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Ionizing radiation induced transformations in materials were monitored through tracking of the generation and degradation processes of radical species. Consequently, the types and quantities of radicals were determined by electron spin resonance (ESR). Subsequently, differential scanning calorimetry (DSC) was utilized to assess the impact of irradiation on the material crystallinity. The effects of gamma rays, X-rays, and electron beams were investigated on different polyamides, which exhibit an ESR signal up to 60 days. DSC results showed no significant effect of irradiation on the melting peak temperature of the materials, indicating that the amount of radicals generated was not large enough to induce a significant alteration of the material's macrostructure.
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The expansion of drug resistant parasites sheds a serious concern on several neglected parasitic diseases. Our recent results on cancer led us to envision the use of peptide-alkoxyamines as a highly selective and efficient new drug against schistosome adult worms, the etiological agents of schistosomiasis. Indeed, the peptide tag of the hybrid compounds can be hydrolyzed by worm's digestive enzymes to afford a highly labile alkoxyamine which homolyzes spontaneously and instantaneously into radicals-which are then used as a drug against Schistosome adult parasites. This approach is nicely summarized as digging their graves with their forks. Several hybrid peptide-alkoxyamines were prepared and clearly showed an activity: two of the tested compounds kill 50% of the parasites in two hours at a concentration of 100 µg/mL. Importantly, the peptide and alkoxyamine fragments that are unable to generate alkyl radicals display no activity. This strong evidence validates the proposed mechanism: a specific activation of the prodrugs by the parasite proteases leading to parasite death through in situ alkyl radical generation.
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Metal-organic frameworks (MOFs) draw increasing attention as nanoenvironments for chemical reactions, especially in the field of catalysis. Knowing the specifics of MOF cavities is decisive in many of these cases; yet, obtaining them in situ remains very challenging. We report the first direct assessment of the apparent polarity and solvent organization inside MOF cavities using a dedicated structurally flexible spin probe. A stable ß-phosphorylated nitroxide radical was incorporated into the cavities of a prospective MOF ZIF-8 in trace amounts. The spectroscopic properties of this probe depend on local polarity, structuredness, stiffness and cohesive pressure and can be precisely monitored by Electron Paramagnetic Resonance (EPR) spectroscopy. Using this approach, we have demonstrated experimentally that the cavities of bare ZIF-8 are sensed by guest molecules as highly non-polar inside. When various alcohols fill the cavities, remarkable self-organization of solvent molecules is observed leading to a higher apparent polarity in MOFs compared to the corresponding bulk alcohols. Accounting for such nanoorganization phenomena can be crucial for optimization of chemical reactions in MOFs, and the proposed methodology provides unique routes to study MOF cavities inside in situ, thus aiding in their various applications.
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The emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite's food vacuoles, our approach is summarized as "to dig its grave with its fork". However, despite promising sub-micromolar IC50 values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity.
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Antimaláricos , Citostáticos , Malaria Falciparum , Malaria , Humanos , Antimaláricos/química , Citostáticos/uso terapéutico , Malaria/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum , Péptidos/farmacología , Péptidos/uso terapéuticoRESUMEN
X-ray and electron-beam (E-beam) sterilization technologies were assessed to supplement gamma sterilization, the most common radiation technology used today for biopharmaceutical product sterilization. The mechanical properties of a PE/EVOH/PE film were studied using tensile tests and dynamical mechanical analysis after each irradiation technology (i.e., gamma, electron beam and X-ray irradiations). The effects of each irradiation were compared using two statistical methods. The results indicate that the three irradiation technologies induce no difference in mechanical properties in the investigated dose range for this material.
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This study analyzes the physico-chemical properties of interpenetrated polymer networks (IPNs) and semi-IPN resulting from cross-linking chitosan with glutaraldehyde and alginate with Ca2+ cations, as a function of the order in which the cross-linking agents are added to the polymer mixture. Three physico-chemical methods were used to assess the differences between systems: rheology, IR spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy. While rheology and IR spectroscopy are commonly used to characterize gel materials, EPR spectroscopy is rarely used, but has the advantage of providing local information about the dynamics of a system. The rheological parameters, which describe the global behavior of the samples, show that semi-IPN systems have a weaker gel behavior and the order of introducing the cross-linker in the polymer systems plays a role. The IR spectra of samples resulting by adding only Ca2+ or Ca2+ as the first cross-linker are similar to that of the alginate gel, while the spectra of samples in which glutaraldehyde is firstly added resemble the chitosan gel spectrum. Using spin-labeled alginate and spin-labeled chitosan, we monitored the changes occurring in the dynamic of the spin labels due to the formation of IPN and semi-IPN. The results show that the order of adding the cross-linking agents influences the dynamic of the IPN network, and that the formation of the alginate network determines the characteristics of the entire IPN system. The EPR data were correlated with the rheological parameters and IR spectra of the analyzed samples.
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Development of very-low field MRI is an active area of research. It aims at reducing operating costs and improve portability. However, the signal-to-noise issue becomes prominent at ultra-low field (<1 mT), especially for molecular imaging purposes that addresses specific biochemical events. In the context of preclinical molecular MRI of abnormal proteolysis the paper describes a MRI system able to produce Overhauser-enhanced MR images in living rats through in situ Dynamic Nuclear Polarization at 206 µT using stable and non-toxic nitroxides. In parallel conventional images are generated at 206 µT following pre-polarization at 20 mT. Results show that nitroxides are visualized in 3D within a few minutes in the lungs, kidneys and bladder post-administration. This system will be used for molecular imaging of inflammation using protease-specific nitroxide probes.
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Pulmón , Imagen por Resonancia Magnética , Ratas , Animales , Espectroscopía de Resonancia Magnética , Imagen por Resonancia Magnética/métodos , Óxidos de Nitrógeno/químicaRESUMEN
The design of smart nanoplatforms presenting well-definite structures able to achieve controlled cascade action remotely triggered by external stimuli presents a great challenge. We report here a new nanosystem consisting of magnetic iron oxide nanoparticles covalently grafted with a thermosensitive radical initiator alkoxyamine, able to provide controlled and localized release of free radicals triggered by an alternating current (ac) magnetic field. These nanoparticles exhibit a high intrinsic loss power of 4.73 nHm2 kg-1 providing rapid heating of their surface under the action of an ac field, inducing the homolysis of alkoxyamine C-ON bond and then the oxygen-independent formation of radicals. This latter was demonstrated by electronic paramagnetic resonance spectroscopy, and the kinetics of homolysis has been investigated allowing a comparison of the temperature of alkoxyamine's homolysis with the one measured during the magnetothermia process.
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Gamma-ray irradiation, using the cobalt-60 isotope, is the most common radiation modality used for medical device and biopharmaceutical products sterilization. Although X-ray and electron-beam (e-beam) sterilization technologies are mature and have been in use for decades, impediments remain to switching to these sterilization modalities because of lack of data on the resulting radiation effects for the associated polymers, as well as a lack of education for manufacturers and regulators on the viability of these sterilization alternatives. For this study, the compatibility of ethylene vinyl acetate (EVA) multilayer films with different ionizing radiation sterilization (X-ray, e-beam, and gamma irradiation) is determined by measuring chemical and physical film properties using high performance liquid chromatography, differential scanning calorimetry, Fourier-Transform InfraRed spectroscopy (FTIR), surface energy measurement, and electron spin resonance techniques. The results indicate that the three irradiation modalities induce no differences in thermal properties in the investigated dose range. Gamma and X-Ray irradiations generate the same level of reactive species in the EVA multilayer film, whereas e-beam generates a reduced quantity of reactive species.
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Current chemotherapies suffer low specificity and sometimes drug resistance. Neutrophil elastase activity in cancer is associated with poor prognosis and metastasis settlement. More generally, tumors harbor various and persistent protease activities unseen in healthy tissues. In an attempt to be more specific, we designed prodrugs that are activatable by neutrophil elastase. Upon activation, these alkoxyamine-based drugs release cytotoxic alkyl radicals that act randomly to prevent drug resistance. As a result, U87 glioblastoma cells displayed high level caspase 3/7 activation during the first hour of exposure in the presence of human neutrophil elastase and the prodrug in vitro. The apoptosis process and cell death occurred between 24 and 48 h after exposure with a half lethal concentration of 150 µM. These prodrugs are versatile and easy to synthetize and can be adapted to many enzymes.
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Antineoplásicos , Glioblastoma , Profármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Glioblastoma/patología , Humanos , Elastasa de Leucocito/metabolismo , Neutrófilos/metabolismo , Profármacos/metabolismoRESUMEN
Two alkylated verdazyl radicals (AlkVZs) were investigated as active compounds for photoinitiated controlled MCF-7 cell death. Observed results unambiguously showed that AlkVZ could be a potential structural moiety for the design of a novel family of photodynamic therapy agents. The main advantage of the proposed substances is an oxygen-independent generation of active radicals, which play a pivotal role in the treatment of oxygen-deficient tumors.
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Muerte Celular/efectos de los fármacos , Células MCF-7/efectos de los fármacos , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Radicales Libres/metabolismo , HumanosRESUMEN
Frataxin (FXN) is a highly conserved mitochondrial protein whose deficiency causes Friedreich's ataxia, a neurodegenerative disease. The precise physiological function of FXN is still unclear; however, there is experimental evidence that the protein is involved in biosynthetic iron-sulfur cluster machinery, redox imbalance, and iron homeostasis. FXN is synthesized in the cytosol and imported into the mitochondria, where it is proteolytically cleaved to the mature form. Its involvement in the redox imbalance suggests that FXN could interact with mitochondrial superoxide dismutase (SOD2), a key enzyme in antioxidant cellular defense. In this work, we use site-directed spin labelling coupled to electron paramagnetic resonance spectroscopy (SDSL-EPR) and fluorescence quenching experiments to investigate the interaction between human FXN and SOD2 in vitro. Spectroscopic data are combined with rigid body protein-protein docking to assess the potential structure of the FXN-SOD2 complex, which leaves the metal binding region of FXN accessible to the solvent. We provide evidence that human FXN interacts with human SOD2 in vitro and that the complex is in fast exchange. This interaction could be relevant during the assembly of iron-sulfur (FeS) clusters and/or their incorporation in proteins when FeS clusters are potentially susceptible to attacks by reactive oxygen species.
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To increase sterilization capacity, X-ray and e-beam irradiation modalities are more and more attractive for the indutrial sterilization of heathcare products (medical devices and biopharmaceutical goods). However, no study comparing these different techniques are available concerning multi-layer films. Thus, with the PE/EVOH/PE multilayer film as a model, we show that, whatever the modality of irradiation, the thermal properties are not significantly changed as shown by DSC, and, as such, the physical and mechanical properties of this material are also expected to behave similarly. On the other hand, chemical properties such as oxidation ability are strikingly modified, i.e., the same oxidation level for X-ray and γ-irradiation and twice weaker for e-beam irradiation.
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Chemically and biologically safe storage of solutions for medical uses is a daily concern for industry since decades and it appeared even more dramatic during the last two years of pandemia. Biological safety is readily reached by sterilization using γ-irradiation process. However, such a type of irradiation induces the degradation and the release of chemicals able to spoil the biological solutions. Surprisingly, there are no investigations on multi-layer films combining multi-technique and multi-method approaches to unveil the events occurring during γ-irradiation. Furthermore, our investigations are focuses on properties/events occurring at product, macromolecular, and molecular levels.
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Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.
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The nature of plasmon interaction with organic molecules is a subject of fierce discussion about thermal and non-thermal effects. Despite the abundance of physical methods for evaluating the plasmonic effects, chemical insight has not been reported yet. In this contribution, we propose a chemical insight into the plasmon effect on reaction kinetics using alkoxyamines as an organic probe through their homolysis, leading to the generation of nitroxide radicals. Alkoxyamines (TEMPO- and SG1-substituted) with well-studied homolysis behavior are covalently attached to spherical Au nanoparticles. We evaluate the kinetic parameters of homolysis of alkoxyamines attached on a plasmon-active surface under heating and irradiation at a wavelength of plasmon resonance. The estimation of kinetic parameters from experiments with different probes (Au-TEMPO, Au-SG1, Au-SG1-TEMPO) allows revealing the apparent differences associated with the non-thermal contribution of plasmon activation. Moreover, our findings underline the dependency of kinetic parameters on the structure of organic molecules, which highlights the necessity to consider the nature of organic transformations and molecular structure in plasmon catalysis.
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Cables, especially their insulation and jacket materials made of polymers, are vulnerable to ageing degradation during normal operation. However, they must remain functional for the entire life of a nuclear power plant, or even in the event of an accident for cables with a safety requirement. This study focuses on models of crosslinked polyethylene (XLPE)-based insulation of cables and deals with the structure modification and the behavior of XLPE for nuclear applications due to the effect of additives. Various additives are added to the polymer formulation to evaluate their impact on ageing. The samples are irradiated at room temperature by several gamma doses, up to 374 kGy, with two dose rates (40 Gy/h and 300 Gy/h) and compared with a non-irradiated sample used as reference. To understand the impact of gamma irradiation on the materials, the principal component analysis (PCA) method is applied on spectra recorded through attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. The results highlight the effects of ageing depending on the dose rate and on the formulation of the materials, with the identification of different degradation products. A curve resolution study compares the effects of different additives on polymer oxidation and shows that the low dose rate leads to a higher degradation than the high dose rate.
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Photodynamic therapy (PDT) is a promising technique to treat different kinds of disease especially cancer. PDT requires three elements: molecular oxygen, a photoactivatable molecule called the photosensitizer (PS), and appropriate light. Under illumination, the PSs generate, in the presence of oxygen, the formation of reactive oxygen species including singlet oxygen, toxic, which then destroys the surrounding tissues. Even if PDT is used with success to treat actinic keratosis or prostate cancer for example, PDT suffers from two major drawbacks: the lack of selectivity of most of the PSs currently used clinically as well as the need for oxygen to be effective. To remedy the lack of selectivity, targeting the tumor neovessels is a promising approach to destroy the vascularization and cause asphyxia of the tumor. KDKPPR peptide affinity for the neuropilin-1 (NRP-1) receptor overexpressed on endothelial cells has already been proven. To compensate for the lack of oxygen, we focused on photoactivatable alkoxyamines (Alks), molecules capable of generating toxic radicals by light activation. In this article, we describe the synthesis of a multifunctional platform combining three units: a PS for an oxygen-dependent PDT, a peptide to target tumor neovessels, and an Alk for an oxygen-independent activity. The synthesis of the compound was successfully carried out, and the study of its photophysical properties showed that the PS retained its capacity to form singlet oxygen and the affinity tests confirmed the affinity of the compound for NRP-1. Thanks to the electron paramagnetic resonance spectroscopy, a technique of choice for radical investigation, the radicals generated by the illumination of the Alk could be detected. The proof of concept was thus successfully established.
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Sistemas de Liberación de Medicamentos , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/uso terapéutico , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Estructura Molecular , Oxígeno , Péptidos , FotoquímicaRESUMEN
Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich's ataxia. Frataxin's actual physiological function has been debated for a long time without reaching a general agreement; however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin in vitro. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we produced evidence that ferric iron causes reversible aggregation without preferred interfaces in a concentration-dependent fashion, starting at relatively low concentrations (micromolar range), whereas ferrous iron binds without inducing aggregation. Moreover, our experiments show that the ferrous binding does not lead to changes of protein conformation. The data reported in this study reveal that the currently reported binding stoichiometries should be taken with caution. The use of a spin label resistant to reduction, as well as the comparison of the binding effect of Fe2+ in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe2+ binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe2+ and Fe3+ might play a relevant role in the context of the proposed FXN physiological functions.
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Dicroismo Circular/métodos , Compuestos Férricos/química , Compuestos Ferrosos/química , Proteínas de Unión a Hierro/química , Hierro/química , Marcadores de Spin , Espectroscopía de Resonancia por Spin del Electrón/métodos , Humanos , Concentración de Iones de Hidrógeno , Agregación Patológica de Proteínas , Unión Proteica , Conformación Proteica , Espectrometría de Fluorescencia/métodos , FrataxinaRESUMEN
In this study, the oxidation of methionine is used as a proxy to model the gamma radiation-induced changes in single-use bags; these changes lead to the formation of acids, radicals, and hydroperoxides. The mechanisms of formation of these reactive species and of methionine oxidation are discussed. With the help of reaction kinetics, the optimal conditions for the use of these single-use bags minimizing the impact of radical chemistry are highlighted. Biopharmaceutical bags gamma irradiated from 0 kGy to 260 kGy and aged from 0 to 36 months were filled with a methionine solution to follow the oxidation of the methionine. The methionine sulfoxide was measured with HPLC after different storage times (0, 3, 10, 14, 17, and 21 days). Three main results were analyzed through a design of experiments: the oxidative induction time, the methionine sulfoxide formation rate, and the maximum methionine sulfoxide concentration detected. A key aspect of the study is that it highlights that methionine is oxidized not necessarily directly by hydro(gen) peroxide but throughperacid, and likely peracetic acid. The answers to the design of experiments were considered to obtain the desirability domain for the optimization of the conditions of use for the single-use bags limiting the oxidation of methionine as well as the release of reactive species thereof.