Diamonsil plus c18 column

GPD quantitative determinations were performed by using the HP 1260 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) with a reverse-phase Diamonsil^® Plus C18 column, (250×4.6 mm, 5 µm; Dikma Technologies, Foothill Range, CA, USA). The mobile phase consisted of acetonitrile and pure water (35:65, v/v) at a flow rate of 1.0 mL/min. The column temperature was maintained at 30°C and the effluent was monitored at 203 nm. Quantified samples were filtered through a 0.45-µm filter membrane prior to automatic injection into the HPLC system.

BRE quantitative determinations were performed by using the HP 1260 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) with a reverse-phase Diamonsil^® Plus C18 column, (250 mm × 4.6 mm, 5 µm; Dikma Technologies, Foothill Range, CA, USA). The mobile phase consisted of methanol, acetonitrile and 20 mmol/L phosphate buffer (17:17:66, v/v) at a flow rate of 0.8 mL/min. The column temperature was maintained at 25°C and the effluent was monitored at 334 nm. Quantified samples were filtered through a 0.45-µm filter membrane prior to automatic injection into the HPLC system.

To quantify the cellular uptake, cells were incubated with PRN-loaded nanocomposites (PRN solution, PRN-LDH nanoparticles, two different DS of GS in CG-GS-PRN-LDH or mixture of CG-GS and PRN solution) at a concentration of 2 μg/mL for 1, 2 and 4 h, respectively. Then the test samples were removed and the cells were washed with DPBS followed by trypsinization and disruption with lysis buffer for 1 h at 4°C. The lysate was then used to assay for PRN with a high-pressure liquid chromatography (HPLC) method and protein content assay (BCA assay kit; KeyGen BioTech, Nanjing, China). Cellular uptake was represented as the amount of PRN normalized with per mg of total cellular protein.
HPLC system employed in the study consisted of LC-2010CHT (Shimadzu, Kyoto, Japan) with an LC-2010CHT variable wavelength UV-vis detector (λ_detection=230 nm). A Diamonsil^® Plus-C18 column (4.6×150 mm, 5 μm) (Dikma, Beijin, China) was operated at 35°C. The mobile phase was composed of NaH₂PO₄-methanol-acetonitrile (7:2:1, v/v/v) and was pumped at a flow rate of 1 mL/min.