Aloïse Mabondzo

Titanium dioxide nanoparticle impact and translocation through ex vivo, in vivo and in vitro gut epithelia

BackgroundTiO2 particles are commonly used as dietary supplements and may contain up to 36% of nano-sized particles (TiO2-NPs). Still impact and translocation of NPs through the gut epithelium is poorly documented.ResultsWe show that, in vivo and ex vivo, agglomerates of TiO2-NPs cross both the regular ileum epithelium and the follicle-associated epithelium (FAE) and alter the paracellular permeability of the ileum and colon epithelia. In vitro, they accumulate in M-cells and mucus-secreting cells, much less in enterocytes. They do not cause overt cytotoxicity or apoptosis. They translocate through a model of FAE only, but induce tight junctions remodeling in the regular ileum epithelium, which is a sign of integrity alteration and suggests paracellular passage of NPs. Finally we prove that TiO2-NPs do not dissolve when sequestered up to 24 h in gut cells.ConclusionsTaken together these data prove that TiO2-NPs would possibly translocate through both the regular epithelium lining the ileum and through Peyer’s patches, would induce epithelium impairment, and would persist in gut cells where they would possibly induce chronic damage.

In vitro evidence of dysregulation of blood–brain barrier function after acute and repeated/long-term exposure to TiO2 nanoparticles

The effects of titanium dioxide nanoparticles (TiO(2) NPs) on blood-brain barrier (BBB) function are unknown. Here, we report such evidence of adverse effects after in vitro exposure of a rat primary cell-based BBB model to NPs. BBB integrity was studied by measuring the flux of sucrose through the monolayer. P-glycoprotein (P-gp) activity was assessed by measuring the passage of vinblastine. Transcription profiles of P-gp and other ABC transporters as well as of cytokines were investigated by real-time PCR. Electron microscopy and particle-induced X-ray emission measurements were performed. We compared several exposure modalities, from early to chronic, mimicking a brain-to-blood transport or a systemic contamination. In the first case, BBB integrity was preserved, but P-gp activity of endothelial cells (BECs) was reduced. In the second case, BBB integrity and P-gp function were impaired from 5 μg/mL for 24 h and expression of tight junction proteins and efflux transporters was modulated. An inflammatory response had repercussions on ABC transporter expression of glial cells. We demonstrate that NPs accumulated in BECs and crossed the cell monolayer. These findings suggest that there is an immunoregulatory loop between inflammatory components, BECs and glial cells in the dysfunction of the BBB during exposure to TiO(2) NPs.

P-glycoprotein, breast cancer resistance protein, Organic Anion Transporter 3, and Transporting Peptide 1a4 during blood-brain barrier maturation: involvement of Wnt/β-catenin and endothelin-1 signaling.

Our current knowledge about drug transporters in the maturational brain is very limited. In this study, we provide a comprehensive overview of the expression and activity profile of P-glycoprotein (P-gp), Breast Cancer Resistance Protein (bcrp), Organic Anion Transporter 3 (oat3), and Transporting Peptide 1a4 (oatp1a4) transporters during blood-brain barrier (BBB) maturation. Gene and protein expressions of the analyzed transporters increase as the brain matures, with no variation in their activity for P-gp and bcrp, while the transport activity of oat3 and oatp1a4 increases during brain maturation from preterm up to adulthood. For the first time, we illustrate a downregulation of nuclear β-catenin expression in brain capillaries when bcrp, P-gp, oat3, and oatp1a4 transporters are at their highest expression levels. In vivo activation of β-catenin in rat brains, by intracerebroventricular (ICV) injection of a GSK-3 inhibitor, enhances the activity of P-gp, bcrp, oat3, and oatp1a4. Interestingly, in an in vitro BBB model consisting of a coculture of primary endothelial brain cells with astrocytes or in vivo, activation of β-catenin enhances the mRNA expression of ET-1. Interestingly, blocking the ETA receptor for endothelin-1 in vivo by ICV injection of a ETA antagonist decreases transporter activity mediated by the activation of β-catenin. These findings shed light on the role of an interaction between β-catenin and endothelin-1 signaling in the regulation of these transporters at the BBB.

Biopersistence and translocation to extrapulmonary organs of titanium dioxide nanoparticles after subacute inhalation exposure to aerosol in adult and elderly rats.

The increasing industrial use of nanoparticles (NPs) has raised concerns about their impact on human health. Since aging and exposure to environmental factors are linked to the risk for developing pathologies, we address the question of TiO2 NPs toxicokinetics in the context of a realistic occupational exposure. We report the biodistribution of titanium in healthy young adults (12-13-week-old) and in elderly rats (19-month-old) exposed to 10mg/m3 of a TiO2 nanostructured aerosol 6h/day, 5days/week for 4 weeks. We measured Ti content in major organs using inductively coupled plasma mass spectrometry immediately and up to 180days after the end of exposure. Large amounts of titanium were initially found in lung which were slowly cleared during the post-exposure period. From day 28, a small increase of Ti was found in the spleen and liver of exposed young adult rats. Such an increase was however never found in their blood, kidneys or brain. In the elderly group, translocation to extra-pulmonary organs was significant at day 90. Ti recovered from the spleen and liver of exposed elderly rats was higher than in exposed young adults. These data suggest that TiO2 NPs may translocate from the lung to extra-pulmonary organs where they could possibly promote systemic health effects.

Rational design of polyarginine nanocapsules intended to help peptides overcoming intestinal barriers

The aim of this work was to rationally design and characterize nanocapsules (NCs) composed of an oily core and a polyarginine (PARG) shell, intended for oral peptide delivery. The cationic polyaminoacid, PARG, and the oily core components were selected based on their penetration enhancing properties. Insulin was adopted as a model peptide to assess the performance of the NCs. After screening numerous formulation variables, including different oils and surfactants, we defined a composition consisting of oleic acid, sodium deoxycholate (SDC) and Span 80. This selected NCs composition, produced by the solvent displacement technique, exhibited the following key features: (i) an average size of 180nm and a low polydispersity (0.1), (ii) a high insulin association efficacy (80-90% AE), (iii) a good colloidal stability upon incubation in simulated intestinal fluids (SIF, FaSSIF-V2, FeSSIF-V2), and (iv) the capacity to control the release of the associated insulin for >4h. Furthermore, using the Caco-2 model cell line, PARG nanocapsules were able to interact with the enterocytes, and reversibly modify the TEER of the monolayer. Both cell adhesion and membrane permeabilization could account for the pronounced transport of the NCs-associated insulin (3.54%). This improved interaction was also visualized by confocal fluorescent microscopy following oral administration of PARG nanocapsulesto mice. Finally, in vivo efficacy studies performed in normoglycemic rats showed a significant decrease in their plasma glucose levels after treatment. In conclusion, here we disclose key formulation elements for making possible the oral administration of peptides.

In vivo effects of zearalenone on the expression of proteins involved in the detoxification of rat xenobiotics

Zearalenone (ZEN) is a lactone derivative of the resorcylic acid produced by various Fusarium species that are widely found in foods and animal feeds. ZEN exerts species‐specific estrogenic effects, possibly because of the metabolism differences arising from reduction, hydroxylation, or glucuro‐conjugation. The main objective of this study was to determine the levels of expression of rat proteins that are involved in the ZEN detoxification pathway upon acute ZEN treatment. This was achieved by monitoring the mRNA associated with 25 genes using RT‐PCR upon ZEN uptake. These genes code for a variety of proteins that are involved in cellular detoxifying pathways, transporters, cytochromes P450 (CYPs), hydroxysteroid dehydrogenases, and transferases, and receptors that are involved in CYP expression or steroid metabolism. Liver samples from rats treated with ZEN were compared to untreated rats or animals treated with classical CYP inducers (phenobarbital, dexamethasone, β‐naphtoflavone, and clofibrate). Significant changes of mRNA expression were observed for the efflux transporter, P‐glycoprotein, monooxygenases (CYP2C7, CYP2E1, CYP3A1, CYP3A2, and aromatase), steroid dehydrogenases, and Uridine diphospho–glucuronyl transferases (UGTs). Following a single ZEN treatment, the initial modifications in mRNA levels indicate a close association with microsomal enzyme activity of the CYP2B, CYP2C, and CYP3A protein families.

Investigation of TiO2 nanoparticles translocation through a Caco-2 monolayer

Nanoparticles (NPs) are introduced in a growing number of commercial products, including food and beverage but their effects on gastrointestinal tract are poorly investigated. Here we focused on the translocation of TiO2 NPs through Caco-2 monolayers exposed to anatase and rutile NPs up to 24 h. Internalization was followed by transmission electronic microscopy and μ-XRF elemental mapping, coupled to XAS analysis of Ti atoms environment. This innovative technique is among the best techniques to get insights on NP fate after internalization. The originality of this project relies on the panel of microscopy techniques implemented to investigate digestive barrier translocation, bringing together biologists, chemists and physicists in a pluridisciplinary research program.

Mineralization of TiO2 nanoparticles for the determination of titanium in rat tissues

In order to draw appropriate conclusions about the possible adverse biological effects of titanium dioxide nanoparticles (TiO2—NPs), the so-called “dose?effect” relationship must be explored. This requires proper quantification of titanium in complex matrices such as animal organs for future toxicological studies. This study presents the method development for mineralizing TiO2—NPs for analysis of biological tissues. We compared the recovery and quantification limits of the four most commonly used mineralization methods for metal oxides. Microwave-assisted dissolution in an HNO3–HF mixture followed by H2O2 treatment produced the best results for a TiO2—NPs suspension, with 96 ± 8% recovery and a limit of quantification as low as 0.9 µg/L. This method was then used for the determination of titanium levels in tissue samples taken from rats. However, our tests revealed that even this method is not sensitive enough for quantifying titanium levels in single olfactory bulbs or hippocampus in control animals.

PEG-PGA enveloped octaarginine-peptide nanocomplexes: An oral peptide delivery strategy

&NA; The objective of this work was the development of a new drug nanocarrier intended to overcome the barriers associated to the oral modality of administration and to assess its value for the systemic or local delivery of peptides. The nanocarrier was rationally designed taking into account the nature of the intestinal barriers and was loaded with insulin, which was selected as a model peptide. The nanocarrier consisted of a complex between insulin and a hydrophobically‐modified cell penetrating peptide (CPP), enveloped by a protecting polymer. The selected CPP was octaarginine (r8), chemically conjugated with cholesterol (Chol) or lauric acid (C12), whereas the protecting polymer was poly (glutamic acid)‐poly (ethylene glycol) (PGA‐PEG). This enveloping material was intended to preserve the stability of the nanocomplex in the intestinal medium and facilitate its diffusion across the intestinal mucus. The enveloped nanocomplexes (ENCPs) exhibited a number of key features, namely (i) a unimodal size distribution with a mean size of 200 nm and a neutral zeta potential, (ii) the capacity to associate insulin (˜100% association efficiency) and protect it from degradation in simulated intestinal fluids, (iii) the ability to diffuse through intestinal mucus and, most importantly, (iv) the capacity to interact with the Caco‐2 model epithelium, resulting in a massive insulin cell uptake (47.59 ± 5.79%). This enhanced accumulation of insulin at the epithelial level was not translated into an enhanced insulin transport. In fact, only 2% of insulin was transported across the monolayer, and this was correlated with a moderate response of insulin following oral administration to healthy rats. Despite of this, the accumulation of the insulin‐loaded nanocarriers in the intestinal mucosa could be verified in vivo upon their labeling with 99mTc. Overall, these data underline the capacity of the nanocarriers to overcome substantial barriers associated to the oral modality of administration and to facilitate the accumulation of the associated peptide at the intestinal level. Graphical abstract Figure. No caption available.