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Abstract

l-Buthionine sulfoximine (l-BSO) is an adjuvant drug that is reported to increase the sensitivity of cancer cells to neoplastic agents. Dendrimers are exceptional drug delivery systems and l-BSO nanoformulations are envisaged as potential chemotherapeutics. The absorption of l-BSO at a low wavelength limits its detection by conventional analytical tools. A simple and sensitive method for l-BSO detection and quantification is now reported. In this study, l-BSO was encapsulated in a folate-targeted generation four polyurea dendrimer (PUREG4-FA2) and its release profile was followed for 24 h at pH 7.4 and 37 °C. The protocol uses in situ l-BSO derivatization, by the formation of a catechol-derived orto-quinone, followed by visible detection of the derivative at 503 nm. The structure of the studied l-BSO derivative was assessed by NMR spectroscopy.

2.1. l-BSO Encapsulation and Release Studies

shows the l-BSO release profile from a l-BSO@PUREG4-FA2 nanoformulation at pH 7.4. The release was followed for 24 h and the results indicate a burst release in the first hours, as expected in this type of delivery system [,]. After 1 h, around 60% of the loaded drug was released to the medium, reaching a plateau after 3 h. After 24 h, 90% of l-BSO was released, meaning that only a residual amount of l-BSO was trapped inside the PUREG4-FA2 dendrimer during this period of time. The quantification of BSO released from the PUREG4-FA2 dendrimer was determined using a calibration curve, obtained from an assay under the same experimental conditions (see ). In more acidic media, a faster release is expected.

3.1. Reagents and Materials

l-Buthionine-(S,R)-sulfoximine (l-BSO) (≥97% purity) was obtained from Sigma-Aldrich. All chemicals and solvents were used as received without further purification. Polyurea (PURE) dendrimer generation four (PUREG4) was synthesized following our reported supercritical-assisted polymerization [].

3.5. Quantification of BSO by UV-Vis Spectroscopy

Using a modified protocol [], l-BSO was derivatized in order to be detected by UV-Vis spectroscopy. l-BSO quantification was performed by adding to the samples 300 µL of catechol 2.25 mM in PBS, followed by 300 µL of sodium periodate 6.75 mM in PBS. After 60 sec, the absorption of the l-BSO derivative (503 nm) was measured in a PerkinElmer Lambda 25 UV-Vis Spectrometer with a slit width of 5 nm at a scan rate of 240 nm min−1 at 25 °C. For the calibration curve, standard solutions of l-BSO were prepared in the concentration range of 0.1–150 µM in PBS and processed using the same protocol. A good correlation coefficient (R2 = 0.997) was obtained (see ).

Abstract

The aim of the present study was to assess the effects of l-buthionine-(S,R)-sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, and GBR 12909, a dopamine reuptake inhibitor, administered alone or in combination to Sprague-Dawley rats during early postnatal development (p5–p16), on the levels of reactive oxygen species (ROS), lipid peroxidation (LP) and the activities of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione disulfide reductase (GR) in peripheral tissues (liver, kidney) and selected brain structures (prefrontal cortex, PFC; hippocampus, HIP; and striatum, STR) of 16-day-old rats. The studied parameters were analyzed with reference to the content of GSH and sulfur amino acids, methionine (Met) and cysteine (Cys) described in our previous study. This analysis showed that treatment with a BSO + GBR 12909 combination caused significant decreases in the lipid peroxidation levels in the PFC and HIP, in spite of there being no changes in ROS. The reduction of lipid peroxidation indicates a weakening of the oxidative power of the cells, and a shift in balance in favor of reducing processes. Such changes in cellular redox signaling in the PFC and HIP during early postnatal development may result in functional changes in adulthood.

2.1. Animals and Treatment

To create the neurodevelopmental model of schizophrenia, pregnant Sprague-Dawley females were delivered to our laboratory by the Charles River Company (Sulzfeld, Germany). They were kept in individual cages, with free access to standard laboratory food and tap water under standard laboratory conditions i.e., at room temperature (22 °C) and under an artificial light/dark cycle (12/12 h). On the day of delivery, the sex of pups was determined. Only male pups which were left with their mothers were treated on the postnatal days p5–p16, with the selective inhibitor of GCL, compound l-buthionine-(S,R)-sulfoximine (BSO) and the DA reuptake inhibitor GBR 12909, alone or in combination. BSO at a dose of 3.8 mmol/kg s.c., was administered once daily, while GBR 12909 at a dose of 5 mg/kg s.c., every second day. Both model compounds were dissolved in 0.9% NaCl. Under the combined treatment regimen, BSO administration proceeded GBR 12909 injection. Control rats instead of the model compounds received a 0.9% NaCl once daily. Every day, pups were weighed and the injected volumes of the studied model compounds were adjusted accordingly with the actual body weight. On postnatal day p16, the rats were killed by decapitation 4 h after the last doses of model compounds, and their liver, kidney and selected brain structures (PFC, HIP, STR) were dissected, immediately frozen on dry ice and stored at −80 °C until further biochemical analysis.