ABSTRACT
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.
Subject(s)
Chitosan/toxicity , Lipids/toxicity , Nanocapsules/toxicity , Spermatozoa/drug effects , Animals , Cattle , Chitosan/chemistry , DNA Damage , Lipid Peroxidation/drug effects , Lipids/chemistry , Male , Membrane Potential, Mitochondrial/drug effects , Nanocapsules/chemistry , Sperm Motility/drug effects , Spermatozoa/metabolism , Spermatozoa/physiologyABSTRACT
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.
Subject(s)
Antioxidants/administration & dosage , Melatonin/administration & dosage , Nanocapsules/administration & dosage , Oocytes/drug effects , Reactive Oxygen Species/metabolism , Animals , Apoptosis/drug effects , Caspase 3/genetics , Cattle , Cell Differentiation/drug effects , Embryo, Mammalian/drug effects , Embryonic Development/drug effects , Female , Fertilization in Vitro , Gene Expression Regulation, Developmental , Glutathione/metabolism , Glutathione Peroxidase/genetics , Oocytes/physiology , Superoxide Dismutase/genetics , bcl-2-Associated X Protein/genetics , Glutathione Peroxidase GPX1ABSTRACT
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.
Subject(s)
Antioxidants/pharmacology , Drug Carriers/pharmacology , Embryo, Mammalian/drug effects , Melatonin/pharmacology , Polyesters/chemistry , Polymethacrylic Acids/chemistry , Animals , Antioxidants/chemistry , Apoptosis/drug effects , Caspase 3/genetics , Caspase 3/metabolism , Catalase/genetics , Catalase/metabolism , Cattle , Culture Media/chemistry , Drug Carriers/chemistry , Drug Compounding , Embryo, Mammalian/physiology , Embryonic Development/drug effects , Female , Fertilization in Vitro , Gene Expression Regulation, Developmental , Male , Melatonin/chemistry , Nanocapsules/chemistry , Pregnancy , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Src Homology 2 Domain-Containing, Transforming Protein 1/genetics , Src Homology 2 Domain-Containing, Transforming Protein 1/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25µM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.