Nanotechnology in reproduction: Vitamin E nanoemulsions for reducing oxidative stress in sperm cells.

Vitamin E is considered a powerful biological antioxidant; however, its characteristics such as high hydrophobicity and low stability limit its application. We propose to use nanotechnology as an innovative tool in spermatology, formulating nanoemulsions (NE) that accommodate vitamin E, protecting it from oxidation and promoting its release into the medium. The protective effect of the NE against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from eleven stags were thawed and extended to 400 × 106 sperm/ml in Bovine Gamete Medium (BGM). Once aliquoted, the samples were supplemented with the NE at different concentrations (0, 6 and 12 mM), with or without induced oxidative stress (100 µM Fe2+/ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. Motility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) were assessed. After 2 and 4 h of incubation, the NE were able to prevent the deleterious effects of oxidative stress, thus improving total and progression motility (P ˂0.05). Moreover, the highest concentration tested (12 mM) improved almost every sperm kinematic variable (P ˂0.05) and preserved sperm viability in samples subjected to oxidative stress. In addition, 12 mM of NE protected the acrosomes integrity, maintained and protected mitochondrial activity, prevented sperm lipoperoxidation and reduced ROS production (P ˂0.05) in samples subjected to oxidative stress. This work indicates for the first time that vitamin E formulated in NE could be a new approach against sperm oxidative damage. This could be highly relevant for sperm physiology preservation in the context of assisted reproduction techniques.

Ascorbyl-dipalmitate-stabilised nanoemulsions as a potential localised treatment of inflammatory bowel diseases.

Current efforts on inflammatory bowel diseases (IBD) treatment are focused on strategies for localised drug delivery at the intestinal mucosa. Despite the potential of curcumin (CC) for IBD treatment, its low solubility and stability limit its application. Thus, the design of nanocarriers that focus CC delivery at the intestinal epithelium is an area of interest. This work proposes α-tocopherol nanoemulsions (NE) stabilised by ascorbyl-2,6-dipalmitate (ADP) as intestinal CC-carriers. The antioxidant capacity of α-tocopherol and ADP could have a synergistic effect on IBD-affected tissues, characterised by an oxidative environment. We obtained nanoemulsions (NE-ADP) with size below 200 nm, negative surface charge, stable in gastrointestinal media and no toxic in the Caco-2 cell model. Intracellular retention of NE-ADP in Caco-2 cells was observed by confocal microscopy. The extremely low Papp values obtained for CC and α-tocopherol indicated the lack of transport across the Caco-2 monolayer. Control nanoemulsion stabilised by lecithin (NE-L) was greatly transported across the Caco-2 cells monolayer, confirming the relevance of ADP on the cellular retention of NE-ADP. The therapeutic potential of NE-ADP was shown by the significant decrease of intracellular ROS levels. Altogether, these results indicate the potential of NE-ADP as a novel approach for the treatment of IBD.

The role of the intestinal-protein corona on the mucodiffusion behaviour of new nanoemulsions stabilised by ascorbyl derivatives.

Nanoemulsions are vesicular systems with great potential for the delivery of drugs, which significantly depends on the appropriate selection of the components that constitute them. In this sense, the use of materials with adequate toxicity profiles for the oral route provides additional advantages in terms of safety concerns avoidance. This work describes the formulation of novel two-component nanoemulsions constituted by α-tocopherol and ascorbyl-palmitate derivatives. Among them, ascorbyl-dipalmitate allowed the formation of nanoemulsions with size values around 170 nm and negative charge; additionally, they showed strong antioxidant capacity. These nanoemulsions are proposed to the oral route, so their behaviour in intestinal conditions was evaluated by incubating the nanoemulsion in simulated intestinal fluid. This process led to the formation of an intestinal-protein corona (I-PC) at the colloidal surface that determined the interaction with the mucus barrier. The I-PC displaced the immobile-hindered particles towards a subdiffusive-diffusive population. These studies report for the first time the effect of the I-PC on the mucodiffusion behaviour of vesicular systems, a finding that may help to comprehend the performance of nanocarriers under intestinal conditions.

Vitamin transporters in mice brain with aging.

Its high metabolic rate and high polyunsaturated fatty acid content make the brain very sensitive to oxidative damage. In the brain, neuronal metabolism occurs at a very high rate and generates considerable amounts of reactive oxygen species and free radicals, which accumulate inside neurons, leading to altered cellular homeostasis and integrity and eventually irreversible damage and cell death. A misbalance in redox metabolism and the subsequent neurodegeneration increase throughout the course of normal aging, leading to several age-related changes in learning and memory as well as motor functions. The neuroprotective function of antioxidants is crucial to maintain good brain homeostasis and adequate neuronal functions. Vitamins E and C are two important antioxidants that are taken up by brain cells via the specific carriers αTTP and SVCT2, respectively. The aim of this study was to use immunohistochemistry to determine the distribution pattern of these vitamin transporters in the brain in a mouse model that shows fewer signs of brain aging and a higher resistance to oxidative damage. Both carriers were distributed widely throughout the entire brain in a pattern that remained similar in 4-, 12-, 18- and 24-month-old mice. In general, αTTP and SVCT2 were located in the same regions, but they seemed to have complementary distribution patterns. Double-labeled cell bodies were detected only in the inferior colliculus, entorhinal cortex, dorsal subiculum, and several cortical areas. In addition, the presence of αTTP and SVCT2 in neurons was analyzed using double immunohistochemistry for NeuN and the results showed that αTTP but not SVCT2 was present in Bergmann's glia. The presence of these transporters in brain regions implicated in learning, memory and motor control provides an anatomical basis that may explain the higher resistance of this animal model to brain oxidative stress, which is associated with better motor performance and learning abilities in old age.

PEGylated Nanoemulsions for Oral Delivery: Role of the Inner Core on the Final Fate of the Formulation.

Since the past decade, there has been growing interest to grant nanoparticles with diffusion properties across mucosae. In this sense, the nonionic block copolymer Pluronic F127 (PF127) has emerged as a promising coating agent to formulate mucus-penetrating particles. In the journey to find efficient coating agents, researchers have focused more on the effect of the coating agent architecture rather than on the role of the physicochemical properties of the nanoparticle used as the substrate. The current knowledge about mucodiffusive particles is in general based on model-like nanoparticles, such as polystyrene or poly(lactic-co-glycolic) acid nanoparticles, but there is a lack of information about the potential of PF127 on other colloidal systems. This work aims to shed some light on this issue by selecting three oils, palm (solid), coconut (semisolid), and wheat germ (liquid), with different physicochemical properties to formulate PF127-coated nanoemulsions. The obtained nanoemulsions were characterized, and their colloidal stability was tested. Their diffusion capacity was determined by particle tracking after challenging the nanoemulsions across an intestinal porcine mucus layer. In accordance with the evidence of model-like nanoparticles, our results state that PF127 allows mucodiffusion, but its effectiveness as a coating agent clearly depends on the physicochemical properties of the nanostructure core over which PF127 is placed. Among other physicochemical properties, the results certainly showed that the hydrophobic character of the nanostructure core emerges as a critical factor in the formulation of successful PF127 coatings.

Chitosan-coated mesoporous MIL-100(Fe) nanoparticles as improved bio-compatible oral nanocarriers.

Nanometric biocompatible Metal-Organic Frameworks (nanoMOFs) are promising candidates for drug delivery. Up to now, most studies have targeted the intravenous route, related to pain and severe complications; whereas nanoMOFs for oral administration, a commonly used non-invasive and simpler route, remains however unexplored. We propose here the biofriendly preparation of a suitable oral nanocarrier based on the benchmarked biocompatible mesoporous iron(III) trimesate nanoparticles coated with the bioadhesive polysaccharide chitosan (CS). This method does not hamper the textural/structural properties and the sorption/release abilities of the nanoMOFs upon surface engineering. The interaction between the CS and the nanoparticles has been characterized through a combination of high resolution soft X-ray absorption and computing simulation, while the positive impact of the coating on the colloidal and chemical stability under oral simulated conditions is here demonstrated. Finally, the intestinal barrier bypass capability and biocompatibility of CS-coated nanoMOF have been assessed in vitro, leading to an increased intestinal permeability with respect to the non-coated material, maintaining an optimal biocompatibility. In conclusion, the preservation of the interesting physicochemical features of the CS-coated nanoMOF and their adapted colloidal stability and progressive biodegradation, together with their improved intestinal barrier bypass, make these nanoparticles a promising oral nanocarrier.

Design of the interface of edible nanoemulsions to modulate the bioaccessibility of neuroprotective antioxidants.

Most frequently the use of bioactive molecules for the supplementation of food and beverages is hampered by stability limitations or inadequate intestinal absorption. This work evaluates in vitro the role that the interface of the nanoemulsion has on the physicochemical properties, the stability behavior and the enzymatic degradation after oral intake. For that purpose three soybean oil (SB) formulations were studied. These formulations were based on the emulsifier lecithin but modified with two non-ionic surfactants Pluronic(®) F68 (PF68) or Pluronic(®) F127 (PF127) yielding (i) SB-NE (only lecithin on the interface), (ii) SB-NE PF68 (lecithin plus PF68) and 9 (iii) SB-NE PF127 (lecithin plus PF127). All the formulations tested were low polydispersed and showed a size of about 200 nm and ζ-potential of -50 mV. The in vitro colloidal stability assay showed that lecithin itself was able to promote that formulations reach unaltered to the small intestine and facilitate the absorption of the antioxidant payload on a tunable fashion there (with in vitro bioaccessibility values from around 40% up to a 70%). PF68 was able to sterically stabilize the formulation against the aggregation induced by the pH and electrolytes of the simulated gastrointestinal track; however, this surfactant was easily displaced by the lipases of the simulated intestinal milieu being unable to modulate the digestion pattern of the oil droplets in the small intestine. Finally, PF127 displayed a strong steric potential that dramatically reduced the interaction of the oil droplets with lipases in vitro, which will compromise the capacity of the formulation to improve the bioaccessibility of the loaded antioxidant.

Optimisation of synthetic vector systems for cancer gene therapy - the role of the excess of cationic dendrimer under physiological conditions.

We have previously demonstrated in a therapeutic study that a single systemic course of DAB-Am16 dendriplexes loaded with plasmid expressing TNFα over a period of time of 10 days led to a regression of 100% of tumours and to long term cures of up to 80% of animals. However, the formulation had a relatively low colloidal stability requiring administration soon after nanoparticle preparation. Similar to other cationic polyplex and dendrimer DNA delivery systems, DAB-AM16 dendrimer formulations contained a substantial proportion of free polymer; this free polymer is present independently of the specific polymer:DNA ratio and increases with increasing proportion of polymer (N:P charge ratio) in the formulation. It has previously been shown for this and other systems that the excess of polymer plays a role in promoting the transfection efficiency of synthetic vectors. This has been linked to effects of the polymer on the efficiency of intracellular processing, e.g. endosomal release. However, the free polymer may have additional effects that are relevant to the efficiency of the formulation. This study therefore considered the effect of free dendrimer on the colloidal stability of the complexes, the interaction of the complex with the formulation medium, and with biological components, i.e. electrolytes and serum proteins after administration. Analysis of the total potential of interaction shows that, even at high N:P ratios, the excess of free dendrimer in the medium is not enough to induce the aggregation of the formulation due to depletion forces. This finding is unusual and can be attributed to the particularly low Mw of these dendrimers (1.6 kDa). On the other hand, formulations are highly sensitive to the strength of the dendrimer:DNA interactions. These can be controlled by the degree of protonation (α) of the dendrimer which is strongly dependent on bulk pH. Modulation of the protonation level to α≥0.4 allows reproducible production of colloidally stable particles. Finally, we have demonstrated that electrolytes and proteins present in physiological media play a crucial role to favour the efficiency of these synthetic vectors reducing the toxicity associated with their cationic groups.

Intracellular delivery of docetaxel using freeze-dried polysaccharide nanocapsules.

This article describes the development of a freeze-dried formulation of chitosan (CS) nanocapsules containing docetaxel (DCX) and the evaluation of its efficacy in the NCI-H460 cancer cell line. More specifically, two prototypes of nanocapsules differing in their coating, CS alone or in combination with poloxamer 188 were developed using the solvent displacement technique. These prototypes (150 nm and +45 mV) exhibited high encapsulation efficiencies of DCX (78%) and very similar release profiles. The nanocapsules made of solely CS could be freeze-dried and reconstituted without altering their particle size distribution. CS nanocapsules were tested for their ability to deliver intracellularly the anticancer drug DCX. The results showed that CS nanocapsules maintained the antiproliferative effect of the drug and that it was not affected by the freeze-drying process. Moreover, it was found that this cytostatic effect of DCX was related to its intracellular delivery in the cancer cells.

Effect of soil and a nonionic surfactant on BTE-oX and MTBE biodegradation kinetics.

The biodegradation kinetics of BTE-oX and MTBE, mixed all together, in the presence of 905 mg/L VSS of BTEX-acclimated biomass was evaluated. Effects of soil and Tergitol NP-10 in aqueous samples on substrate biodegradation rates were also evaluated. Biodegradation kinetics was evaluated for 36 hours, every 6 hours. MTBE biodegradation followed a first-order one-phase kinetic model in all samples, whereas benzene, toluene and ethylbenzene biodegradation followed a first-order two-phase kinetic model in all samples. O-xylene biodegradation followed a first-order two-phase kinetic model in the presence of biomass only. Interestingly, o-xylene biodegradation was able to switch to a first-order one-phase kinetic model when either soil or soil and Tergitol NP-10 were added. The presence of soil in aqueous samples retarded benzene, toluene and ethylbenzene removal rates. O-xylene and MTBE removal rates were enhanced by soil. The addition of Tergitol NP-10 to aqueous samples containing soil had a positive effect on substrate removal rate in all samples. Substrate percent removals ranged 77-99.8% for benzene, toluene and ethylbenzene. O-xylene and MTBE percent removals ranged 50.1-65.3% and 9.9-43.0%, respectively.

BTE-OX biodegradation kinetics with MTBE through bioaugmentation.

The biodegradation kinetics of BTE-oX and MTBE, mixed all together, in the presence of bioaugmented bacterial populations as high as 880 mg/L VSS was evaluated. The effect of soil in aqueous samples and the effect of Tergitol NP-10 on substrate biodegradation rates were also evaluated. Biodegradation kinetics was evaluated for 36 hours, every 6 hours. Benzene and o-xylene biodegradation followed a first-order one-phase kinetic model, whereas toluene and ethylbenzene biodegradation was well described by a first-order two-phase kinetic model in all samples. MTBE followed a zero-order removal kinetic model in all samples. The presence of soil in aqueous samples retarded BTE-oX removal rates, with the highest negative effect on o-xylene. The presence of soil enhanced MTBE removal rate. The addition of Tergitol NP-10 to aqueous samples containing soil had a positive effect on substrate removal rate in all samples. Substrate percent removals ranged from 95.4-99.7% for benzene, toluene and ethylbenzene. O-xylene and MTBE percent removals ranged from 55.9-90.1% and 15.6-30.1%, respectively.

[Long-term effects of cardiac pacing on natriuretic atrial peptide levels in patients with AV block]. / Efectos crónicos de la estimulación cardíaca sobre los niveles de hormona auricular natriurética en pacientes con bloqueo AV.

In this work we intend to analyze the long term effects of the classic modes of definitive cardiac pacing (VVI and DDD) on the atrial natriuretic peptide levels. A priori hypothesis is: the AV asynchrony raises the hormonal level. The design is a cross-sectional study, and the setting is the pacemaker doctor's room of a tertiary hospital. In 24 patients (17 VVI and 7 DDD) the atrial natriuretic peptide was determined by RIA. A multiple regression model was fitted to analysis. The basic result is: when several factors were controlled (age, sex, hypertension, and cardiac failure story), the atrial natriuretic peptide levels are increased in the VVI group. In conclusion, the lack of AV synchrony induced the increase of atrial natriuretic levels. For an endocrine viewpoint the DDD is more physiological pacing as well.