Model vcx 750

Hot homogenization and ultra-sonication technique was employed for the preparation of LSP-NLCs [18 (link)]. Accurately weighed solid and liquid lipids were melted at 70 °C followed by the addition of LSP to the lipid melt to allow its complete dissolution. The aqueous phase was prepared by dissolving surfactants in distilled water and heated up to 70 °C. This hot aqueous phase was then poured into the lipid phase and homogenized at 15,000 rpm for 5 min (Homogenizer HG-15D, DAIHAN Scientific, Wonju, Republic of Korea). The obtained coarse O/W emulsion was further sonicated for 3 min at an amplitude of 50% and a power of 100 W using a probe sonicator at 65 °C (Model VCX750, Sonics and Materials Inc., Newtown, CT, USA). Finally, the obtained nano-emulsion was rapidly cooled down in an ice bath to form LSP-NLCs.

Three weeks after SE, 10 SE mice and 11 No SE mice were injected with [U-¹³C]glucose (0.3 mol/L i.p., 558 mg/kg; 99% ¹³C; Cambridge Isotope Laboratories). To denature brain enzymes and other proteins immediately, mice were killed by focal microwave fixation to the head at 5 kW for 0.79–0.83 s (Model MMW-05, Muromachi) 15 min after [U-¹³C]glucose injections. Mice were then decapitated, and hippocampal formations were dissected out and stored at –80°C until extracted. Samples were sonicated in 1 ml of methanol using a Vibra Cell sonicator (Model VCX 750, Sonics and Materials) with 4 μL of a 1 mm azidothymidine (AZT) solution added as an internal standard. Polar metabolites were extracted from samples using a modified Bligh–Dyer water/methanol/chloroform extraction procedure at a 2/2/3 ratio as previously described (Le Belle et al., 2002 (link)). Samples were lyophilized, reconstituted, and stored at –80°C until analyzed.

The pullulan/carrageenan-based film was prepared using a solution casting method [29 (link),30 (link)], as shown schematically in Scheme 1. For the preparation of film solution, DL (5 wt.% based on biopolymer) was mixed with 150 mL distilled water with vigorous mixing using a magnetic stirrer. The CuSNP (0.5 wt.% based on biopolymer) was dispersed in 150 mL of distilled water using a magnetic stirrer and then ultrasonicated at 60% amplitude for 3 min with pulses 5 s on and 2 s off in a probe ultrasonicator (Model VCX 750, Sonics & Materials, Inc., New Town, CT, USA). Mixed solutions of CuSNP (0.5 wt.%) and DL (5 wt.%) were also prepared. To the CuSNP and DL dispersed solutions, 1.2 g of glycerol (30 wt.% based on polymers) was added with continuous stirring. Then, 4 g of biopolymers (2 g each of pullulan and carrageenan) was dissolved slowly and heated for 20 min at 95 °C with constant stirring. The film-forming solution was cast on a flat Teflon film-coated glass plate and dried at room temperature for 48 h. The dried film was peeled from the container and conditioned at 25 °C and 50% RH for at least 48 h. For comparison, control pullulan/carrageenan was prepared following the same procedure without adding CuSNP and DL. The produced films were designated as Pul/Carr, Pul/Carr/DL, Pul/Carr/CuSNP, and Pul/Carr/DL/CuSNP depending on the type of biopolymer and filler material.

All Fmoc-protected amino acids,
Wang resin, and hexafluorophosphate azabenzotriazole tetramethyl uronium
(HATU) were purchased from Matrix Innovation (Quebec, Canada). N,N′-dimethylformamide
(DMF), dichloromethane (DCM), N,N′-diisopropylethylamine (DIPEA),
piperidine, methanol, trifluoroacetic acid (TFA), diethyl ether, and
ethanol were purchased from Bio-Lab (Jerusalem, Israel). Triisopropylsilane
(TIPS), thioanisole, 1,2-ethanedithiol (EDT), acetic anhydride, hydroxybenzotriazole
(HOBT), N,N′-diisopropylcarbodiimide (DIC), 5(6)-carboxyfluorescein
[5(6)-FAM], and phenol were purchased from Sigma-Aldrich (St. Louis,
Missouri, USA). Paraffin oil (puriss meets the analytical specification
of Ph. Eur., BP, a viscous liquid), dimethylaminopropyl-N′-ethylcarbodiimide
hydrochloride (EDC), and MES hydrate were purchased from Sigma-Aldrich.
HPLC-grade water was purchased from Alfa Aesar and was used as received
without further purification. The Sonics Vibra-cell ultrasonic liquid
processor, Model-VCX 750 (Newtown, CT, USA) was used for ultrasonication.

The CHX solution was lyophilized and used in the powder form. Thin-film hydration technique was used for the preparation of liposomes according to the previous work reported by our group [14 (link),15 (link)].
Briefly, HSPC and cholesterol (90:10; 100 mg of total lipids) were dissolved in 10 mL of chloroform in a round-bottomed flask. The organic solvent was evaporated in a rotavapor under vacuum to obtain a thin film. The dried thin film was hydrated by a phosphate buffer solution of pH 7.4 containing 30 mg of CHX. After hydration, the dispersion was subjected to sonication using a probe sonicator (Model-VCX750, Sonics & Materials, Inc., Newtown, CT, USA) for 20 min at 40% amplitude (750 watt) and 6 s pulse. The dispersion was further subjected to high-speed centrifugation (at 22,000 rpm and 4 °C for 45 min) to separate the free drug. The liposomal pellet was re-dispersed in 5 mL of water and stored in a refrigerator. A similar liposomal formulation was prepared by incorporating rhodamine B dye (1% w/v solution) in the chloroform lipid solution to visualize under a confocal laser scanning microscope. The composition of different batches of liposomes is provided in Table 1. The control sample of liposomal formulation, without the drug, was also prepared.