Publications by authors named "Eudald Casals"

48 Publications

Circumventing Drug Treatment? Intrinsic Lethal Effects of Polyethyleneimine (PEI)-Functionalized Nanoparticles on Glioblastoma Cells Cultured in Stem Cell Conditions.

Cancers (Basel) 2021 May 27;13(11). Epub 2021 May 27.

Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, 20520 Turku, Finland.

Glioblastoma (GB) is the most frequent malignant tumor originating from the central nervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remains largely irremediable due to the high degree of intratumoral heterogeneity, infiltrative growth, and intrinsic resistance towards multiple treatments. A sub-population of GB cells, glioblastoma stem cells (GSCs), act as a reservoir of cancer-initiating cells and consequently, constitute a significant challenge for successful therapy. In this study, we discovered that PEI surface-functionalized mesoporous silica nanoparticles (PEI-MSNs), without any anti-cancer drug, very potently kill multiple GSC lines cultured in stem cell conditions. Very importantly, PEI-MSNs did not affect the survival of established GB cells, nor other types of cancer cells cultured in serum-containing medium, even at 25 times higher doses. PEI-MSNs did not induce any signs of apoptosis or autophagy. Instead, as a potential explanation for their lethality under stem cell culture conditions, we demonstrate that the internalized PEI-MSNs accumulated inside lysosomes, subsequently causing a rupture of the lysosomal membranes. We also demonstrate blood-brain-barrier (BBB) permeability of the PEI-MSNs in vitro and in vivo. Taking together the recent indications for the vulnerability of GSCs for lysosomal targeting and the lethality of the PEI-MSNs on GSCs cultured under stem cell culture conditions, the results enforce in vivo testing of the therapeutic impact of PEI-functionalized nanoparticles in faithful preclinical GB models.
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http://dx.doi.org/10.3390/cancers13112631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198814PMC
May 2021

Mesoporous silica coated CeO nanozymes with combined lipid-lowering and antioxidant activity induce long-term improvement of the metabolic profile in obese Zucker rats.

Nanoscale 2021 May 14;13(18):8452-8466. Epub 2021 Apr 14.

Service of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Carrer de Villarroel, 170, 08036 Barcelona, Spain.

Obesity is one of the most important public health problems that is associated with an array of metabolic disorders linked to cardiovascular disease, stroke, type 2 diabetes, and cancer. A sustained therapeutic approach to stop the escalating prevalence of obesity and its associated metabolic comorbidities remains elusive. Herein, we developed a novel nanocomposite based on mesoporous silica coated cerium oxide (CeO) nanozymes that reduce the circulating levels of fatty acids and remarkably improve the metabolic phenotype in a model of obese Zucker rats five weeks after its administration. Lipidomic and gene expression analyses showed an amelioration of the hyperlipidemia and of the hepatic and adipose metabolic dysregulations, which was associated with a down-regulation of the hepatic PI3K/mTOR/AKT pathway and a reduction of the M1 proinflammatory cytokine TNF-α. In addition, the coating of the CeO maximized its cell antioxidant protective effects and minimized non-hepatic biodistribution. The one-pot synthesis method for the nanocomposite fabrication is implemented entirely in aqueous solution, room temperature and open atmosphere conditions, favoring scalability and offering a safe and translatable lipid-lowering and antioxidant nanomedicine to treat metabolic comorbidities associated with obesity. This approach may be further applied to address other metabolic disorders related to hyperlipidemia, low-grade inflammation and oxidative stress.
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http://dx.doi.org/10.1039/d1nr00790dDOI Listing
May 2021

Preclinical studies conducted on nanozyme antioxidants: shortcomings and challenges based on US FDA regulations.

Nanomedicine (Lond) 2021 Jun 11;16(13):1133-1151. Epub 2021 May 11.

Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.

The wide prevalence of oxidative stress-induced diseases has led to a growing demand for antioxidant therapeutics worldwide. Nanozyme antioxidants are drawing enormous attention as practical alternatives for conventional antioxidants. The considerable body of research over the last decade and the promising results achieved signify the potential of nanozyme antioxidants to secure a place in the expanding market of antioxidant therapeutics. Nonetheless, there is no report on clinical trials for their further evaluation. Through analyzing in-depth selected papers which have conducted studies on nanozyme antioxidants, this review aims to pinpoint and discuss possible reasons impeding development of research toward clinical studies and to offer some practical solutions for future studies to bridge the gap between preclinical and clinical stages.
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http://dx.doi.org/10.2217/nnm-2021-0030DOI Listing
June 2021

Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and β-Hydroxybutyrate/Acetoacetate in Biological Samples.

Int J Mol Sci 2021 Apr 30;22(9). Epub 2021 Apr 30.

Service of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Carrer de Villarroel 170, 08036 Barcelona, Spain.

The metabolic ratios lactate/pyruvate and β-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, β-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency's Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and β-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.
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http://dx.doi.org/10.3390/ijms22094752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125771PMC
April 2021

Cerium Oxide Nanoparticles: A New Therapeutic Tool in Liver Diseases.

Antioxidants (Basel) 2021 Apr 24;10(5). Epub 2021 Apr 24.

Service of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d'Investigacions, Biomèdiques August Pi i Sunyer (IDIBAPS), 08905 Barcelona, Spain.

Oxidative stress induced by the overproduction of free radicals or reactive oxygen species (ROS) has been considered as a key pathogenic mechanism contributing to the initiation and progression of injury in liver diseases. Consequently, during the last few years antioxidant substances, such as superoxide dismutase (SOD), resveratrol, colchicine, eugenol, and vitamins E and C have received increasing interest as potential therapeutic agents in chronic liver diseases. These substances have demonstrated their efficacy in equilibrating hepatic ROS metabolism and thereby improving liver functionality. However, many of these agents have not successfully passed the scrutiny of clinical trials for the prevention and treatment of various diseases, mainly due to their unspecificity and consequent uncontrolled side effects, since a minimal level of ROS is needed for normal functioning. Recently, cerium oxide nanoparticles (CeONPs) have emerged as a new powerful antioxidant agent with therapeutic properties in experimental liver disease. CeONPs have been reported to act as a ROS and reactive nitrogen species (RNS) scavenger and to have multi-enzyme mimetic activity, including SOD activity (deprotionation of superoxide anion into oxygen and hydrogen peroxide), catalase activity (conversion of hydrogen peroxide into oxygen and water), and peroxidase activity (reducing hydrogen peroxide into hydroxyl radicals). Consequently, the beneficial effects of CeONPs treatment have been reported in many different medical fields other than hepatology, including neurology, ophthalmology, cardiology, and oncology. Unlike other antioxidants, CeONPs are only active at pathogenic levels of ROS, being inert and innocuous in healthy cells. In the current article, we review the potential of CeONPs in several experimental models of liver disease and their safety as a therapeutic agent in humans as well.
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http://dx.doi.org/10.3390/antiox10050660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146351PMC
April 2021

Phase separation of a nonionic surfactant aqueous solution in a standing surface acoustic wave for submicron particle manipulation.

Lab Chip 2021 02;21(4):660-667

State Key Laboratory of Precision Measuring Technology & Instruments, Tianjin University, Tianjin 300072, China.

Acoustic manipulation of submicron particles in a controlled manner has been challenging to date because of the increased contribution of acoustic streaming, which leads to fluid mixing and homogenization. This article describes the patterning of submicron particles and the migration of their patterned locations from pressure nodes to antinodes in a non-ionic surfactant (Tween 20) aqueous solution in a conventional standing surface acoustic wave field with a wavelength of 150 μm. Phase separation of the aqueous surfactant solution occurs when they are exposed to acoustic waves, probably due to the "clouding behavior" of non-ionic surfactant. The generated surfactant precipitates are pushed to the pressure antinodes due to the negative acoustic contrast factor relative to water. Compared with the mixing appearance in pure water media, the patterning behavior of submicron particles with a diameter of 300 nm dominated by acoustic radiation force is readily apparent in an aqueous solution with 2% volumetric concentration of Tween 20 surfactant, thanks to the suppression effect of acoustic streaming in inhomogeneous fluids. These submicron particles are first pushed to acoustic pressure nodes and then are migrated to antinodes where the surfactant precipitates stay. More attractively, the migration of acoustically patterned locations is not only limited to submicron particles, but also occurs to micrometer-sized particles in solutions with higher surfactant concentrations. These findings open up a novel avenue for controllable acoustic manipulation.
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http://dx.doi.org/10.1039/d0lc00986eDOI Listing
February 2021

Simple spectroscopic determination of the hard protein corona composition in AuNPs: albumin at 75.

Nanoscale 2020 Aug 21;12(29):15832-15844. Epub 2020 Jul 21.

Vall d'Hebron Institut de Recerca (VHIR), 08035, Barcelona, Spain.

We analyzed the different spectroscopic profiles of nanoparticle hard protein corona formation using two model proteins, albumin and immunoglobulin. When compared to serum, this served for the analysis of the hard protein corona main components. To do that, we employed time-resolved UV-Visible light absorption spectroscopy, dynamic light scattering, and zeta potential measurements during nanoparticle-protein incubation. Under the tested experimental conditions, the expected evolution from a non-stable (soft) to a stable (hard) protein corona was confirmed for serum and albumin. At the same time, immunoglobulin incubation inevitably failed to form a corona and led to nanoparticle aggregation. The formation profiles of the protein corona were similar in the case of albumin and serum, indicating the dominance of albumin coating the nanoparticle surface when exposed to plasma. This was confirmed by mass spectrometry. Chemical digestion of the nanoparticles bearing different protein coronas gave indications of the density of the different protein coatings. Overall, this study of the protein corona by determining the adsorption kinetics finger-print enables the development of precise nanotechnologies avoiding cumbersome processes and delaying proteomics analysis.
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http://dx.doi.org/10.1039/d0nr02379eDOI Listing
August 2020

Cerium Oxide Nanoparticles: Advances in Biodistribution, Toxicity, and Preclinical Exploration.

Small 2020 05 24;16(20):e1907322. Epub 2020 Apr 24.

Service of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, 08036, Spain.

Antioxidant nanoparticles have recently gained tremendous attention for their enormous potential in biomedicine. However, discrepant reports of either medical benefits or toxicity, and lack of reproducibility of many studies, generate uncertainties delaying their effective implementation. Herein, the case of cerium oxide is considered, a well-known catalyst in the petrochemistry industry and one of the first antioxidant nanoparticles proposed for medicine. Like other nanoparticles, it is now described as a promising therapeutic alternative, now as threatening to health. Sources of these discrepancies and how this analysis helps to overcome contradictions found for other nanoparticles are summarized and discussed. For the context of this analysis, what has been reported in the liver is reviewed, where many diseases are related to oxidative stress. Since well-dispersed nanoparticles passively accumulate in liver, it represents a major testing field for the study of new nanomedicines and their clinical translation. Even more, many contradictory works have reported in liver either cerium-oxide-associated toxicity or protection against oxidative stress and inflammation. Based on this, finally, the intention is to propose solutions to design improved nanoparticles that will work more precisely in medicine and safely in society.
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http://dx.doi.org/10.1002/smll.201907322DOI Listing
May 2020

Bespoken Nanoceria: An Effective Treatment in Experimental Hepatocellular Carcinoma.

Hepatology 2020 10;72(4):1267-1282

Service of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

Background And Aims: Despite the availability of new-generation drugs, hepatocellular carcinoma (HCC) is still the third most frequent cause of cancer-related deaths worldwide. Cerium oxide nanoparticles (CeO NPs) have emerged as an antioxidant agent in experimental liver disease because of their antioxidant, anti-inflammatory, and antisteatotic properties. In the present study, we aimed to elucidate the potential of CeO NPs as therapeutic agents in HCC.

Approach And Results: HCC was induced in 110 Wistar rats by intraperitoneal administration of diethylnitrosamine for 16 weeks. Animals were treated with vehicle or CeO NPs at weeks 16 and 17. At the eighteenth week, nanoceria biodistribution was assessed by mass spectrometry (MS). The effect of CeO NPs on tumor progression and animal survival was investigated. Hepatic tissue MS-based phosphoproteomics as well as analysis of principal lipid components were performed. The intracellular uptake of CeO NPs by human ex vivo perfused livers and human hepatocytes was analyzed. Nanoceria was mainly accumulated in the liver, where it reduced macrophage infiltration and inflammatory gene expression. Nanoceria treatment increased liver apoptotic activity, while proliferation was attenuated. Phosphoproteomic analysis revealed that CeO NPs affected the phosphorylation of proteins mainly related to cell adhesion and RNA splicing. CeO NPs decreased phosphatidylcholine-derived arachidonic acid and reverted the HCC-induced increase of linoleic acid in several lipid components. Furthermore, CeO NPs reduced serum alpha-protein levels and improved the survival of HCC rats. Nanoceria uptake by ex vivo perfused human livers and in vitro human hepatocytes was also demonstrated.

Conclusions: These data indicate that CeO NPs partially revert the cellular mechanisms involved in tumor progression and significantly increase survival in HCC rats, suggesting that they could be effective in patients with HCC.
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http://dx.doi.org/10.1002/hep.31139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702051PMC
October 2020

Cerium Oxide Nanoparticles Protect against Oxidant Injury and Interfere with Oxidative Mediated Kinase Signaling in Human-Derived Hepatocytes.

Int J Mol Sci 2019 Nov 27;20(23). Epub 2019 Nov 27.

Biochemistry and Molecular Genetics Service, Hospital Clínic Universitari, IDIBAPS, CIBERehd, 08036 Barcelona, Spain.

Cerium oxide nanoparticles (CeONPs) possess powerful antioxidant properties, thus emerging as a potential therapeutic tool in non-alcoholic fatty liver disease (NAFLD) progression, which is characterized by a high presence of reactive oxygen species (ROS). The aim of this study was to elucidate whether CeONPs can prevent or attenuate oxidant injury in the hepatic human cell line HepG2 and to investigate the mechanisms involved in this phenomenon. The effect of CeONPs on cell viability and ROS scavenging was determined, the differential expression of pro-inflammatory and oxidative stress-related genes was analyzed, and a proteomic analysis was performed to assess the impact of CeONPs on cell phosphorylation in human hepatic cells under oxidative stress conditions. CeONPs did not modify HepG2 cell viability in basal conditions but reduced HO- and lipopolysaccharide (LPS)-induced cell death and prevented HO-induced overexpression of MPO, PTGS1 and iNOS. Phosphoproteomic analysis showed that CeONPs reverted the HO-mediated increase in the phosphorylation of peptides related to cellular proliferation, stress response, and gene transcription regulation, and interfered with HO effects on mTOR, MAPK/ERK, CK2A1 and PKACA signaling pathways. In conclusion, CeONPs protect HepG2 cells from cell-induced oxidative damage, reducing ROS generation and inflammatory gene expression as well as regulation of kinase-driven cell survival pathways.
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http://dx.doi.org/10.3390/ijms20235959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928882PMC
November 2019

Cerium oxide nanoparticles improve liver regeneration after acetaminophen-induced liver injury and partial hepatectomy in rats.

J Nanobiotechnology 2019 Oct 31;17(1):112. Epub 2019 Oct 31.

Biochemistry and Molecular Genetics Department, Hospital Clínic of Barcelona, IDIBAPS, CIBERehd, 170 Villarroel St., 08036, Barcelona, Spain.

Background And Aims: Cerium oxide nanoparticles are effective scavengers of reactive oxygen species and have been proposed as a treatment for oxidative stress-related diseases. Consequently, we aimed to investigate the effect of these nanoparticles on hepatic regeneration after liver injury by partial hepatectomy and acetaminophen overdose.

Methods: All the in vitro experiments were performed in HepG2 cells. For the acetaminophen and partial hepatectomy experimental models, male Wistar rats were divided into three groups: (1) nanoparticles group, which received 0.1 mg/kg cerium nanoparticles i.v. twice a week for 2 weeks before 1 g/kg acetaminophen treatment, (2) N-acetyl-cysteine group, which received 300 mg/kg of N-acetyl-cysteine i.p. 1 h after APAP treatment and (3) partial hepatectomy group, which received the same nanoparticles treatment before partial hepatectomy. Each group was matched with vehicle-controlled rats.

Results: In the partial hepatectomy model, rats treated with cerium oxide nanoparticles showed a significant increase in liver regeneration, compared with control rats. In the acetaminophen experimental model, nanoparticles and N-acetyl-cysteine treatments decreased early liver damage in hepatic tissue. However, only the effect of cerium oxide nanoparticles was associated with a significant increment in hepatocellular proliferation. This treatment also reduced stress markers and increased cell cycle progression in hepatocytes and the activation of the transcription factor NF-κB in vitro and in vivo.

Conclusions: Our results demonstrate that the nanomaterial cerium oxide, besides their known antioxidant capacities, can enhance hepatocellular proliferation in experimental models of liver regeneration and drug-induced hepatotoxicity.
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http://dx.doi.org/10.1186/s12951-019-0544-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822381PMC
October 2019

Cerium oxide nanoparticles display antilipogenic effect in rats with non-alcoholic fatty liver disease.

Sci Rep 2019 09 6;9(1):12848. Epub 2019 Sep 6.

Biochemistry and Molecular Genetics Service, Hospital Clínic Universitari, IDIBAPS, CIBERehd, Barcelona, Spain.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide, ranging from steatosis to non-alcoholic steatohepatitis (NASH). Recently, cerium oxide nanoparticles (CeONPs) have emerged as a new antioxidant agent with hepatoprotective properties in experimental liver disease. The aim of the current investigation was to elucidate whether CeONPs display beneficial effects in an experimental model of NAFLD.Therefore, fifteen Wistar rats were subjected to a methionine and choline deficient diet (MCDD) for 6 weeks and intravenously treated with CeONP or vehicle during the weeks three and four of the diet. The effect of CeONPs on serum biochemistry, hepatic steatosis, inflammation, fatty acid content and expression of reactive oxygen species (ROS) and lipid metabolism related genes was assessed. MCDD fed rats showed increased inflammation, enhanced hepatic lipid accumulation of both saturated and unsaturated fatty acids (FAs) and overexpression of genes related to fatty liver and ROS metabolism. Treatment with CeONPs was able to reduce the size and content of hepatocyte lipid droplets, the hepatic concentration of triglyceride- and cholesterol ester-derived FAs and the expression of several genes involved in cytokine, adipokine and chemokine signaling pathways. These findings suggest that CeONPs could be of beneficial value in NAFLD.
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http://dx.doi.org/10.1038/s41598-019-49262-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731222PMC
September 2019

Beyond the Scavenging of Reactive Oxygen Species (ROS): Direct Effect of Cerium Oxide Nanoparticles in Reducing Fatty Acids Content in an In Vitro Model of Hepatocellular Steatosis.

Biomolecules 2019 08 29;9(9). Epub 2019 Aug 29.

Service of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08905 Barcelona, Spain.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic accumulation of lipids. Antisteatotic effects of cerium oxide nanoparticles (CeONPs) have recently been shown in animal models of liver disease. However, it is unclear whether the activity of CeONPs is related solely to the decrease in oxidative stress or, in addition, they directly decrease liver fatty acid accumulation. To address this question, in this work, we used an in vitro model of hepatocellular steatosis, exposing HepG2 cells to oleic and palmitic acid. Cell uptake of CeONPs and their effect on oxidative stress and viability of hepatic cells cultured with HO were also evaluated. Results show that CeONPs were uptaken by HepG2 cells and reduced oxidative stress and improved cell viability. Treatment with oleic and palmitic acid increased lipogenesis and the content of different fatty acids. CeONPs reduced palmitic and stearic acid and most fatty acids consisting of more than 18 carbon atoms. These effects were associated with significant changes in elongase and desaturase activity. In conclusion, CeONPs directly protected HepG2 cells from cell injury in oxidative stress conditions and reduced fatty acid content in steatotic conditions by inducing specific changes in fatty acid metabolism, thus showing potential in the treatment of NAFLD.
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http://dx.doi.org/10.3390/biom9090425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770635PMC
August 2019

Functionalized cerium oxide nanoparticles mitigate the oxidative stress and pro-inflammatory activity associated to the portal vein endothelium of cirrhotic rats.

PLoS One 2019 24;14(6):e0218716. Epub 2019 Jun 24.

Biochemistry and Molecular Genetics Department, Hospital Clínic of Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain.

Background And Aims: The occurrence of endothelial alterations in the liver and in the splanchnic vasculature of cirrhotic patients and experimental models of liver diseases has been demonstrated. However, the pathological role of the portal vein endothelium in this clinical context is scarcely studied and, therefore, deserves attention. In this context, we aimed to investigate whether pathological endothelial activation occurs in the portal vein of cirrhotic rats.

Methods: Cirrhosis was induced in wistar rats by CCl4 inhalation. We generated immortalized endothelial cells from the portal vein of control (CT-iPVEC) and cirrhotic rats (CH-iPVEC) by retroviral transduction of the SV40 T antigen. We assessed differential gene expression and intracellular reactive oxygen species (ROS) levels in iPVECs and in portal veins of control and cirrhotic rats. Finally, we assessed the therapeutic effectiveness of cerium oxide nanoparticles (CeO2NP) on reversing PVEC activation and macrophage polarization.

Results: CH-iPVECs overexpressed collagen-I, endothelin-1, TIMP-1, TIMP-2, IL-6 and PlGF genes. These results were consistent with the differential expression showed by whole portal veins from cirrhotic rats. In addition, CH-iPVECs showed a significant increase in intracellular ROS and the capacity of potentiating M1 polarization in macrophages. The treatment of CH-iPVECs with CeO2NPs blocked intracellular ROS formation and IL-6 and TIMP-2 gene overexpression. In agreement with the in vitro results, the chronic treatment of cirrhotic rats with CeO2NPs also resulted in the blockade of both ROS formation and IL-6 gene overexpression in whole portal veins.

Conclusions: Endothelial cells from portal vein of cirrhotic rats depicted an abnormal phenotype characterized by a differential gene expression and the induction of M1 polarization in macrophages. We identified the excess of intracellular reactive oxygen species (ROS) as a major contributor to this altered phenotype. In addition, we demonstrated the utility of the nanomaterial cerium oxide as an effective antioxidant capable of reverse some of these pathological features associated with the portal vein in the cirrhosis condition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218716PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590813PMC
February 2020

Characterization of modified mesoporous silica nanoparticles as vectors for siRNA delivery.

Asian J Pharm Sci 2018 Nov 21;13(6):592-599. Epub 2018 Feb 21.

Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, BioCity, Artillerigatan 6A, FI 20520 Turku, Finland.

Gene therapy using siRNA molecules is nowadays considered as a promising approach. For successful therapy, development of a stable and reliable vector for siRNA is crucial. Non-viral and non-organic vectors like mesoporous silica nanoparticles (MSN) are associated with lack of most viral vector drawbacks, such as toxicity, immunogenicity, but also generally a low nucleic acid carrying capacity. To overcome this hurdle, we here modified the pore walls of MSNs with surface-hyperbranching polymerized poly(ethyleneimine) (hbPEI), which provides an abundance of amino-groups for loading of a larger amount of siRNA molecules via electrostatic adsorption. After loading, the particles were covered with a second layer of pre-polymerized PEI to provide better protection of siRNA inside the pores, more effective cellular uptake and endosomal escape. To test the transfection efficiency of PEI covered siRNA/MSNs, MDA-MB 231 breast cancer cells stably expressing GFP were used. We demonstrate that PEI-coated siRNA/MSN complexes provide more effective delivery of siRNAs compared to unmodified MSNs. Thus, it can be concluded that appropriately surface-modified MSNs can be considered as prospective vectors for therapeutic siRNA delivery.
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http://dx.doi.org/10.1016/j.ajps.2018.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032096PMC
November 2018

Corrigendum: Intrinsic and Extrinsic Properties Affecting Innate Immune Responses to Nanoparticles: The Case of Cerium Oxide.

Front Immunol 2017 22;8:1891. Epub 2017 Dec 22.

Vall d'Hebron Institut of Research (VHIR), Barcelona, Spain.

[This corrects the article on p. 970 in vol. 8, PMID: 28855907.].
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http://dx.doi.org/10.3389/fimmu.2017.01891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743895PMC
December 2017

Bacterial endotoxin (lipopolysaccharide) binds to the surface of gold nanoparticles, interferes with biocorona formation and induces human monocyte inflammatory activation.

Nanotoxicology 2017 Nov - Dec;11(9-10):1157-1175. Epub 2017 Dec 1.

a Institute of Protein Biochemistry , National Research Council , Napoli , Italy.

Nanoparticles (NPs) are easily contaminated by bacterial endotoxin (lipopolysaccharide [LPS]). The presence of LPS can be responsible for many immune/inflammatory effects attributed to NPs. In this study, we examined the effects of LPS adsorption on the NP surface on the formation of a biocorona in biological fluids and on the subsequent inflammation-inducing activity of NPs. Different gold (Au) NPs with sizes ranging from 10 to 80 nm and with different surface functionalization (sodium citrate, lipoic acid, and branched polyethyleneimine (BPEI), or polyethylene glycol (PEG)) were exposed to E. coli LPS under different conditions. The binding capacity of LPS to the surface of AuNPs was dose- and time-dependent. LPS attached to sodium citrate and lipoic acid coatings, but did not adhere to BPEI- or PEG-coated NPs. By computational simulation, the binding of LPS to AuNPs seems to follow the Langmuir absorption isotherm. The presence of LPS on AuNP surface interfered and caused a decrease in the formation of the expected biomolecular corona upon incubation in human plasma. LPS-coated AuNPs, but not the LPS-free NPs, induced significant inflammatory responses in vitro. Notably, while free LPS did also induce an anti-inflammatory response, LPS bound to NPs appeared unable to do so. In conclusion, the unintentional adsorption of LPS onto the NP surface can affect the biocorona formation and the inflammatory properties of NPs. Thus, for an accurate interpretation of NP interactions with cells, it is extremely important to be able to distinguish the intrinsic NP biological effects from those caused by biologically active contaminants such as endotoxin.
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http://dx.doi.org/10.1080/17435390.2017.1401142DOI Listing
April 2018

Cancer resistance to treatment and antiresistance tools offered by multimodal multifunctional nanoparticles.

Cancer Nanotechnol 2017 26;8(1). Epub 2017 Oct 26.

Vall d'Hebron Research Institute (VHIR), Passeig Vall d'Hebron 119-129, 08035 Barcelona, Spain.

Chemotherapeutic agents have limited efficacy and resistance to them limits today and will limit tomorrow our capabilities of cure. Resistance to treatment with anticancer drugs results from a variety of factors including individual variations in patients and somatic cell genetic differences in tumours. In front of this, multimodality has appeared as a promising strategy to overcome resistance. In this context, the use of nanoparticle-based platforms enables many possibilities to address cancer resistance mechanisms. Nanoparticles can act as carriers and substrates for different ligands and biologically active molecules, antennas for imaging, thermal and radiotherapy and, at the same time, they can be effectors by themselves. This enables their use in multimodal therapies to overcome the wall of resistance where conventional medicine crash as ageing of the population advance. In this work, we review the cancer resistance mechanisms and the advantages of inorganic nanomaterials to enable multimodality against them. In addition, we comment on the need of a profound understanding of what happens to the nanoparticle-based platforms in the biological environment for those possibilities to become a reality.
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http://dx.doi.org/10.1186/s12645-017-0030-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658477PMC
October 2017

Formation of the Protein Corona: The Interface between Nanoparticles and the Immune System.

Semin Immunol 2017 12 21;34:52-60. Epub 2017 Oct 21.

Institut Català de Nanociència i Nanotecnologia (ICN2), CSIC and The Barcelona Institute of Science and Technology (BIST), Campus UAB, 08193, Bellaterra, Barcelona, Spain; Vall d'Hebron Institut de Recerca (VHIR), 08035 Barcelona, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), P. Lluís Companys 23, 08010 Barcelona, Spain. Electronic address:

The interaction of inorganic nanoparticles and many biological fluids often withstands the formation of a Protein Corona enveloping the nanoparticle. This Protein Corona provides the biological identity to the nanoparticle that the immune system will detect. The formation of this Protein Corona depends not only on the composition of the nanoparticle, its size, shape, surface state and exposure time, but also on the type of media, nanoparticle to protein ratio and the presence of ions and other molecular species that interfere in the interaction between proteins and nanoparticles. This has important implications on immune safety, biocompatibility and the use of nanoparticles in medicine.
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http://dx.doi.org/10.1016/j.smim.2017.10.001DOI Listing
December 2017

Intrinsic and Extrinsic Properties Affecting Innate Immune Responses to Nanoparticles: The Case of Cerium Oxide.

Front Immunol 2017 14;8:970. Epub 2017 Aug 14.

Vall d'Hebron Institut of Research (VHIR), Barcelona, Spain.

We review the apparent discrepancies between studies that report anti-inflammatory effects of cerium oxide nanoparticles (CeO NPs) through their reactive oxygen species-chelating properties and immunological studies highlighting their toxicity. We observe that several underappreciated parameters, such as aggregation size and degree of impurity, are critical determinants that need to be carefully addressed to better understand the NP biological effects in order to unleash their potential clinical benefits. This is because NPs can evolve toward different states, depending on the environment where they have been dispersed and how they have been dispersed. As a consequence, final characteristics of NPs can be very different from what was initially designed and produced in the laboratory. Thus, aggregation, corrosion, and interaction with extracellular matrix proteins critically modify NP features and fate. These modifications depend to a large extent on the characteristics of the biological media in which the NPs are dispersed. As a consequence, when reviewing the scientific literature, it seems that the aggregation state of NPs, which depends on the characteristics of the dispersing media, may be more significant than the composition or original size of the NPs. In this work, we focus on CeO NPs, which are reported sometimes to be protective and anti-inflammatory, and sometimes toxic and pro-inflammatory.
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http://dx.doi.org/10.3389/fimmu.2017.00970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557789PMC
August 2017

Analyses in zebrafish embryos reveal that nanotoxicity profiles are dependent on surface-functionalization controlled penetrance of biological membranes.

Sci Rep 2017 08 21;7(1):8423. Epub 2017 Aug 21.

Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, FI-20520, Turku, Finland.

Mesoporous silica nanoparticles (MSNs) are extensively explored as drug delivery systems, but in depth understanding of design-toxicity relationships is still scarce. We used zebrafish (Danio rerio) embryos to study toxicity profiles of differently surface functionalized MSNs. Embryos with the chorion membrane intact, or dechoroniated embryos, were incubated or microinjected with amino (NH-MSNs), polyethyleneimine (PEI-MSNs), succinic acid (SUCC-MSNs) or polyethyleneglycol (PEG-MSNs) functionalized MSNs. Toxicity was assessed by viability and cardiovascular function. NH-MSNs, SUCC-MSNs and PEG-MSNs were well tolerated, 50 µg/ml PEI-MSNs induced 100% lethality 48 hours post fertilization (hpf). Dechoroniated embryos were more sensitive and 10 µg/ml PEI-MSNs reduced viability to 5% at 96hpf. Sensitivity to PEG- and SUCC-, but not NH-MSNs, was also enhanced. Typically cardiovascular toxicity was evident prior to lethality. Confocal microscopy revealed that PEI-MSNs penetrated into the embryos whereas PEG-, NH2- and SUCC-MSNs remained aggregated on the skin surface. Direct exposure of inner organs by microinjecting NH-MSNs and PEI-MSNs demonstrated that the particles displayed similar toxicity indicating that functionalization affects the toxicity profile by influencing penetrance through biological barriers. The data emphasize the need for careful analyses of toxicity mechanisms in relevant models and constitute an important knowledge step towards the development of safer and sustainable nanotherapies.
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http://dx.doi.org/10.1038/s41598-017-09312-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566213PMC
August 2017

Conserved effects and altered trafficking of Cetuximab antibodies conjugated to gold nanoparticles with precise control of their number and orientation.

Nanoscale 2017 May;9(18):6111-6121

Institut Català de Nanotecnologia (ICN2), Campus UAB, 08193 Cerdanyola del Vallés, Spain.

Gold nanoparticles (17 nm) have been functionalized with the antiangiogenic monoclonal antibody drug Cetuximab at a well-defined orientation and coverage density of antibodies. Functionalization has been carried out through site-directed chemistry via the selective oxidation of the carbohydrate moiety of antibodies linked to a thiolated hydrazide. A431 tumor cells have been exposed to these conjugates for in vitro evaluation of their effects. In addition to epithelial growth factor receptor blocking, trafficking and signaling alterations were also observed. Thus, the blocking effects of Cetuximab were increased and sustained for a longer time when associated with the nanoparticles. Enhancing antibody therapy effects by decreasing the needed dose and prolonging its effect by avoiding receptor recycling may serve to obtain increased therapeutic benefits for immunotherapy.
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http://dx.doi.org/10.1039/c7nr00947jDOI Listing
May 2017

Stimuli-responsive hybrid nanocarriers developed by controllable integration of hyperbranched PEI with mesoporous silica nanoparticles for sustained intracellular siRNA delivery.

Int J Nanomedicine 2016;11:6591-6608. Epub 2016 Dec 8.

Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University.

Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with the challenge being to deliver it in a sustained manner. The combination of mesoporous silica nanoparticles (MSNs) and polycations in the confined pore space allows for incorporation and controlled release of therapeutic siRNA payloads. We hereby constructed MSNs with expanded mesopores and pore-surface-hyperbranched poly(ethyleneimine) (PEI) tethered with redox-cleavable linkers that could carry a high payload of siRNA (120 mg·g). The developed nanocarriers were efficiently taken up by cancer cells and were subsequently able to escape to the cytoplasm from the endosomes, most likely owing to the integrated PEI. Triggered by the intracellular redox conditions, the siRNA was sustainably released inside the cells over a period of several days. Functionality of siRNAs was demonstrated by using cell-killing siRNA as cargo. Despite not being the aim of the developed system, in vitro experiments using cell-killing siRNAs showed that the efficacy of siRNA transfection was comparable to the commercial in vitro transfection agent Lipofectamine. Consequently, the developed MSN-based delivery system offers a potential approach to hybrid nanocarriers for more efficient and long-term siRNA delivery and, in a longer perspective, in vivo gene silencing for RNA interference (RNAi) therapy.
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http://dx.doi.org/10.2147/IJN.S120611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154729PMC
February 2017

Assessing the Immunosafety of Engineered Nanoparticles with a Novel in Vitro Model Based on Human Primary Monocytes.

ACS Appl Mater Interfaces 2016 Oct 13;8(42):28437-28447. Epub 2016 Oct 13.

Institute of Protein Biochemistry, National Research Council , 80131 Napoli, Italy.

The possibility that nanomaterials could perturb the normal course of an inflammatory response is a key issue when assessing nanoimmunosafety. The alteration of the normal progress of an inflammatory response may have pathological consequences, since inflammation is a major defensive mechanism and its efficiency maintains the body's health. The immunosafety of engineered nanoparticles at nontoxic concentrations was investigated with the use of a human primary monocyte-based in vitro system, which reproduces in a simplified fashion the full course of the physiological inflammatory response, from initiation and development to resolution. The kinetics of expression and production of inflammatory and anti-inflammatory cytokines and the proteomic profiles were used for describing the inflammatory defensive response. We assessed the ability of gold and silver nanoparticles to trigger inflammation and to interfere with the course of an ongoing defensive reaction. While neither nanoparticle type was able to directly activate monocytes, silver nanoparticles could exacerbate the inflammatory response of monocytes but did not interfere with the resolution of the inflammatory reaction. These findings support the use of human primary monocyte-based in vitro assays for realistically investigating the effects of engineered nanoparticles on human innate immune responses, in order to predict the immunological risk of nanomaterials and implement safe nanoparticle-based applications.
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http://dx.doi.org/10.1021/acsami.6b06278DOI Listing
October 2016

Pharmacokinetics and Tissue Disposition of Nanosystem-Entrapped Betulin After Endotracheal Administration to Rats.

Eur J Drug Metab Pharmacokinet 2017 Apr;42(2):327-332

Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, BioCity (3rd Floor), Tykistökatu 6A, 20520, Turku, Finland.

Background And Objectives: Betulin is a triterpene extracted from the cork layer of the outer bark of Betula spp. It has a wide spectrum of pharmacological activities, including being lung protective; however, its bioavailability is low. To increase its bioavailability, betulin was entrapped in a nanosystem (BN). In this study, we investigated the pharmacokinetics and tissue distribution of nanosystem-entrapped betulin after single dose endotracheal administration to rats.

Method: Betulin was nanosystem-entrapped using a solvent exchange technique. The surface morphology and size of the nanosystem were characterized by transmission electron microscopy and dynamic light scattering. The plasma and tissue concentrations of betulin were determined using a validated high-performance liquid chromatography method.

Results: The highest concentration of betulin was found in lungs and liver, and the lowest in the heart. Betulin did not penetrate highly vascularized tissues or tissue with an average degree of vascularization, nor did it cross the blood-brain barrier. Tissue availability in the lungs was 1.3 times higher for BN than for free betulin. Betulin was detected in the bloodstream at 15 min after administration of BN compared with only at 1 h after administration of free betulin. Penetration of betulin in the liver tissue was characterized by a high degree of intensity both for BN and free betulin. Betulin in the heart tissue was detected in much smaller quantities than in the liver.

Conclusion: Entrapment of betulin in nanosystem form shows promise as a novel strategy in the treatment of pulmonary diseases.
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http://dx.doi.org/10.1007/s13318-016-0340-7DOI Listing
April 2017

Chitosan functionalisation of gold nanoparticles encourages particle uptake and induces cytotoxicity and pro-inflammatory conditions in phagocytic cells, as well as enhancing particle interactions with serum components.

J Nanobiotechnology 2015 Nov 18;13:84. Epub 2015 Nov 18.

Department of Molecular Biology, Division of Allergy and Immunology, Paris-Lodron University Salzburg, Hellbrunnerstr. 34, 5020, Salzburg, Austria.

Background: Gold nanoparticles (AuNPs) are a popular choice for use in medical and biomedical research applications. With suitable functionalisation AuNPs can be applied in drug delivery systems, or can aid in disease diagnosis. One such functionalisation is with chitosan, which enables efficient interaction and permeation of cellular membranes, providing an effective adjuvant. As both AuNPs and chitosan have been shown to have low toxicity and high biocompatibility their proposed use in nanomedicine, either individually or combined, is expanding. However, further toxicological and immunological assessments of AuNP-chitosan conjugates are still needed. Therefore, we have evaluated how AuNP functionalisation with chitosan can affect uptake, cytotoxicity, and immunological responses within mononuclear cells, and influence the interaction of AuNPs with biomolecules within a complex biofluid. The AuNPs used were negatively charged through citrate-coating, or presented either low or high positive charge through chitosan-functionalisation. Uptake by THP-1 cells was assessed via transmission electron microscopy and electron energy loss spectroscopy, pro-inflammatory responses by ELISA and qRT-PCR, and cell death and viability via lactate dehydrogenase release and mitochondrial activity, respectively. Interactions of AuNPs with protein components of a frequently used in vitro cell culture medium supplement, foetal calf serum, were investigated using mass spectrometry.

Results: Although cells internalised all AuNPs, uptake rates and specific routes of intracellular trafficking were dependent upon chitosan-functionalisation. Accordingly, an enhanced immune response was found to be chitosan-functionalisation-dependent, in the form of CCL2, IL-1β, TNF-α and IL-6 secretion, and expression of IL-1β and NLRP3 mRNA. A corresponding increase in cytotoxicity was found in response to chitosan-coated AuNPs. Furthermore, chitosan-functionalisation was shown to induce an increase in unique proteins associating with these highly charged AuNPs.

Conclusions: It can be concluded that functionalisation of AuNPs with the perceived non-toxic biocompatible molecule chitosan at a high density can elicit functionalisation-dependent intracellular trafficking mechanisms and provoke strong pro-inflammatory conditions, and that a high affinity of these NP-conjugates for biomolecules may be implicit in these cellular responses.
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http://dx.doi.org/10.1186/s12951-015-0146-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652435PMC
November 2015

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

J Hepatol 2016 Mar 28;64(3):691-8. Epub 2015 Oct 28.

Biochemistry and Molecular Genetics Service, Hospital Clínic Universitari, IDIBAPS, CIBERehd, Barcelona, Spain; Department Ciencies Fisiologiques I, University of Barcelona, Barcelona, Spain. Electronic address:

Background & Aims: Cerium oxide nanoparticles (CeO2NPs) have proven to behave as free radical scavengers and/or anti-inflammatory agents. The aim of the study was to determine whether CeO2NPs display hepatoprotective properties in experimental chronic liver disease.

Methods: Systemic and hepatic effects of nanoparticles were assessed in CCl4-treated rats receiving CeO2NPs or vehicle twice weekly for two weeks and CCl4 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 CeO2NPs 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 CeO2NPs. This was associated with reduced macrophage infiltration and reduced abundance of caspase-3, α-SMA and inflammatory cytokines.

Conclusions: CeO2NPs administration to CCl4-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 CeO2NPs may be of therapeutic value in chronic liver disease.
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http://dx.doi.org/10.1016/j.jhep.2015.10.020DOI Listing
March 2016

Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response following zinc oxide nanoparticle exposure under different cell culture conditions.

Part Fibre Toxicol 2015 Sep 29;12:29. Epub 2015 Sep 29.

Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020, Salzburg, Austria.

Background: Stably transfected lung epithelial reporter cell lines pose an advantageous alternative to replace complex experimental techniques to monitor the pro-inflammatory response following nanoparticle (NP) exposure. Previously, reporter cell lines have been used under submerged culture conditions, however, their potential usefulness in combination with air-liquid interface (ALI) exposures is currently unknown. Therefore, the aim of the present study was to compare a panel of interleukin-8 promoter (pIL8)-reporter cell lines (i.e. green or red fluorescent protein (GFP, RFP), and luciferase (Luc)), originating from A549 lung epithelial type II-like cells cells, following NPs exposure under both submerged and ALI conditions.

Methods: All cell lines were exposed to zinc oxide (ZnO) NPs at 0.6 and 6.2 μg/cm(2) for 3 and 16 hours under both submerged and ALI conditions. Following physicochemical characterization, the cytotoxic profile of the ZnO-NPs was determined for each exposure scenario. Expression of IL-8 from all cell types was analyzed at the promoter level and compared to the mRNA (qRT-PCR) and protein level (ELISA).

Results: In summary, each reporter cell line detected acute pro-inflammatory effects following ZnO exposure under each condition tested. The pIL8-Luc cell line was the most sensitive in terms of reporter signal strength and onset velocity following TNF-α treatment. Both pIL8-GFP and pIL8-RFP also showed a marked signal induction in response to TNF-α, although only after 16 hrs. In terms of ZnO-NP-induced cytotoxicity pIL8-RFP cells were the most affected, whilst the pIL8-Luc were found the least responsive.

Conclusions: In conclusion, the use of fluorescence-based reporter cell lines can provide a useful tool in screening the pro-inflammatory response following NP exposure in both submerged and ALI cell cultures.
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http://dx.doi.org/10.1186/s12989-015-0104-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587722PMC
September 2015

The oxidative potential of differently charged silver and gold nanoparticles on three human lung epithelial cell types.

J Nanobiotechnology 2015 Jan 16;13. Epub 2015 Jan 16.

Department of Molecular Biology, Paris Lodron-University of Salzburg, Hellbrunnerstr. 34, A-5020, Salzburg, Austria.

Background: Nanoparticle (NPs) functionalization has been shown to affect their cellular toxicity. To study this, differently functionalized silver (Ag) and gold (Au) NPs were synthesised, characterised and tested using lung epithelial cell systems.

Methods: Monodispersed Ag and Au NPs with a size range of 7 to 10 nm were coated with either sodium citrate or chitosan resulting in surface charges from -50 mV to +70 mV. NP-induced cytotoxicity and oxidative stress were determined using A549 cells, BEAS-2B cells and primary lung epithelial cells (NHBE cells). TEER measurements and immunofluorescence staining of tight junctions were performed to test the growth characteristics of the cells. Cytotoxicity was measured by means of the CellTiter-Blue ® and the lactate dehydrogenase assay and cellular and cell-free reactive oxygen species (ROS) production was measured using the DCFH-DA assay.

Results: Different growth characteristics were shown in the three cell types used. A549 cells grew into a confluent mono-layer, BEAS-2B cells grew into a multilayer and NHBE cells did not form a confluent layer. A549 cells were least susceptible towards NPs, irrespective of the NP functionalization. Cytotoxicity in BEAS-2B cells increased when exposed to high positive charged (+65-75 mV) Au NPs. The greatest cytotoxicity was observed in NHBE cells, where both Ag and Au NPs with a charge above +40 mV induced cytotoxicity. ROS production was most prominent in A549 cells where Au NPs (+65-75 mV) induced the highest amount of ROS. In addition, cell-free ROS measurements showed a significant increase in ROS production with an increase in chitosan coating.

Conclusions: Chitosan functionalization of NPs, with resultant high surface charges plays an important role in NP-toxicity. Au NPs, which have been shown to be inert and often non-cytotoxic, can become toxic upon coating with certain charged molecules. Notably, these effects are dependent on the core material of the particle, the cell type used for testing and the growth characteristics of these cell culture model systems.
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http://dx.doi.org/10.1186/s12951-014-0062-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304186PMC
January 2015

Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles.

Nanotoxicology 2015 May 14;9(4):462-73. Epub 2014 Aug 14.

Institute of Biomedical Technologies, National Research Council , Pisa , Italy .

Engineered nanoparticles (NP) are generally contaminated by bacterial endotoxin, a ubiquitous bacterial molecule with significant toxic and inflammatory effects. The presence of endotoxin, if not recognised, can be responsible for many of the in vitro and in vivo effects attributed to NPs. The Limulus Amoebocyte Lysate (LAL) assay, the test requested by regulatory authorities for assessing endotoxin contamination in products for human use, is not immediately applicable for testing endotoxin in NP preparations, mainly due to the possible interference of NPs with the assay readouts and components. In this study, we have compared different commercially available LAL assays for detecting endotoxin in gold, silver and iron oxide NPs. Different NP chemistry, concentrations and surface coatings could differently interfere with the LAL assays' results. After accurate testing of the possible interaction/interference of NPs with the various assay components, the modified chromogenic LAL assay proved the most suitable assay for measuring endotoxin in NP samples, provided the appropriate controls are performed. Thus, endotoxin determination can be performed in NP preparation with commercial LAL assays only after assay validation, i.e. once possible interference of NPs with the assay components and readouts has been excluded.
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http://dx.doi.org/10.3109/17435390.2014.948090DOI Listing
May 2015