Characterization of NLC

The size and polydispersity index (PDI) of NLCs were determined using Zetasizer (PCS, Nano ZS90, Malvern Instruments, United Kingdom). Samples were prepared by diluting the final NLC suspension 10–15 times using deionized water kept in polystyrene cuvettes and observed at a fixed angle of 90° at 25 ± 0.1°C. The zeta potential of the formulated NLCs was determined in folded capillary cells by laser Doppler anemometry using Malvern Zetasizer. Measurements were carried out at 25 ± 0.1°C with the samples properly dispersed in deionized water kept in the electrophoretic cell at an electric field of 15.24 V/cm to determine the zeta potential (Eratte et al., 2014).

The surface morphology of the prepared NLC formulation was confirmed by transmission electron microscopy (TEM) and is in accordance with the findings of Garg et al. (2016c, 2017) and Chauhan et al. (2020). Scanning electron microscopy (SEM) analysis provides high-resolution imaging useful to determine surface fractures, flaws, contaminants, or corrosion. A drop of diluted NLC suspension was taken on a coverslip which was fixed on to a brass stub using a double-sided adhesive tape and analyzed using SEM. It was then dried and made electrically conducive by coating it with a thin layer of gold; the sample was then loaded onto the microscope to take images. TEM analysis was conducted by placing a drop of diluted sample on a membrane-coated grid surface and further stained using a drop of 1% phosphotungstic acid and immediately added to the grid surface for 1 min, then the excess fluid was removed and the grid surface air dried at room temperature. The sample was then examined using high-resolution (HR)–TEM, Tecnai 200 Kv TEM at 10 × 15,000 magnification (Garg et al., 2016c; Garg et al., 2017; Chauhan et al., 2020).

The drug encapsulation efficiency was determined by using the direct lysis method as reported by Garg et al. (2017). Briefly, ultracentrifugation was performed at 40,000 rpm for 20 min at 4°C using 20 ml solution of NLCs, and pellets were washed three times using double-distilled water to separate the unentrapped drug. Next, the pellets were lysed using chloroform diluted with methanol followed by filtration through 0.22-mm filters (Millipore, United States). The drug was quantified from the filtrate using the HPLC method (Sharma et al., 2016a). The HPLC system (Shimadzu LC-2010C HT ver. 3.01 system, M/s Shimadzu Inc., Tokyo, Japan) was connected with a UV-vis detector (equipped with a quaternary pump, mobile phase degasser, column thermostat controller, and SPD-10AVP column oven). C18 columns from Thermo Fisher Scientific Inc., United States, were used for the separation. Isocratic elution (methanol:water, 70:30% v/v having 0.02% orthophosphoric acid) was used as the mobile phase at 275 nm. The flow rate was kept at 1 ml/min at room temperature for 20–30 μl of injection volume.

The drug content was quantified by using the HPLC system (Shimadzu, Japan), equipped with a double reciprocating pump and photodiode array detector, operating at 254 nm, and with a Thermo Hypersil BDS C18 column (250 mm × 4.6 mm, 5 µ), Thermo Scientific, United States. The sample was analyzed by the isocratic elution mode with mobile phase, methanol: 0.02% orthophosphoric acid at 70:30 v/v ratio. The injection volume was kept at 20–30 µl with a flow rate of 1 ml/min at room temperature (Garg et al., 2016c).