Internalization and toxicological mechanisms of uncoated and PVP-coated cerium oxide nanoparticles in the freshwater alga Chlamydomonas reinhardtii

Due to the wide range of applications of cerium oxide nanoparticles (CeO2NPs), a risk assessment of their biological effects using environmentally relevant species becomes highly important. There are contradictory reports on the effects of CeO2NPs, which may be related to the use of different types of nanoparticles (NPs) and coatings. CeO2NPs may act as an oxidant causing toxicity or as an antioxidant able to scavenge free radicals. As a consequence of such complexity, the toxicological behaviour of these NPs is still poorly understood. Moreover, little is known about the internalization process of CeO2NPs in algae. There is evidence of CeO2NP-internalization by the green alga Chlamydomonas reinhardtii, but the mechanism and route of uptake are still unknown. In this study, we used uncoated and different polyvinylpyrrolidone (PVP)-coated CeO2NPs with the aim of identifying their toxicological mechanisms to C. reinhardtii and exploring their possible internalization. Our results showed that PVP coated-CeO2NPs significantly increased the formation of reactive oxygen species in exposed cells, indicating that oxidative stress is an important toxicity mechanism for these particles. Direct contact and damage of the cellular membrane was identified as the mechanism causing the toxicity of uncoated NPs. From experiments with endocytosis inhibitors, clathrin-dependent endocytosis was revealed as the main internalization route for all NPs. However, as uncoated CeO2NPs led to severe cellular membrane damage, direct passage of NPs through membrane holes could not be discarded. To our knowledge, this is the first report with evidence of direct linking between NP internalization and a specific endocytic pathway. The results presented here will help to unravel the toxicological mechanism and behaviour of CeO2NPs and provide input information for the environmental health and safety assessment of CeO2NPs.This research was supported by CTM2013-45775-C2-1,2-R and CTM2016-74927-C2-1,2-R grants from MINECO. NanoMILE (Grant Agreement no 310451 to EVJ & SMB) and the Endeavour Scholarship Scheme (Group B) (to SMB) are acknowledged.Peer reviewe

Parameter optimization of milk pulsation homogenizer

Homogenization is one of the normative processing stages in dairy foods production. One of the topical problems of the modern food industry is to design energy effective equipment for homogenization of milk emulsions. The perspective method to intensify milk fat phase dispersing is to improve pulsation homogenization. Such machines combine high energy efficiency and dispersion degree which exceed the indexes of valve homogenizer. The aim of researches is to decline power consumption of the process of milk pulsation homogenization by optimizing its parameters. Experimental pulsation homogenizer has a piston with openings which performs sinusoidal oscillations in the cylindrical chamber and is driven by crank gear. The product is supplied into the homogenizer by the volumetric pump. Creating high velocity relative slip between a fat globule and milk plasma causes size reduction of the milk fat. To find out the optimal parameters of the process the graphic methods of the local optimization are used. Emulsion dispersion degree was determined by measuring fat globules sizes by optical microscope with digital camera. Conducted researches on the pulsation homogenizer showed high correlation between the acceleration of emulsion and fat globules average size. The analytically got results are experimentally confirmed on increasing dispersion degree whereas the specific energy consumption declines and piston oscillation frequency increases. Using conical openings of the piston compared with cylindrical ones makes it possible to reduce energy consumption of the homogenizer. Decreasing coefficient of the piston open area negatively affects milk dispersion degree. Using piston with the conical openings at its oscillation amplitude of 10 mm and vibrations frequency of 150 s-1 and emulsion acceleration of about 105 m/s2 it is possible to get milk emulsion with the average dispersion of the fat phase of 0.8 μm. Specific energy consumption here does not exceed 3400 J/kg. The received results prove high potential of further research- and-developments of the industrial prototype of the pulsation homogenizer

Synthesis, kinetic control and properties engineering of cerium oxide nanoparticles for biomedical applications

La presente Tesis Doctoral es un fruto de colaboración entre el Instituto Catalán de Nanociencia y Nanotecnología (ICN2) y el Hospital Clínic de Barcelona, implicados en el proyecto “Marató TV3 2012”, con el objetivo de utilizar las nanopartículas de óxido de Cerio (CeO2 Nps o “nanoceria”) como una nueva herramienta terapéutica en la regeneración del tejido hepático en las enfermedades del hígado. CeO2NPs son un material inorgánico fascinante, con una gran variedad de aplicaciones y muchas más por llegar. Lo que las hace tan interesantes es su alta capacidad de hacer de buffer de electrones en un entorno oxidante/reductor, gracias a su estructura electrónica incompleta en la capa 4f. Ésto hace posible que presente una habilidad de ser oxidada o reducida, seguida de captura o liberación de oxígeno o especies reactivas de oxígeno (ROS y los radicales libres, tales como OH·). Como consecuencia, nanoceria se comporta como una esponga natural de electrones libres. El desequilibrio de ROS tiene lugar en un gran número de enfermedades humanas. También, la sobreproducción de ROS es crítica en la neurodegeneración. A pesar de la atractiva capacidad antioxidante de CeO2 NPs, una controversia importante, en cuanto a su función biológica, fue descrita en la literatura actual. A lo largo de ésta Tesis, los métodos existentes de síntesis de nanoceria han sido analizados en detalle, cómo también la calidad de los productos obtenidos y los aspectos toxicológicos de ámbos (los procesos y los productos). Éste trabajo de investigación fue enfocado en sobrepasar las problemáticas existentes de la toxicidad de nanoceria (debida a la agregación de nanopartículas, la toxicidad del surfactante o el solvente, o a la contaminación de las muestras con endtoxina) y en diseño de soluciones útiles, con el objetivo de sacar el provecho máximo de las propiedades antioxidantes de CeO2NPs en recerca y aplicaciones biomédicos. Asimismo, el presente trabajo fue centrado en el estudio de las propiedades físico-químicas y bio-quimicas de nanoceria, para optimizar su preparación y tamaño (Capitulo 2), evaluar su reactividad (Capitulo 3), biodistribución (Capitulo 4) y no-toxicidad (Capitulo 2, Anexo 2). Finalmente, la biodistribución de nanoceria y sus efectos sobre los mediadores fibrogénicos e inflamatorios fueron evaluados a nivel molecular y celular, demostrando que la administración de CeO2NPs podría ser de interés terapéutico en enfermedades del hígado (Anexo 3).The current Doctoral Thesis is the fruit of collaboration between the Catalan Institute of Nanoscience and Nanotechnology (ICN2) and the Hospital Clinic of Barcelona, involved in the project “Marató TV3 2012”, with the objective to apply Cerium oxide nanoparticles (CeO2 NPs) as a new therapeutic tool for tissue regeneration in liver diseases. CeO2NPs is a fascinating inorganic material with many applications and more to come. What makes nanoceria very appealing is its high capacity to buffer electrons from an oxidant/reducing environment due to the unfilled 4f electronic structure. This is due to its easy ability of being oxidized and reduced, followed by the capture or release of oxygen or reactive oxygen species (ROS and free radicals as OH·). As a result, nanoceria behaves as a natural electron sponge. Note that ROS disbalance takes place in an enormous number of human diseases. Also, the overproduction of ROS is critical in neurodegeneration. Despite the appealing redox catalytic capacity of CeO2 NPs, an important controversy upon biological effects of CeO2 has been numerously reported. During this Thesis, the existent methods of nanoceria preparation have been analyzed in detail, as well as the quality of the obtained products and the toxicological aspects of both (the processes and the products). This Doctoral research has been focused in overtaking the existent problematics of the nanoceria toxicity (due to aggregation of NPs, toxic surfactant or solvent, or contamination with endotoxin) and offering suitable solutions, in order to take full advantage of the antioxidant CeO2NPs properties in biomedical research and applications. Thus, the current work has been focused on the study of physicochemical and biochemical properties of CeO2 NPs, to optimize the preparation methods and the obtained product, in an environmentally-friendly way (Chapter 2, Annex 2). The optimization of the NPs size and monodispersity (Chapter 2); as well as the evaluation of the correspondent antioxidant activity (Chapter 3) were also performed. Finally, the in-vivo biodistribution study of CeO2 NPs, as well as their effects on inflammatory and fibrogenic mediators were evaluated at molecular and cellular level, demonstrating that administration of CeO2 NPs could be of therapeutic value in liver diseases (Annex 3)

Cerium oxide nanoparticles reduce steatosis, portal hypertension and display anti-inflammatory properties in rats with liver fibrosis

et al.[Background & Aims]: Cerium oxide nanoparticles (CeONPs) have proven to behave as free radical scavengers and/or anti-inflammatory agents. The aim of the study was to determine whether CeONPs display hepatoprotective properties in experimental chronic liver disease. [Methods]: Systemic and hepatic effects of nanoparticles were assessed in CCl-treated rats receiving CeONPs or vehicle twice weekly for two weeks and CCl treatment was continued for 8 additional weeks. Thereafter, mean arterial pressure and portal pressure (PP) were assessed and serum samples obtained to measure standard hepatic and renal function tests. Organ and subcellular distribution of NPs were assessed using mass spectrometry (ICP-MS) and transmission electron microscopy. Liver samples were obtained to evaluate steatosis, α-SMA expression, macrophage infiltration, apoptosis and mRNA expression of oxidative stress, inflammatory or vasoactive related genes. [Results]: Most CeONPs were located in the liver and it reduced hepatic steatosis, ameliorated systemic inflammatory biomarkers and improved PP without affecting mean arterial pressure. In addition, a marked reduction in mRNA expression of inflammatory cytokines (TNFα, IL1β, COX-2, iNOS), ET-1 and messengers related to oxidative (Epx, Ncf1, Ncf2) or endoplasmic reticulum (Atf3, Hspa5) stress signaling pathways was observed in the liver of rats receiving CeONPs. This was associated with reduced macrophage infiltration and reduced abundance of caspase-3, α-SMA and inflammatory cytokines. [Conclusions]: CeONPs administration to CCl-treated rats protects against chronic liver injury by reducing liver steatosis and portal hypertension and markedly attenuating the intensity of the inflammatory response, thereby suggesting that CeONPs may be of therapeutic value in chronic liver disease.This work was supported by grants to W. Jiménez from Ministerio de Economia y Competitividad (SAF12-35979), Cofinanced by FEDER, European Union, a way of making Europe, Agència de Gestió d’Ajuts Universitaris i de Recerca (SGR 2014/219) and Fundació La Maratò de TV3 (Maratò 120930). The Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by the Instituto de Salud Carlos III.Peer Reviewe

Synthesis, kinetic control and properties engineering of cerium oxide nanoparticles for biomedical applications

La presente Tesis Doctoral es un fruto de colaboración entre el Instituto Catalán de Nanociencia y Nanotecnología (ICN2) y el Hospital Clínic de Barcelona, implicados en el proyecto "Marató TV3 2012", con el objetivo de utilizar las nanopartículas de óxido de Cerio (CeO2 Nps o "nanoceria") como una nueva herramienta terapéutica en la regeneración del tejido hepático en las enfermedades del hígado. CeO2NPs son un material inorgánico fascinante, con una gran variedad de aplicaciones y muchas más por llegar. Lo que las hace tan interesantes es su alta capacidad de hacer de buffer de electrones en un entorno oxidante/reductor, gracias a su estructura electrónica incompleta en la capa 4f. Ésto hace posible que presente una habilidad de ser oxidada o reducida, seguida de captura o liberación de oxígeno o especies reactivas de oxígeno (ROS y los radicales libres, tales como OH·). Como consecuencia, nanoceria se comporta como una esponga natural de electrones libres. El desequilibrio de ROS tiene lugar en un gran número de enfermedades humanas. También, la sobreproducción de ROS es crítica en la neurodegeneración. A pesar de la atractiva capacidad antioxidante de CeO2 NPs, una controversia importante, en cuanto a su función biológica, fue descrita en la literatura actual. A lo largo de ésta Tesis, los métodos existentes de síntesis de nanoceria han sido analizados en detalle, cómo también la calidad de los productos obtenidos y los aspectos toxicológicos de ámbos (los procesos y los productos). Éste trabajo de investigación fue enfocado en sobrepasar las problemáticas existentes de la toxicidad de nanoceria (debida a la agregación de nanopartículas, la toxicidad del surfactante o el solvente, o a la contaminación de las muestras con endtoxina) y en diseño de soluciones útiles, con el objetivo de sacar el provecho máximo de las propiedades antioxidantes de CeO2NPs en recerca y aplicaciones biomédicos. Asimismo, el presente trabajo fue centrado en el estudio de las propiedades físico-químicas y bio-quimicas de nanoceria, para optimizar su preparación y tamaño (Capitulo 2), evaluar su reactividad (Capitulo 3), biodistribución (Capitulo 4) y no-toxicidad (Capitulo 2, Anexo 2). Finalmente, la biodistribución de nanoceria y sus efectos sobre los mediadores fibrogénicos e inflamatorios fueron evaluados a nivel molecular y celular, demostrando que la administración de CeO2NPs podría ser de interés terapéutico en enfermedades del hígado (Anexo 3).The current Doctoral Thesis is the fruit of collaboration between the Catalan Institute of Nanoscience and Nanotechnology (ICN2) and the Hospital Clinic of Barcelona, involved in the project "Marató TV3 2012", with the objective to apply Cerium oxide nanoparticles (CeO2 NPs) as a new therapeutic tool for tissue regeneration in liver diseases. CeO2NPs is a fascinating inorganic material with many applications and more to come. What makes nanoceria very appealing is its high capacity to buffer electrons from an oxidant/reducing environment due to the unfilled 4f electronic structure. This is due to its easy ability of being oxidized and reduced, followed by the capture or release of oxygen or reactive oxygen species (ROS and free radicals as OH·). As a result, nanoceria behaves as a natural electron sponge. Note that ROS disbalance takes place in an enormous number of human diseases. Also, the overproduction of ROS is critical in neurodegeneration. Despite the appealing redox catalytic capacity of CeO2 NPs, an important controversy upon biological effects of CeO2 has been numerously reported. During this Thesis, the existent methods of nanoceria preparation have been analyzed in detail, as well as the quality of the obtained products and the toxicological aspects of both (the processes and the products). This Doctoral research has been focused in overtaking the existent problematics of the nanoceria toxicity (due to aggregation of NPs, toxic surfactant or solvent, or contamination with endotoxin) and offering suitable solutions, in order to take full advantage of the antioxidant CeO2NPs properties in biomedical research and applications. Thus, the current work has been focused on the study of physicochemical and biochemical properties of CeO2 NPs, to optimize the preparation methods and the obtained product, in an environmentally-friendly way (Chapter 2, Annex 2). The optimization of the NPs size and monodispersity (Chapter 2); as well as the evaluation of the correspondent antioxidant activity (Chapter 3) were also performed. Finally, the in-vivo biodistribution study of CeO2 NPs, as well as their effects on inflammatory and fibrogenic mediators were evaluated at molecular and cellular level, demonstrating that administration of CeO2 NPs could be of therapeutic value in liver diseases (Annex 3)

Exploring the Long-Term Tissue Accumulation and Excretion of 3 nm Cerium Oxide Nanoparticles after Single Dose Administration

Nanoparticle (NP) pharmacokinetics significantly differ from traditional small molecule principles. From this emerges the need to create new tools and concepts to harness their full potential and avoid unnecessary risks. Nanoparticle pharmacokinetics strongly depend on size, shape, surface functionalisation, and aggregation state, influencing their biodistribution, accumulation, transformations, and excretion profile, and hence their efficacy and safety. Today, while NP biodistribution and nanoceria biodistribution have been studied often at short times, their long-term accumulation and excretion have rarely been studied. In this work, 3 nm nanoceria at 5.7 mg/kg of body weight was intravenously administrated in a single dose to healthy mice. Biodistribution was measured in the liver, spleen, kidney, lung, brain, lymph nodes, ovary, bone marrow, urine, and faeces at different time points (1, 9, 30, and 100 days). Biodistribution and urinary and faecal excretion were also studied in rats placed in metabolic cages at shorter times. The similarity of results of different NPs in different models is shown as the heterogeneous nanoceria distribution in organs. After the expectable accumulation in the liver and spleen, the concentration of cerium decays exponentially, accounting for about a 50% excretion of cerium from the body in 100 days. Cerium ions, coming from NP dissolution, are most likely excreted via the urinary tract, and ceria nanoparticles accumulated in the liver are most likely excreted via the hepatobiliary route. In addition, nanoceria looks safe and does not damage the target organs. No weight loss or apathy was observed during the course of the experiments