Nuno Faria

Origin and fate of dietary nanoparticles and microparticles in the gastrointestinal tract

Humans have evolved with oral exposure to dietary microparticles and nanoparticles as a normal occurrence but the ever-growing exploitation of nanotechnology is likely to increase exposure further, both qualitatively and quantitatively. Moreover, unlike the situation with respirable particles, relatively little is known about gastrointestinal intake and handling of nanoparticles. With a long term interest in gut exposure and responses to dietary microparticles, our group is now applying its expertise to nanoparticles in the gastrointestinal tract. Here we aim to address (i) the current challenges associated with the characterisation of particle-host or particle-cell interactions, (ii) the origin and mechanisms of uptake of particles in the gastrointestinal tract, especially via the Peyers patch and (iii) potential cellular effects of nanoparticles in the generation of reactive oxygen species and inflammasome activation, or microparticles in their adjuvant activity in pro-inflammatory signalling and immune responsiveness.

Influence of simulated gastrointestinal conditions on particle-induced cytotoxicity and interleukin-8 regulation in differentiated and undifferentiated Caco-2 cells

Abstract Novel aspects of engineered nanoparticles offer many advantages for optimising food products and packaging. However, their potential hazards in the gastrointestinal tract require further investigation. We evaluated the toxic and inflammatory potential of two types of particles that might become increasingly relevant to the food industry, namely SiO2 and ZnO. The materials were characterised for their morphology, oxidant generation and hydrodynamic behaviour. Cytotoxicity and interleukin-8 mRNA and protein expression were evaluated in human intestinal Caco-2 cells. Particle pretreatment under simulated gastric and intestinal pH conditions resulted in reduced acellular ROS formation but did not influence cytotoxicity (WST-1 assay) or IL-8 expression. However, the differentiation status of the cells markedly determined the cytotoxic potency of the particles. Further research is needed to determine the in vivo relevance of our current observations regarding the role of particle aggregation and the stage of intestinal epithelial cell differentiation in determining the hazards of ingested particles.

Caco-2 Cell Acquisition of Dietary Iron(III) Invokes a Nanoparticulate Endocytic Pathway

Dietary non-heme iron contains ferrous [Fe(II)] and ferric [Fe(III)] iron fractions and the latter should hydrolyze, forming Fe(III) oxo-hydroxide particles, on passing from the acidic stomach to less acidic duodenum. Using conditions to mimic the in vivo hydrolytic environment we confirmed the formation of nanodisperse fine ferrihydrite-like particles. Synthetic analogues of these (~ 10 nm hydrodynamic diameter) were readily adherent to the cell membrane of differentiated Caco-2 cells and internalization was visualized using transmission electron microscopy. Moreover, Caco-2 exposure to these nanoparticles led to ferritin formation (i.e., iron utilization) by the cells, which, unlike for soluble forms of iron, was reduced (p=0.02) by inhibition of clathrin-mediated endocytosis. Simulated lysosomal digestion indicated that the nanoparticles are readily dissolved under mildly acidic conditions with the lysosomal ligand, citrate. This was confirmed in cell culture as monensin inhibited Caco-2 utilization of iron from this source in a dose dependent fashion (p<0.05) whilet soluble iron was again unaffected. Our findings reveal the possibility of an endocytic pathway for acquisition of dietary Fe(III) by the small intestinal epithelium, which would complement the established DMT-1 pathway for soluble Fe(II).

Nanoparticulate iron(III) oxo-hydroxide delivers safe iron that is well absorbed and utilised in humans

Iron deficiency is the most common nutritional disorder worldwide with substantial impact on health and economy. Current treatments predominantly rely on soluble iron which adversely affects the gastrointestinal tract. We have developed organic acid-modified Fe(III) oxo-hydroxide nanomaterials, here termed nano Fe(III), as alternative safe iron delivery agents. Nano Fe(III) absorption in humans correlated with serum iron increase (P < 0.0001) and direct in vitro cellular uptake (P = 0.001), but not with gastric solubility. The most promising preparation (iron hydroxide adipate tartrate: IHAT) showed ~80% relative bioavailability to Fe(II) sulfate in humans and, in a rodent model, IHAT was equivalent to Fe(II) sulfate at repleting haemoglobin. Furthermore, IHAT did not accumulate in the intestinal mucosa and, unlike Fe(II) sulfate, promoted a beneficial microbiota. In cellular models, IHAT was 14-fold less toxic than Fe(II) sulfate/ascorbate. Nano Fe(III) manifests minimal acute intestinal toxicity in cellular and murine models and shows efficacy at treating iron deficiency anaemia. From the Clinical Editor This paper reports the development of novel nano-Fe(III) formulations, with the goal of achieving a magnitude less intestinal toxicity and excellent bioavailability in the treatment of iron deficiency anemia. Out of the tested preparations, iron hydroxide adipate tartrate met the above criteria, and may become an important tool in addressing this common condition.

A nano-disperse ferritin-core mimetic that efficiently corrects anemia without luminal iron redox activity

The 2-5 nm Fe(III) oxo-hydroxide core of ferritin is less ordered and readily bioavailable compared to its pure synthetic analogue, ferrihydrite. We report the facile synthesis of tartrate-modified, nano-disperse ferrihydrite of small primary particle size, but with enlarged or strained lattice structure (~ 2.7 Å for the main Bragg peak versus 2.6 Å for synthetic ferrihydrite). Analysis indicated that co-precipitation conditions can be achieved for tartrate inclusion into the developing ferrihydrite particles, retarding both growth and crystallization and favoring stabilization of the cross-linked polymeric structure. In murine models, gastrointestinal uptake was independent of luminal Fe(III) reduction to Fe(II) and, yet, absorption was equivalent to that of ferrous sulphate, efficiently correcting the induced anemia. This process may model dietary Fe(III) absorption and potentially provide a side effect-free form of cheap supplemental iron. From the Clinical Editor Small size tartrate-modified, nano-disperse ferrihydrite was used for efficient gastrointestinal delivery of soluble Fe(III) without the risk for free radical generation in murine models. This method may provide a potentially side effect-free form iron supplementation.

Extracellular vesicles are independent metabolic units with asparaginase activity.

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. While it has been shown that cells can traffic metabolic enzymes via EVs much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Both our metabolomics and functional analyses revealed that EVs harbour L-asparaginase activity catalysed by the enzyme Asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC-derived EVs traffic ASRGL1. Our results demonstrate for the first time that NSC EVs function as independent, extracellular metabolic units able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Ferroportin mediates the intestinal absorption of iron from a nanoparticulate ferritin core mimetic in mice

The ferritin core is composed of fine nanoparticulate Fe3+ oxohydroxide, and we have developed a synthetic mimetic, nanoparticulate Fe3+ polyoxohydroxide (nanoFe3+). The aim of this study was to determine how dietary iron derived in this fashion is absorbed in the duodenum. Following a 4 wk run‐in on an Fe‐deficient diet, mice with intestinal‐specific disruption of the Fpn‐1 gene (Fpn‐KO), or littermate wild‐type (WT) controls, were supplemented with Fe2+ sulfate (FeSO4), nanoFe3+, or no added Fe for a further 4 wk. A control group was Fe sufficient throughout. Direct intestinal absorption of nanoFe3+ was investigated using isolated duodenal loops. Our data show that FeSO4 and nanoFe3+ are equally bioavailable in WT mice, and at wk 8 the mean ± sem hemoglobin increase was 18 ± 7 g/L in the FeSO4 group and 30 ± 5 g/L in the nanoFe3+ group. Oral iron failed to be utilized by Fpn‐KO mice and was retained in enterocytes, irrespective of the iron source. In summary, although nanoFe3+ is taken up directly by the duodenum its homeostasis is under the normal regulatory control of dietary iron absorption, namely via ferroportin‐dependent efflux from enterocytes, and thus offers potential as a novel oral iron supplement.—Aslam, M. F., Frazer, D. M., Faria, N., Bruggraber, S. F. A., Wilkins, S. J., Mirciov, C., Powell, J. J., Anderson, G. J., Pereira, D. I. A. Ferroportin mediates the intestinal absorption of iron from a nanoparticulate ferritin core mimetic in mice. FASEB J. 28, 3671–3678 (2014). www.fasebj.org

Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate

AIM To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles. MATERIAL & METHODS The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. RESULTS Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. CONCLUSION In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes.

Development of DRC-ICP-MS methodology for the rapid determination of 58Fe erythrocyte incorporation in human iron absorption studies

Iron deficiency is the only major nutritional deficiency that still exists in the developed world and iron deficiency anaemia affects nearly one billion people worldwide (Benoist et al., Worldwide prevalence on anaemia 1993–2005, WHO, 2008). However, iron supplements are still not optimally formulated so cheap, side effect-free and well absorbed iron supplements are sought. The development of these requires the determination of iron absorption in animals, volunteers or patients, typically by determining iron isotopic enrichment in blood after administering isotopically labelled iron supplements. Current analytical techniques for isotope ratio work, such as thermal ionisation mass spectrometry (TIMS) and multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS), have a low throughput, due to the requirements of sample pre-treatment and, generally, are not present in clinical or nutritional laboratories. Here we describe a novel, more accessible, dynamic reaction cell inductively coupled plasma mass spectrometry (DRC-ICP-MS) method for the determination of 58Fe enrichment in samples from nutritional or clinical studies. This is a high throughput method, in which the samples require no pre-treatment other than dilution, that was validated against MC-ICP-MS, and was shown to be fit-for-purpose.

Synthetic mimetics of the endogenous gastrointestinal nanomineral: Silent constructs that trap macromolecules for intracellular delivery

Amorphous magnesium-substituted calcium phosphate (AMCP) nanoparticles (75-150 nm) form constitutively in large numbers in the mammalian gut. Collective evidence indicates that they trap and deliver luminal macromolecules to mucosal antigen presenting cells (APCs) and facilitate gut immune homeostasis. Here, we report on a synthetic mimetic of the endogenous AMCP and show that it has marked capacity to trap macromolecules during formation. Macromolecular capture into AMCP involved incorporation as shown by STEM tomography of the synthetic AMCP particle with 5 nm ultra-fine iron (III) oxohydroxide. In vitro, organic cargo-loaded synthetic AMCP was taken up by APCs and tracked to lysosomal compartments. The AMCP itself did not regulate any gene, or modify any gene regulation by its cargo, based upon whole genome transcriptomic analyses. We conclude that synthetic AMCP can efficiently trap macromolecules and deliver them to APCs in a silent fashion, and may thus represent a new platform for antigen delivery.