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

Eliza Rossi Komninou,Ruy Carlos Ruver Beck,Fabiana Kömmling Seixas,William Borges Domingues,Andrea Cristina Basso,Adriana Raffin Pohlmann,Mariana Härter Remião,Tiago Collares,Denise Soledade Jornada,Vinicius Farias Campos,João Carlos Deschamps,Caroline Gomes Lucas,Silvia Stanisçuaski Guterres,Vilceu Bordignon

doi:10.1371/journal.pone.0157561

Abstract

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.

Highlights

In vitro culture conditions including the energy source, growth factors, pH, atmospheric oxygen concentration or transient light exposure, are associated with detrimental factors to the embryonic development, caused by enhanced levels of reactive oxygen species (ROS) [1]
Considering that i) melatonin loaded-Eudragit1 S100 nanocapsules (Mel-NC) can improve the antioxidant effects of melatonin, when compared to the non-encapsulated melatonin, that ii) lipid-core nanocapsules (LNC) are promising intracellular carriers to melatonin, and that iii) no previous studies have been conducted to evaluate the effects of melatonin associated with nanoparticulated systems on cultured embryos, in this study, we examined the effects of melatonin associated with NC or LNC on in vitro development of bovine embryos
Our result demonstrates that both melatonin-loaded in polymeric (Mel-NC) as melatonin-loaded lipid-core nanocapsules (Mel-LNC) did not induce aberrant expression of pluripotency genes, OCT4, SOX2, and NANOG, maintaining the pluripotency potential in bovine embryonic cells

Summary

Introduction

In vitro culture conditions including the energy source, growth factors, pH, atmospheric oxygen concentration or transient light exposure, are associated with detrimental factors to the embryonic development, caused by enhanced levels of reactive oxygen species (ROS) [1]. These molecules are highly reactive and can oxidize DNA, proteins, and lipids resulting in mitochondrial alterations, ATP depletion, embryonic developmental arrest, low blastocyst production and defective embryo development [1, 2]. The direct scavenger activity against toxic oxygen derivatives, and the ability to stimulate detoxifying enzymes like superoxide dismutase and glutathione peroxidase, have been described as the main melatonin mechanisms to intercept and prevent ROS production in embryos [16, 17]

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