For the in vivo study, C57BL/6J 8-week old mice were utilized. Animals were housed and maintained in a 12-h light/dark cycle and fed ad libitum. All procedures were performed according to the European Union policy (Directive 86/609/EEC) (carried out in compliance with Greek Government guidelines) and institutionally approved protocols (Veterinary Directorate of Prefecture of Heraklion (Crete) and FORTH ethics committee (License number: EL91-BIOexp-02)). Mice were anesthetized using an intraperitoneal injection of a ketamine (Nerketan 10, 100 mg/mL) and xylazine (Xylapan, 20 mg/mL) cocktail. Once the hind-limp was lost, the mice were fixed in the supine position, 25 µL of each formulation was administered to each mouse in 60 s intervals, via 1–2 µL dose alternatively into each nostril [26 (link)]. Animals were sacrificed 60 and 120 min after the completion of the administration. Brains were quickly dissected out of the cranium; excess blood was wiped off, and the brain was snap frozen until further processing.
After weighing, brain samples were homogenized in 300 µL ice-cold distilled water/methanol solution (25/75 v/v) and sonicated for 20 min at 4 °C. Next, three volumes of ice-cold acetonitrile were added, followed by sonication for 10 min and centrifugation at 14,000× g/15 min/4 °C. After an extra addition of 100 µL ice-cold acetonitrile to the supernatant, a 10 min-sonication and centrifugation at 14,000× g/15 min/4 °C were carried out. The supernatant was vacuum-dried at a SpeedVac without heating. Prior to analysis, samples were stored at −80 °C.
To quantify the BNN27 levels, the detection limit of the enzymatic method used for BNN27 quantification in the formulations (>2 ppm) was not low enough; therefore, a liquid chromatography mass spectrometry (LC-MSn) method was developed using deuteriated pregnenolone (pregnenolone 17,21,21,21-D4) as the internal standard (70 ng/mL). The analysis was performed on an LTQ-Orbitrap Velos mass spectrometer (MS) (Thermo Fisher Scientific, Bremen, Germany) connected to an Accela ultra-high-performance LC (UHPLC) system. An Acquity UPLC BEH C18 VanGuard pre-column (130 Å, 1.7 µm, 2.1 mm × 100 mm) coupled to an Acquity UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 5 mm) was used. Quality control samples were prepared at three concentrations (low, medium, high) to monitor the instruments’ performance and chromatographic integrity over time. Monitoring occurred in positive ion mode. The standard curve concentration range was 1–2000 ng/mL (Y = 0.000546519 + 0.000897604∙X; R2 = 0.9673; W:1/x). The injection volume was set at 5 µL, and the mobile phase flow rate was set at 0.2 mL/min. Mobile phase solvents were A (95% H2O, 5% methanol, 0.1% formic acid) and B (methanol, 0.1% formic acid). The eluting gradient program was the following: 0–0.1 min (40% A, 60% B), 0.1–0.5 min (20% A, 80% B), 0.6–6.5 min (5% A, 95% B), 6.51–8.0 min (40% A, 60% B). Data were processed with Xcalibur software (version 2.1, Thermo Scientific, Waltham, MA, USA) and data analysis was conducted using the R programming language. In order to calculate the BNN27 levels in brain tissue, expressed as administered dose percent (ID%), the following formula was applied: ID%/g brain = (Xbrain/X in dose) × 100; where: Xbrain = BNN27 (mg) per g of weighted brain tissue, and X in dose = BNN27 (mg) in 25 µL solution for intranasal administration.
After weighing, brain samples were homogenized in 300 µL ice-cold distilled water/methanol solution (25/75 v/v) and sonicated for 20 min at 4 °C. Next, three volumes of ice-cold acetonitrile were added, followed by sonication for 10 min and centrifugation at 14,000× g/15 min/4 °C. After an extra addition of 100 µL ice-cold acetonitrile to the supernatant, a 10 min-sonication and centrifugation at 14,000× g/15 min/4 °C were carried out. The supernatant was vacuum-dried at a SpeedVac without heating. Prior to analysis, samples were stored at −80 °C.
To quantify the BNN27 levels, the detection limit of the enzymatic method used for BNN27 quantification in the formulations (>2 ppm) was not low enough; therefore, a liquid chromatography mass spectrometry (LC-MSn) method was developed using deuteriated pregnenolone (pregnenolone 17,21,21,21-D4) as the internal standard (70 ng/mL). The analysis was performed on an LTQ-Orbitrap Velos mass spectrometer (MS) (Thermo Fisher Scientific, Bremen, Germany) connected to an Accela ultra-high-performance LC (UHPLC) system. An Acquity UPLC BEH C18 VanGuard pre-column (130 Å, 1.7 µm, 2.1 mm × 100 mm) coupled to an Acquity UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 5 mm) was used. Quality control samples were prepared at three concentrations (low, medium, high) to monitor the instruments’ performance and chromatographic integrity over time. Monitoring occurred in positive ion mode. The standard curve concentration range was 1–2000 ng/mL (Y = 0.000546519 + 0.000897604∙X; R2 = 0.9673; W:1/x). The injection volume was set at 5 µL, and the mobile phase flow rate was set at 0.2 mL/min. Mobile phase solvents were A (95% H2O, 5% methanol, 0.1% formic acid) and B (methanol, 0.1% formic acid). The eluting gradient program was the following: 0–0.1 min (40% A, 60% B), 0.1–0.5 min (20% A, 80% B), 0.6–6.5 min (5% A, 95% B), 6.51–8.0 min (40% A, 60% B). Data were processed with Xcalibur software (version 2.1, Thermo Scientific, Waltham, MA, USA) and data analysis was conducted using the R programming language. In order to calculate the BNN27 levels in brain tissue, expressed as administered dose percent (ID%), the following formula was applied: ID%/g brain = (Xbrain/X in dose) × 100; where: Xbrain = BNN27 (mg) per g of weighted brain tissue, and X in dose = BNN27 (mg) in 25 µL solution for intranasal administration.
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