Xl 2000

A method described previously by Shehata et al. was followed to formulate niosomal preparation using thin film hydration method [36 (link)]. Accordingly, non-ionic surfactant (span 60) and cholesterol with a molar ratio (1:1) were mixed together, followed by adding sufficient quantity of DSPE-PEG 2000 and then dissolved in chloroform using a round bottom flask. Afterward, under reduced pressure, the mixture was dried at 60 °C using rotary evaporator (Cole-Parmer, T-1602-21, Tokyo, Japan). A dried lipid film was formed on the wall of the flask, which was subjected to hydration using 5 mL dist. water containing colchicine (25 mg), while keeping agitation in water bath at 60 °C for 1 h to get niosomal solution. Consequently, the attained niosome was exposed to probe sonicator (XL-2000, Qsonica, Newtown, CT, USA) for 30 s to obtain the anticipated particle size.

Thin film hydration method that was previously described by Knudsen et al. was executed in order to develop different liposomal formulations [48 (link)]. Fifty milligrams of Brucine, ten milligrams of DSPE-PEG 2000 and a specified amount of EPC and cholesterol, as mentioned in Table 1, was added to a round bottom flask and mixed with a 6 mL ethanol: chloroform mixture (1:2). The flask was attached to a rotary evaporator (Heidolph, GmbH, Co. KG, Schwabach, Germany) for 2 h at 100 rpm maintained at 60 °C and operated till complete evaporation of the solvent and formation of thin lipid film on the inner wall of the round-bottom flask. The obtained lipid film was rehydrated with 5 mL phosphate buffer pH 7.4 for 30 min while vortexing using a classic advanced vortex mixer (VELP Scintifica, Usmate Velate, Italy). Next, the resultant dispersion was subjected to sonication for 30 s utilizing probe sonicator (XL-2000, Qsonica, Newtown, CT, USA) to obtain proper particle size. Eleven experimental formulations were fabricated using (CCD) alongside the values of their observed response as clarified in Table 1.

ATP was measured by luciferin–luciferase assay (Enliten ATP Assay System, Promega). The retina (including RPE) was dissected from enucleated eyes of WT and obe^td15 fish and placed in Krebs solution. The samples were transferred to 2.5% trichloroacetic acid (TCA), then homogenized by sonication (3 × 10 seconds, XL-2000 Qsonica LLC) on ice. Cell debris was removed by centrifugation at 13,000 rpm for 30 minutes at 4 °C. The supernatant was collected and the TCA was neutralized with 1 M Tris–acetate buffer (pH 7.75, final TCA concentration 0.0625%). Protein concentration was measured using the BCA Protein Assay Kit (Pierce), a plate reader (Tecan Safire) and Magellan Software. To analyze ATP levels, 10 µl of the neutralized samples was added to 100 µl of luciferin–luciferase in fresh buffer and ATP was measured using a Glomax-20/20 luminometer (Promega) and data normalized to concentration of protein.