Up100h ultrasonic processor

Liver and pancreas samples were homogenized (homogenization buffer: 20 mM TrisBase; 150 mM NaCl; pH 7.8; 0.05% TritonX100) with a sonicator (4 °C; 3 × 10 s; Hielscher UP100H Ultrasonic Processor [Hielscher Ultrasonics, Teltow, Germany]) and centrifuged (1000 g; 5 min; 4 °C). Tissue cholesterol and triglyceride were measured from the supernatant by enzymatic colorimetric assays according to the manufacturer’s instructions (CHOD-PAP and GPO-PAP assays; Cobas-Roche, West Sussex, England).

A UP100H Ultrasonic Processor (Hielscher) with a 2-mm probe was used to sonicate the samples, using pulse settings with 0.5-s cycles and 27% power. The samples underwent n × 30 s sonication cycles, with 30 s of sonication followed by 30 s on ice for each cycle. The numbers (n) of sonication cycles used in this study for different amounts of input were optimized and can be found in Supplementary Table 1. For mouse liver samples, 10 ng and 100 pg were used to construct input libraries after sonication. RNA from pools of zygotes was used for input controls for single zebrafish zygotes (Supplementary Table 1). In the case of single mouse oocytes and early embryos, 10% of multiple oocyte/embryo RNA was removed and served as input control (Supplementary Table 1). To the samples consisting of 80 μl, 20 μl of 5× IP buffer (50 mM Tris-HCl (pH 7.5), 750 mM NaCl, 0.5% (vol/vol) NP-40 and 5 U μl⁻¹ RiboLock RNase inhibitor) was added to make a final volume of 100 μl for sonication.

Ternary phase diagrams composed of S—surfactant (CAPB or CB), O—oil (OA or LA), and W—water, i.e., S:O:W systems were prepared via a composed aqueous titration-ultrasound method to examine the phase behavior of the samples. The phase boundaries were determined by the stepwise addition of water to the weighted mixture of the S:O:W components. After the addition of each portion of water, the samples were sonicated by a tip sonicator (UP100H ultrasonic processor developed by Hielscher Ultrasonics GmbH, Teltow, Germany; cycle, 0.9; amplitude, 80%) for 2 min and kept in thermostated baths at 25 °C to equilibrate. The amount of aqueous phase added was varied to obtain a water concentration ranging from 5% to 99% of the total volume in about 5% intervals. The boundary lines drawn on the phase diagram lie at equal distances between consecutive experimental measurements on both sides of the phase boundary. Visual observation was made after each 5% addition of the aqueous phase to the S:O mixture and its equilibration. Based on the visual assessment, the obtained formulations were categorized as follows: (i) transparent and translucent with a blue sheen: oil-in-water (o/w) nanoemulsion (NE); (ii) milky or cloudy: emulsion (E); (iii) two separate phases (2Ph). Selected formulations were subjected to further physicochemical characterization.

To facilitate the suspension of the different ONCs (with and without GMA or EGDMA), the pH of the suspension was adjusted to 11 with NaOH 0.5 M obtaining the salt form of the carboxylic acid (more hydrophilic). ONCs were further suspended by sonication with an immersed probe at 0 °C (UP100H Ultrasonic Processor, Hielscher, Teltow, Germany), with an output power of 100% (100 Watt ultrasonicator) for one hour (with a stop of a few minutes every 15 min).
The suspension was used for preliminary characterization (DLS, TEM) and for mortar preparation. After ultrasonication, an aliquot was lyophilized to obtain the yield percentage for each reaction, which varied between 60 and 70%, and to conduct IR and SEM analyses.