Denise Soledade Jornada

Incorporation in polymeric nanocapsules improves the antioxidant effect of melatonin against lipid peroxidation in mice brain and liver.

It has been recently shown that the association of melatonin with polymeric nanoparticles causes a significant increase of the in vitro effect against lipid peroxidation. Hence, the aim of the present study was to compare the in vivo acute antioxidant effect of intraperitoneal administration of melatonin-loaded polysorbate 80-coated nanocapsules with that of melatonin aqueous solution in mice brain (frontal cortex and hippocampus) and liver. The lipid peroxidation through thiobarbituric acid reactive substance levels, the total antioxidant reactivity (luminol-enhanced chemiluminescence) and the free radical levels (formed dichlorofluorescein) has been carried out. Our results show that a single melatonin aqueous solution injection exerted no antioxidant activity in the evaluated range, while the administration of the melatonin-loaded polysorbate 80-coated nanocapsules caused a marked reduction on lipid peroxidation levels in all studied tissues. No differences on free radical content were found in the tissues. The melatonin-loaded nanocapsules also increased the total antioxidant reactivity in the hippocampus. These in vivo results are in accordance with our previous in vitro findings and confirm the hypothesis that polymeric nanocapsules improve the antioxidant effect of melatonin against lipid peroxidation.

Lipid-core nanocapsules: mechanism of self-assembly, control of size and loading capacity

Nanotechnology in pharmaceutics has the potential to improve drug efficacy by influencing drug distribution in tissues. Nanocarriers have been developed as drug delivery systems to be administered by different biological routes. To ensure the nanotechnological properties, pre-formulation studies are especially critical in determining the influence of the process parameters on the size and polydispersity of particles. Thus, the objective of this work was to establish the mechanism of self-assembly, by determining the influence of the critical aggregation concentration of the materials in the organic phase on the final average particle size and polydispersity of polymeric lipid-core nanocapsules obtained by interfacial deposition of polymer. Measurements of the surface tension and viscosity of the organic and aqueous phases were correlated with the particle size and the concentration of raw materials. We demonstrated that the lipid-core nanocapsules are formed on the nanoscopic scale as unimodal distributions, if the aggregation state of raw materials in the organic phase tends to infinite dilution. The strategy for controlling the particle size distribution is a valuable tool in producing lipid-core nanocapsule formulations with different loading capacities intended for therapeutics.

Caenorhabditis elegans as an alternative in vivo model to determine oral uptake, nanotoxicity, and efficacy of melatonin-loaded lipid-core nanocapsules on paraquat damage

Caenorhabditis elegans is an alternative in vivo model that is being successfully used to assess the pharmacological and toxic effects of drugs. The exponential growth of nanotechnology requires the use of alternative in vivo models to assess the toxic effects of theses nanomaterials. The use of polymeric nanocapsules has shown promising results for drug delivery. Moreover, these formulations have not been used in cases of intoxication, such as in treatment of paraquat (PQ) poisoning. Thus, the use of drugs with properties improved by nanotechnology is a promising approach to overcome the toxic effects of PQ. This research aimed to evaluate the absorption of rhodamine B-labeled melatonin (Mel)-loaded lipid-core nanocapsules (LNC) by C. elegans, the application of this model in nanotoxicology, and the protection of Mel-LNC against PQ damage. The formulations were prepared by self-assembly and characterized by particle sizing, zeta potential, drug content, and encapsulation efficiency. The results demonstrated that the formulations had narrow size distributions. Rhodamine B-labeled Mel-LNC were orally absorbed and distributed in the worms. The toxicity assessment of LNC showed a lethal dose 50% near the highest dose tested, indicating low toxicity of the nanocapsules. Moreover, pretreatment with Mel-LNC significantly increased the survival rate, reduced the reactive oxygen species, and maintained the development in C. elegans exposed to PQ compared to those worms that were either untreated or pretreated with free Mel. These results demonstrated for the first time the uptake and distribution of Mel-LNC by a nematode, and indicate that while LNC is not toxic, Mel-LNC prevents the effects of PQ poisoning. Thus, C. elegans may be an interesting alternative model to test the nanocapsules toxicity and efficacy.

Vitamin K1-loaded lipid-core nanocapsules: physicochemical characterization and in vitro skin permeation.

The incorporation of substances in nanocarriers can modulate and/or manage their delivery profiles (immediate or sustained) and permeation through skin. Consequently, drug nanencapsulation intended for topical treatment can reduce the systemic absorption of the substance.

Melatonin delivery by nanocapsules during in vitro bovine oocyte maturation decreased the reactive oxygen species of oocytes and embryos

In this work, a promising approach to increase the advantageous properties of melatonin through its encapsulation into lipid-core nanocapsules (LNC) was examined. Oocytes were treated during in vitro maturation with non-encapsulated melatonin (Mel), melatonin-loaded lipid-core nanocapsules (Mel-LNC), and unloaded LNC. Cytotoxicity, meiotic maturation rate, development to the blastocyst stage, reactive oxygen species (ROS) and glutathione levels, mean cell number and apoptotic cell/blastocyst, and mRNA quantification were evaluated. Both Mel and Mel-LNC enhanced in vitro embryo production, however, Mel-LNC proved to be more effective at decreasing ROS levels and the apoptotic cell number/blastocyst, increasing the cleavage and blastocyst rates, up-regulating the GPX1 and SOD2 genes, and down-regulating the CASP3 and BAX genes. Mel-LNC could penetrate into oocytes and remain inside the cells until they reach the blastocyst stage. In conclusion, when melatonin was encapsulated in LNC and applied during in vitro oocyte maturation, some quality aspects of the blastocysts were improved.

Pantoprazole-loaded Eudragit blended microparticles: preparation, characterization, in vitro gastro-resistance and in vivo anti-ulcer evaluation

Pantoprazole is a proton-pump inhibitor pro-drug used in the treatment of gastric ulcers and gastro-esophageal reflux disease. Pantoprazole must be absorbed intact in the gastrointestinal tract, because it is labile in the stomach environment, indicating that enteric delivery systems are required. The purpose of this study was to prepare pantoprazole-loaded microparticles by spray-drying using Eudragit S100 (M1), Eudragit RS30D (M3), or a Eudragit S100/Eudragit RS30D blend (M2). All microparticles were successfully obtained. The in vitro dissolution evaluation showed slower release from Eudragit S100 microparticles and Eudragit S100/Eudragit RS30D blended microparticles than the pure drug. The in vitro gastro-resistance assay showed that M1 and M2 microparticles were effective in protecting the drug in acid medium. After oral administration, in vivo anti-ulcer evaluation showed that the microparticles were able to protect the rat stomachs against ulcer formation by ethanol, while the drug aqueous solution did not present any activity.

Effects of chitosan-coated lipid-core nanocapsules on bovine sperm cells

Toxicology studies have a pivotal role for selection of new nanosystems. As lipid-core nanocapsules (LNC) rise as a potential system not only for drug delivery but also for immunotherapy and gene therapy, the demand for models of toxic screening increases, and sperm arises as a promising model due to the easiness to evaluate its viability parameters. LNCs were coated with chitosan, chitosan-coated lipid-core nanocapsules (LNC-CS), in order to modify the nanocapsule surface. We evaluated the toxicity of LNC and LNC-CS after incubation with bovine sperm in different concentrations (2.5%, 5%, 10%, 20%, 40% and 80%) (v/v) and periods of exposure (0h and 1h). CASA parameters and flow cytometry assays were performed to assess LNC and LNC-CS effects. The results corroborated with previous studies indicating that there is no toxicity from LNCs and LNC-CS below 40% (v/v) concentration.

Antimicrobial effect and physicochemical properties of an adhesive system containing nanocapsules

OBJECTIVE To incorporate indomethacin and triclosan-loaded nanocapsules into primer and adhesive, and evaluate its properties. METHODS Indomethacin and triclosan were encapsulated by deposition of preformed polymer and subsequently characterized regarding morphology, particle size, drug content and cytotoxicity. Nanocapsules (NCs) were incorporated into primer at 2% and into adhesive at 1, 2, 5, and 10% concentrations. Degree of conversion (DC) and softening in ethanol of the adhesive were evaluated. Drug release and drug diffusion through dentin was quantified by high performance liquid chromatography. Antimicrobial test was performed until 96h. RESULTS Spherical and biocompatible NCs presented mean size of 159nm. Drugs content was 3mg indomethacin/g powder and 2mg triclosan/g powder. Incorporating NCs in adhesive showed no influence in DC (p=0.335). The addition of 2% of NCs showed no influence in softening in ethanol (p>0.05). After 120h, 93% of indomethacin and 80% of triclosan were released from primer, 20% of indomethacin and 17% of triclosan were released from adhesive with 10% of NCs. Indomethacin showed diffusion through dentin. In 24h, adhesive containing 2 and 5% of NCs using primer with NCs showed antimicrobial effect. In 96h, adhesives containing different concentration of NCs promoted antimicrobial effect. CONCLUSIONS Indomethacin and triclosan-loaded nanocapsules were successfully incorporated into primer and adhesive, promoting controlled drugs release, indomethacin diffusion through dentin and antimicrobial effect without compromising its physicochemical properties. SIGNIFICANCE Indomethacin and triclosan-loaded nanocapsules have potential to prevent recurrent caries and to be used in deep cavities controlling pulpar inflammatory process.

Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model

Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10−6, 10−9, and 10−12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10−9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10−9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.

In vivo gastroprotective effect of nanoparticles: influence of chemical composition and volume fraction.

In nanomedicine, different nanomaterials and nanoparticles have been proposed as therapeutic agents or adjuvants, as well as diagnosis devices. Considering that the principal cause of the ulcerations is the imbalance among the gastric juice secretion and the protection provided by the mucosal barrier and the neutralization of the gastric acid, as well as that nanoparticles are able to accumulate in the gastro-intestinal tissues, we proposed a 2(2) factorial design to evaluate the influence of the chemical composition and the volume fraction of the dispersed phase on the gastric protective effect against ulceration induced by ethanol. Cocoa-theospheres (CT) and lipid-core nanocapsules (LNC) (two different kinds of surfaces: lipid and polymeric, respectively) prepared at two different concentrations of soft materials: 4% and 12% (w/v) were produced by high pressure homogenization and solvent displacement methods, respectively. Laser diffraction showed volume-weighted mean diameters ranging from 133 to 207 nm, number median diameters lower than 100 nm and specific surfaces between 41.2 and 51.2 m(2) g(-1). The formulations had pH ranging from 4.7 to 6.3; and zeta potential close to -9 mV due to their coating with polysorbate 80. The ulcer indexes were 0.40 (LNC(4)) and 0.48 (CT(4)) for the lower total administered areas (3.3 and 4.1 m(2)g(-1), respectively), and 0.09 (LNC(12) and CT(12)) for the higher administered areas (10.0 and 12.0 m(2) g(-1), respectively). LNC(4), LNC(12) and CT(12) showed lower levels in the lipid peroxidation assay when compared either to the negative control (saline) or to CT(4). LNC(12) and CT(12) showed similar TBARS levels, as well as CT(4) was similar to the negative control. SEM analysis of the stomach mucosa showed coatings more homogenous and cohesive when LNC formulations were administered compared to the correspondent CT formulations. The higher total area of administered nanoparticles showed film formation. Moreover, LNC(12) provided a more thick and cohesive film, completely covering the mucosal surface. In conclusion, both kinds of formulations are able to prevent ulceration induced by ethanol in rats. The 2(2) factorial design showed that the chemical composition had a strong influence when the lower areas of nanoparticles are administered, while when the higher areas are used this is the more influencing parameter on the gastroprotection.