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

<div><p>Melatonin has been used as a supplement in culture medium to improve the efficiency of <i>in vitro</i> 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 <i>in vitro</i> cultured embryos are exposed during early development. <i>In vivo</i> and <i>in vitro</i> 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 <i>in vitro</i> 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 <i>BAX</i>, <i>CASP3</i>, and <i>SHC1</i> genes, and up-regulated mRNA levels of <i>CAT</i> and <i>SOD2</i> genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during <i>in vitro</i> embryo culture in bovine species.</p></div

Effects of free and nanoencapsulated melatonin (10⁻⁹ M) on the relative mRNA abundance of pluripotency-related genes in bovine blastocysts.

<p>Data represent mean ± S.E.M.</p

ROS levels in 4–8 cell stage embryos cultured in the presence of free and nanoencapsulated melatonin.

<p>Embryos were cultured in the presence of 10⁻⁹ M melatonin non-encapsulated (Mel) or encapsulated in nanocapsules (Mel-NC) or lipid-core nanocapsules (Mel-LNC). In the control group embryos were cultured in SOFaa BSA medium alone. Data represent mean ± S.E.M. Different letters above the error bars indicate significant differences between groups (P< 0.05). Representative fluorescent (B, C, D, E) and corresponding bright field (b, c, d, e) images of control, Mel, Mel-NC, and Mel-LNC embryos, respectively. Scale bar = 50 μm. Magnification = 100X.</p

Hatching rates of bovine embryos cultured in the presence of free or nanoencapsulated melatonin.

<p>Mel = Non-encapsulated melatonin, Mel-NC = melatonin-loaded polymeric nanocapsules, Mel-LNC = melatonin-loaded lipid-core nanocapsules, NC = drug-unloaded nanocapsules, LNC = drug-unloaded lipid-core nanocapsules. Melatonin was used at 10⁻¹² M, 10⁻⁹ M or 10⁻⁶ M concentrations. Control group was cultured in SOFaa BSA alone. The numerators represent the numbers of hatched blastocysts and the denominators represent total blastocysts in each group. Different letters (a-c) above the bars indicate significant differences between groups (<i>P</i> < 0.05).</p

Effects of free and nanoencapsulated melatonin (10⁻⁹ M) on the relative mRNA abundance of oxidative stress-related genes.

<p>Data represent mean ± S.E.M. Different letters (a, b and c) indicate significant differences between groups (P< 0.05).</p