Sónia Fraga

Effect of surface coating on the biodistribution profile of gold nanoparticles in the rat

Successful application of gold nanoparticles (AuNPs) in biomedicine requires extensive safety assessment for which biokinetic studies are crucial. We evaluated the biodistribution of AuNPs (∼20 nm) with different surface coatings: citrate, 11-MUA and 3 pentapeptides, CALNN, CALND and CALNS, after i.v. administration to rats (0.6-1 mg Au/kg). Biodistribution was evaluated based on Au tissue content measured by GFAAS. Citrate-AuNPs were rapidly removed from circulation with 60% of the injected dose depositing in the liver. Thirty minutes post-injection, the lungs presented about 6% of the injected dose with levels decreasing to 0.7% at 24 h. Gold levels in the spleen were of 2.6%. After 24 h, liver presented the highest Au level, followed by spleen and blood. A similar biodistribution profile was observed for MUA-coated AuNPs compared to Cit-AuNPs at 24h post-injection, while significantly higher levels of peptide-capped AuNPs were found in the liver (74-86%) accompanied by a corresponding decrease in blood levels. TEM analysis of liver slices showed AuNPs in Kupffer cells and hepatocytes, trapped inside endosomes. Our data demonstrate that AuNPs are rapidly distributed and that the liver is the preferential accumulation organ. Peptide capping significantly increased hepatic uptake, showing the influence of AuNPs functionalization in biodistribution.

Impaired synthesis or cellular storage of norepinephrine, dopamine, and 5-hydroxytryptamine in human inflammatory bowel disease.

The present study was aimed at evaluating the extent of dysfunction of the enteroendocrine and enteric nervous system, as indicated by changes in tissue levels of monoamines (dopamine, DA; norepinephrine, NE; 5-hydroxytryptamine, 5-HT) and their precursors and metabolites in the colonic mucosa of patients afflicted with ulcerative colitis (UC, N = 21) and Crohns disease (CD, N = 22). In CD, but not in UC, NE tissue levels in both the noninflamed and inflamed colonic mucosa were markedly lower than in control subjects (N = 16). In the inflamed mucosa of CD and in UC patients levels of l-DOPA were twice those in controls. DA levels in the inflamed mucosa of CD and UC patients were markedly lower than in controls. This resulted in significant reductions in DA/l-DOPA tissue ratios, a rough measure of l-amino acid decarboxylase activity. 5-HT levels in the inflamed mucosa of CD and UC patients were markedly lower than in controls. In conclusion, intestinal cellular structures responsible for the synthesis and storage of DA, NE, and 5-HT may have been affected by the associated inflammatory process in both CD and UC.

Influence of the surface coating on the cytotoxicity, genotoxicity and uptake of gold nanoparticles in human HepG2 cells

The toxicological profile of gold nanoparticles (AuNPs) remains controversial. Significant efforts to develop surface coatings to improve biocompatibility have been carried out. In vivo biodistribution studies have shown that the liver is a target for AuNPs accumulation. Therefore, we investigated the effects induced by ~20 nm spherical AuNPs (0–200 μM Au) with two surface coatings, citrate (Cit) compared with 11‐mercaptoundecanoic acid (11‐MUA), in human liver HepG2 cells. Cytotoxicity was evaluated using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release assays after 24 to 72 h of incubation. DNA damage was assessed by the comet assay, 24 h after incubation with the capped AuNPs. Uptake and subcellular distribution of the tested AuNPs was evaluated by quantifying the gold intracellular content by graphite furnace atomic absorption spectrometry (GFAAS) and transmission electron microscopy (TEM), respectively. The obtained results indicate that both differently coated AuNPs did not induce significant cytotoxicity. An inverse concentration‐dependent increase in comet tail intensity and tail moment was observed in Cit‐AuNPs‐ but not in MUA‐AuNPs‐exposed cells. Both AuNPs were internalized in a concentration‐dependent manner. However, no differences were found in the extent of the internalization between the two types of NPs. Electron‐dense deposits of agglomerates of Cit‐ and MUA‐AuNPs were observed either inside endosomes or in the intercellular spaces. In spite of the absence of cytotoxicity, DNA damage was observed after exposure to the lower concentrations of Cit‐ but not to MUA‐AuNPs. Thus, our data supports the importance of the surface properties to increase the biocompatibility and safety of AuNPs. Copyright

Short- and long-term distribution and toxicity of gold nanoparticles in the rat after a single-dose intravenous administration

UNLABELLED Surface chemistry plays an important role in gold nanoparticles (AuNPs) stability and biocompatibility, which are crucial for their implementation into the clinical setting. We evaluated short- (30 min) and long-term (28 days) biodistribution and toxicity of ~20 nm citrate- and pentapeptide CALNN-coated AuNPs after a single intravenous injection in rats. The pattern of AuNPs distribution in Cit- and CALNN-AuNPs-injected rats was very similar in the assessed time-points. Both AuNPs were quickly removed from the bloodstream and preferentially accumulated in the liver. At 28 days liver remained the main accumulation site but at significantly lower levels compared to those found at 30 min. Spleen atrophy and hematological findings compatible with mild anemia were observed in CALNN-AuNPs-administered rats. Under our experimental conditions, surface coating had more impact on toxicity rather than on biodistribution of the AuNPs. Improvements in the design of capping peptides need to be done to increase biomedical applicability of peptide-coated AuNPs. FROM THE CLINICAL EDITOR The biodistribution and toxicity of ~ 20 nm citrate- and pentapeptide CALNN-coated gold nanoparticles was investigated after a single intravenous injection in rats. Rapid clearance and hepatic accumulation was found at 30-minutes, whereas mild anemia and spleen atrophy was seen 28 days post injection. The authors also concluded that the toxicity was related to the capping proteins as opposed to the biodistribution of the particles, providing important suggestion for future design of gold nanoparticles.

Are iron oxide nanoparticles safe? Current knowledge and future perspectives.

Due to their unique physicochemical properties, including superparamagnetism, iron oxide nanoparticles (ION) have a number of interesting applications, especially in the biomedical field, that make them one of the most fascinating nanomaterials. They are used as contrast agents for magnetic resonance imaging, in targeted drug delivery, and for induced hyperthermia cancer treatments. Together with these valuable uses, concerns regarding the onset of unexpected adverse health effects following exposure have been also raised. Nevertheless, despite the numerous ION purposes being explored, currently available information on their potential toxicity is still scarce and controversial data have been reported. Although ION have traditionally been considered as biocompatible - mainly on the basis of viability tests results - influence of nanoparticle surface coating, size, or dose, and of other experimental factors such as treatment time or cell type, has been demonstrated to be important for ION in vitro toxicity manifestation. In vivo studies have shown distribution of ION to different tissues and organs, including brain after passing the blood-brain barrier; nevertheless results from acute toxicity, genotoxicity, immunotoxicity, neurotoxicity and reproductive toxicity investigations in different animal models do not provide a clear overview on ION safety yet, and epidemiological studies are almost inexistent. Much work has still to be done to fully understand how these nanomaterials interact with cellular systems and what, if any, potential adverse health consequences can derive from ION exposure.

Expression of LAT1 and LAT2 amino acid transporters in human and rat intestinal epithelial cells

Summary.The present study evaluated the presence of LAT1 and LAT2 amino acid transporters in human Caco-2 cells and rat IEC-6 cells along the mucosa of the rat digestive tract. The LAT1 cDNA was amplified by PCR using two sets of primers (one specific for rat LAT1 and another simultaneously specific for human, rat and mice). The LAT2 cDNA was amplified by PCR using one set of primers simultaneously specific for human, rat and mice LAT2. The presence of LAT1 and LAT2 protein was examined by means of immunoblotting using an antibody raised against the rat LAT1 and mouse LAT2. Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 transporter transcripts and protein. LAT1 protein is most abundant in IEC-6 cells, which is in agreement with functional data previously reported. The findings in the rat intestinal mucosa indicate that LAT1 protein is most abundant in the colon and its abundance markedly decreases at the level of jejunum and ileum, which contrast with relative homogeneous presence of LAT2 across the digestive tract. In conclusion, Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 amino acid transporter transcripts and protein.

The L-3,4-dihydroxyphenylalanine transporter in human and rat epithelial intestinal cells is a type 2 hetero amino acid exchanger.

Information on the intestinal transport of L-3,4-dihydroxyphenylalanine (L-DOPA) is scarce. We present here the functional characteristics and regulation of the apical inward L-DOPA transport in two intestinal epithelial cell lines (human Caco-2 and rat IEC-6). The inward transfer of L-DOPA and L-leucine was promoted through an energy-driven system but with different sensitivity to extracellular Na(+) concentration: a minor component of L-leucine uptake (approximately 25%) was found to require extracellular Na(+) in comparison with L-DOPA transport which was Na(+)-independent. L-DOPA and L-leucine uptake was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). L- and D-neutral amino acids, but not acidic and basic amino acids, markedly inhibited L-DOPA and [(14)C]L-leucine accumulation in both cell lines. The [(14)C]L-DOPA and [14C]L-leucine outward were markedly increased by L-leucine and BCH present in extracellular medium, but not by L-arginine. In both cell lines, L-DOPA transport was stimulated by acidic pH in comparison with [(14)C]L-leucine inward which was pH-independent. In conclusion, it is likely that system B(0) might be responsible for the Na(+)-dependent uptake of L-leucine in Caco-2 and IEC-6 cells, whereas sodium-independent uptake of L-leucine and L-DOPA may include system type 1 and type 2 L-amino acid transporter (LAT1 and LAT2), the activation of which results in trans-stimulation of substrates outward transfer.

Intestinal 5-hydroxytryptamine and mast cell infiltration in rat experimental colitis.

The present study evaluated the extent of dysfunction of the 5-hydroxytryptaminergic system in inflamed (distal colon) and noninflamed segments (jejunum and ileum) after intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in the rat. Dexamethasone was administered to control rats and TNBS-treated animals before and for 7 days after TNBS treatment. Tissue levels of 5-hydroxytryptamine (5-HT) in the inflamed colon were higher than in controls. The densitmast cells was also markedly increased. Treatment with dexamethasone attenuated the inflammatory response but did not prevent the increase in colonic 5-HT. In the noninflamed jejunum and terminal ileum, 5-HT tissue levels were markedly increased, but treatment with dexamethasone prevented this increase. It is concluded that the increase in 5-HT levels in the inflamed colon in TNBS-induced colitis may result fmast cell infiltration. In the noninflamed intestine, 5-HT tissue levels were also increased, favoring the view of a generalized mast cell infiltration.

Transport of the organic cations gonyautoxin 2/3 epimers, a paralytic shellfish poison toxin, through the human and rat intestinal epitheliums.

The aim of this work is to study the mechanisms involved in gonyautoxins (GTXs) intestinal absorption. For this purpose, we studied the transport of GTX 2/3 epimers by intestinal epithelial cell lines (IEC-6 and Caco-2) cultured on polycarbonate filters. Specific transport was calculated by subtracting from the flux of GTX 2/3 measured at 37 degrees C that occurring at 4 degrees C, this being an indication of transcellular transport. The transcellular apical-to-basolateral (A-B) flux in Caco-2 cell monolayers, was greater than that in the opposite direction, suggesting the involvement of an active transport system favoring the absorption of the toxin. However, in IEC-6 cells the transcellular basolateral-to-apical (B-A) specific transport of the toxin was greater than that in the opposite direction. The A-B and B-A fluxes were, respectively, 127 +/- 26 and 205 +/- 23 nmol/min, suggesting the presence of a prevalent secretive process of the toxin in IEC-6 cells. The A-B transport of GTX 2/3 epimers in Caco-2 cells, but not in IEC-6 cells, was partially Na(+)-dependent and significantly inhibited by adenosine. TEA and verapamil in both Caco-2 and IEC-6 cells failed to affect the A-B and B-A transport of GTX 2/3 epimers. Cyanine in IEC-6 cells, but not in Caco-2 cells, increased the A-B flux of the toxin, suggesting the involvement of the organic cation transporter in the absorption of GTX 2/3 epimers. The mitochondrial energetic uncoupler 2,4-dinitrophenol significantly inhibited the A-B and the B-A transport in both cell lines. In conclusion, IEC-6 cells secrete actively the toxins, whereas Caco-2 cells were found to absorb the toxins in a process that was inhibited in the presence of adenosine and the absorption was dependent of Na(+).

Role of H2O2 in hypertension, renin‐angiotensin system activation and renal medullary disfunction caused by angiotensin II

BACKGROUND AND PURPOSE Activation of the intrarenal renin‐angiotensin system (RAS) and increased renal medullary hydrogen peroxide (H2O2) contribute to hypertension. We examined whether H2O2 mediated hypertension and intrarenal RAS activation induced by angiotensin II (Ang II).