range of medical applications such as, controlled release, drug delivery systems or imagery, theranostics, implants…. For the moment, there is no legal definition of nanoparticles or nanomaterials for therapeutic use. The specific case of gold nanoparticles is not an exception: their current definition as nanoparticle material does not correspond to classic pharmaceutical ingredients as described in Pharmacopoeias. In this study, more than 30 different batches of citrate stabilized gold nanoparticles (AuNP) were synthesized and analyzed thanks to both classical approaches (UV-Vis spectrophotometry, dynamic light scattering coupled or not to electrophoresis …) and capillary zone electrophoresis (CZE) coupled to diode array detection to assess their purity and impurity profiles. These techniques led to the beginning of defined specifications, a key step for the use of gold nanoparticles as pharmaceutical ingredients. CZE was demonstrated suitable to evaluate a batch-to-batch quality control, to monitor the purification processes and to follow the stability of 18 different batches for 20 days. Finally, commercially available AuNP samples were tested and the results compared to the provided certificates of analysis.

Metallic bismuth nanoparticles: Towards a robust, productive and ultrasound assisted synthesis from batch to flow-continuous chemistry.

Bismuth is a highly biocompatible and inexpensive metal with a high atomic number, which confers an important X-rays opacity. While bismuth oxide or bismuth sulphide have been extensively studied in imaging, little is known about metallic bismuth nanoparticles. The latter are more attractive for X-rays imaging because they contain neither oxygen nor sulfur, so that a high amount of metal atoms is contained within the nanoparticles. We report here a robust, efficient and green ultrasound assisted synthesis to obtain metallic bismuth NPs. The procedure, which has been optimized to get a reproducible synthesis, will also tend to minimize chemical hazards to health and environment. By applying the green chemistry principles, several experimental parameters have been studied such as reaction time, reactants stoichiometry, temperature, starting material quantities and purification steps number. Two energy delivery system (classical heating and sonication) were compared. The production of small metallic bismuth NPs on a large scale by flow chemistry coupled to sonication was showed for the first time. These optimizations of the process were completed by a comparison of two purification methods (centrifugation and ultrafiltration) to isolate purified thin black powders of d-glucose-coated bismuth NPs. Several analytical techniques were used to characterize products (structures, sizes and morphology) such as Fourier Transform InfraRed (FT-IR) spectroscopy, Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray spectrometry (EDX) and X-Ray Diffraction (XRD). All these analyses corroborated well with the structure of metallic bismuth NPs coated with a d-glucose shell.

Quantification of hydroxyl radicals and solvated electrons produced by irradiated gold nanoparticles suggests a crucial role of interfacial water.

The potential benefit of gold nanoparticles (GNP) to radiotherapy has been demonstrated in a range of cell lines and radiation sources as well as in rodent models, sometimes with contradictory results. Few experimental studies have explored the involved deleterious species, hydroxyl radical being so far the most cited, whereas theoretical studies have usually focused on secondary electrons emitted from GNP, making comparison between these two approaches difficult. Here we focus on the physico-chemical step (i.e. radical production) and report the first experimental determination of both hydroxyl radicals and solvated electrons yields of formation in the presence of GNP. We also compare these yields between X- and γ-rays under different atmospheres. Our main finding is a massive and equivalent production of both species under X- and more surprisingly γ-rays. For concentration as low as 1 nM (0.02% wt of gold), both radiations lead to 3 to 4 times more radicals than water radiolysis without GNP. This is in contradiction with a physical prediction of dose enhancement. Supported by our whole set of experiments the key role of water molecules at the nanoparticle interface in the radical production emerges. This leads us to propose the paramount importance of the physico-chemical step compared to the physical one. Classical approaches based on energy-absorption coefficients and electron ejections should therefore be revisited.

Hydroxyl radical production induced by plasma hydrogenated nanodiamonds under X-ray irradiation.

For the first time, overproduction of hydroxyl radicals (HO˙) induced by plasma hydrogenated detonation nanodiamonds (H-NDs) under X-ray irradiation is reported. Using coumarin (COU) as a fluorescent probe, we reveal a significant increase of 40% of the HO˙ production in the presence of H-NDs (6-100 µg ml-1) compared with water alone. This effect is related to the negative electron affinity of the hydrogenated nanodiamonds and illustrates the ability of H-NDs to produce reactive oxygen species probably via electron emission in water under X-ray irradiation.

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.

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.

How protein structure affects redox reactivity: example of Human centrin 2.

Electron transfer inside proteins plays a central role in their reactivity and biological functions. Herein, we developed a combined approach by gamma radiolysis and electrochemistry which allowed a deep insight into the reactivity of Human centrin 2, a protein very sensitive to oxidative stress and involved in several key biological processes. This protein bears a single terminal tyrosine and was observed to be extremely sensitive to ionizing radiation sources, leading to a tyrosine dimer. By cyclic voltammetry in the 100-1000 V s(-1) range, its redox potential and dimerization rate could be evaluated. Accordingly, reaction in solution with a redox mediator revealed an efficient catalysis. Finally, protein denaturation by a progressive increase in temperature was proportional to a decrease of dimerization radiolytic yield. Our results thus demonstrated that the protein structure plays a major role in oxidation sensitivity. This leads to meaningful results to understand protein redox reactivity.

Could nanoparticle corona characterization help for biological consequence prediction?

As soon as they enter a biological medium (cell culture medium for in vitro, blood or plasma for in vivo studies), nanoparticles, in most cases, see their surface covered by biomolecules, especially proteins. What the cells see is thus not the ideal nanoparticle concocted by chemists, meaning the biomolecular corona could have great biological and physiological repercussions, sometimes masking the expected effects of purposely grafted molecules. In this review, we will mainly focus on gold nanoparticles. In the first part, we will discuss the fate of these particles once in a biological medium, especially in terms of size, and the protein composition of the corona. We will highlight the parameters influencing the quantity and the identity of the adsorbed proteins. In a second part, we will resume the main findings about the influence of a biomolecular corona on cellular uptake, toxicity, biodistribution and targeting ability. To be noticed is the need for standardized experiments and very precise reports of the protocols and methods used in the experimental sections to extract informative data. Given the biological consequences of this corona, we suggest that it should be taken into account in theoretical studies dealing with nanomaterials to better represent the biological environment.

A new mechanism for hydroxyl radical production in irradiated nanoparticle solutions.

The absolute yield of hydroxyl radicals per unit of deposited X-ray energy is determined for the first time for irradiated aqueous solutions containing metal nanoparticles based on a "reference" protocol. Measurements are made as a function of dose rate and nanoparticle concentration. Possible mechanisms for hydroxyl radical production are considered in turn: energy deposition in the nanoparticles followed by its transport into the surrounding environment is unable to account for observed yield whereas energy deposition in the water followed by a catalytic-like reaction at the water-nanoparticle interface can account for the total yield and its dependence on dose rate and nanoparticle concentration. This finding is important because current models used to account for nanoparticle enhancement to radiobiological damage only consider the primary interaction with the nanoparticle, not with the surrounding media. Nothing about the new mechanism appears to be specific to gold, the main requirements being the formation of a structured water layer in the vicinity of the nanoparticle possibly through the interaction of its charge and the water dipoles. The massive hydroxyl radical production is relevant to a number of application fields, particularly nanomedicine since the hydroxyl radical is responsible for the majority of radiation-induced DNA damage.

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.

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.

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.

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.