## 3 Results
### Intracellular Uptake by Macrophages  
To evaluate the influence of glutathione on the EFA uptake, cells were treated with a pure form of edavirenz with and without GSH, (Figure 1). Later, a liposomal form of EFA and GSH were examined (Figure 2). It was observed that the addition of GSH along with EFA demonstrated an almost two-fold increase in the intracellular concentration of pure EFA in comparison to pure EFA alone Figure 1A. To further confirm the uptake, confocal images were captured, which revealed that the viability was improved, as evidenced by an increase in the number of cells (Figure 1B,C).  
Further, the incorporation of EFA into liposomes was investigated and compared with pure EFA at various concentrations. As represented in Figure 2A, the initial dose-escalation study was carried out using 100 uM to 400 uM of EFA in the pure form and liposomal form. After 24 h of incubation, intracellular lysate concentrations revealed a significantly higher amount of EFA taken up by the cells from EFA liposomes compared to pure EFA. A dose-dependent increase in the uptake of EFA was observed. Nevertheless, at all concentrations evaluated, we observed that encapsulation of EFA into the liposomes increased its uptake by the macrophages. The concentration of 400 uM resulted in the highest uptake when liposome loaded EFA was administered, which was selected for further evaluation.  
Further, the influence of the immunomododulatory agent GSH on the uptake of EFA encapsulated into liposomes was examined. Formulation composed of EFA + GSH (F2) was incubated with the THP-1 cells. As depicted in Figure 2B, the inclusion of GSH into  
Figure 1: (**A**) Comparison of intracellular lysate concentrations of pure EFA with and without GSH (*** indicate \(p\) < 0.001). Values are means \(\pm\) S.D. of three independent experiments. (**B,C**) represent the uptake of rhodamine123-containing pure EFA without and with GSH by the THP-1 monocyte/macrophage cells respectively. Cells were incubated with sample preparation for 24 h at 37 \({}^{\circ}\)C. Fluorescent images were acquired at \(\times\)40 original magnification. Nucleus is stained with DAPI (blue); R123-loaded EFA shown as green fluorescence.  
Figure 2: **(A) Intracellular lysate concentrations of pure EFA and liposomal EFA after 24 h at 37 \({}^{\circ}\)C incubation of THP-1 monocyte/macrophage cells with liposomal EFA. (A) Intracellular lysate concentrations of EFA with varying concentrations of pure (pure EFA) and liposomal EFA (L-EFA), significant difference between uptake of pure EFA and L-EFA was observed at 200, 300 and 400 \(\upmu\)M (*** indicate \(p\) < 0.0001). (B) Significant difference between uptake of liposomal EFA with and without GSH was observed (* indicate \(p\) < 0.05). Values are means \(\pm\) S.D. of three independent experiments (C,D) represent the uptake of rhodamine 123-containing liposomal EFA with and without GSH by the THP-1 monocyte/macrophage cells. Cells were incubated with sample preparation for 24 h at 37 \({}^{\circ}\)C. Fluorescent images were acquired at \(\times\)40 original magnification. Nucleus is stained with DAPI (blue); R123-loaded EFA liposomes shown as green fluorescence.**  
incubation, the microscopic images displayed intact cells, and the respective fluorescent images visualized the uptake of RH123 labelled liposomes by these cells. The nuclei were stained with DAPI (blue). As noticed in Figure 2C,D, the images reveal that a higher amount of EFA from liposomes was internalized by the THP-1 Mo/Mac cells. In comparison to the pure form of EFA, liposomal EFA demonstrated an enhanced fluorescent Rhodamine uptake by the cells, which is clearly evidence of intracellular localization of drug, further intensified in the presence of GSH.