RESUMO
Phototherapies are promising for noninvasive treatment of aggressive tumors, especially when combining heat induction and oxidative processes. Herein, we show enhanced phototoxicity of gold shell-isolated nanorods conjugated with toluidine blue-O (AuSHINRs@TBO) against human colorectal tumor cells (Caco-2) with synergic effects of photothermal (PTT) and photodynamic therapies (PDT). Mitochondrial metabolic activity tests (MTT) performed on Caco-2 cell cultures indicated a photothermal effect from AuSHINRs owing to enhanced light absorption from the localized surface plasmon resonance (LSPR). The phototoxicity against Caco-2 cells was further increased with AuSHINRs@TBO where oxidative processes, such as hydroperoxidation, were also present, leading to a cell viability reduction from 85.5 to 39.0%. The molecular-level mechanisms responsible for these effects were investigated on bioinspired tumor membranes using Langmuir monolayers of Caco-2 lipid extract. Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) revealed that the AuSHINRs@TBO incorporation is due to attractive electrostatic interactions with negatively charged groups of the Caco-2 lipid extract, resulting in the expansion of surface pressure isotherms. Upon irradiation, Caco-2 lipid extract monolayers containing AuSHINRs@TBO (1:1 v/v) exhibited ca. 1.0% increase in surface area. This is attributed to the generation of reactive oxygen species (ROS) and their interaction with Caco-2 lipid extract monolayers, leading to hydroperoxide formation. The oxidative effects are facilitated by AuSHINRs@TBO penetration into the polar groups of the extract, allowing oxidative reactions with carbon chain unsaturations. These mechanisms are consistent with findings from confocal fluorescence microscopy, where the Caco-2 plasma membrane was the primary site of the cell death induction process.
RESUMO
Giant unilamellar vesicles (GUVs) are composed of lipophilic layers and are sensitive to the action of reactive oxygen species (ROS). The use of GUVs as microcarriers of biological macromolecules is particularly interesting since ROS produced by gametes or embryos during in vitro culture can induce the opening of pores in the membrane of these vesicles and cause the release of their content. This study investigated the behavior of GUVs [composed of 2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)] in co-culture with in vitro produced bovine embryos, as well as their embryotoxicity and effectiveness as cysteine carriers in culture medium. Embryonic developmental rates were unaffected, demonstrating the absence of toxicity of GUVs co-cultured with the embryos. No increase of intracellular ROS levels was observed in the embryos co-cultured with GUVs, indicating that the higher lipid content of the culture environment resulting from the lipid composition of the GUV membrane itself did not increase oxidative stress. Variations in the diameter and number of GUVs demonstrated their sensitivity to ROS produced by embryos cultured under conditions that generate oxidative stress. Encapsulation of cysteine in GUVs was found to be more effective in controlling the production of ROS in embryonic cells than direct dilution of this antioxidant in the medium. In conclusion, the use of GUVs in in vitro culture was found to be safe since these vesicles did not promote toxic effects nor did they increase intracellular ROS concentrations in the embryos. GUVs were sensitive to oxidative stress, which resulted in structural changes in response to the action of ROS. The possible slow release of cysteine into the culture medium by GUV rupture would therefore favor the gradual supply of cysteine, prolonging its presence in the medium. Thus, the main implication of the use of GUVs as cysteine microcarriers is the greater effectiveness in preventing the intracytoplasmic increase of ROS in in vitro produced bovine embryos.