HPLC–FLD and spectrofluorometer apparatus: How to best detect fluorescent probe-loaded niosomes in biological samples

The analytical tools allow the detection of bioactive compounds, diagnostic agents and chemotherapeutics. Recently, new methods have been developed to analyze pharmaceutical samples and ingredients. In this attempt, analytical parameters, e.g., the lack of trueness, robustness and sensitivity, play a pivotal role to quantify and analyze molecules, both for diagnostic applications as well as therapeutic treatments. Spectrophotometers and spectrofluorometers are apparatus for easy and rapid quantification of molecular probes and chemotherapeutics into cells, plasma and tissues. However, they lack accuracy and precision. Conversely, HPLC provides the maximum resolution to detect and separate fluorescent probes and chemotherapeutics after their incubation in cells, plasma and tissues. The aim of this work was to develop an HPLC method that easily detects molecular and fluorescent probes, e.g., Nile Red, in biological samples. To improve the robustness of the method, Nile Red was analyzed before and after loading into niosomes made from Tween 20 and 21, respectively. A significant difference was further obtained by comparing the entrapment efficacy percentage of niosomes made from Tween 21 (42.23%) and Tween 20 (53.25%). The comparison between HPLC and spectrofluorometer assays showed differences between the two methods in terms of limit of detection, linearity and accuracy. The resulting data demonstrated that the HPLC-FLD provides a limit of detection for Nile Red of 0.1 ng/mL, and a good linearity up to 62.5 ng/mL. The HPLC-FLD analysis showed a limit of quantification value for a total mass of Nile Red 1200-folds better than data previously reported in studies; and 312-folds better than the spectrofluorometer analysis. Additionally, results show that the HPLC-FLD increases the sensitivity for biological samples compared to the spectrofluorometer. The Nile Red-loaded niosomes were also incubated at different times with HEK-293 cells. In vitro results demonstrated that the HPLC-FLD apparatus detects Nile Red-loaded niosomes at higher concentrations into HEK-293 cells than the spectrofluorometer. The intracellular uptake of Nile Red was increased at 120 and 24 min using niosomes made from Tween 20 and 21, respectively, and its intracellular accumulation shows a time-dependent internalization over 120 min of incubation time.