## 3 Results
### Measurement of ROS-Induced Fluorescence by Flow Cytometry  
The results of the flow cytometry analysis using THP-1 cells treated with pure forms EFA and GSH alone and in combination is depicted in Figure 4, and when encapsulated into liposomal carriers is shown in Figure 5. Comparison of mean fluorescence data when cells are incubated with pure EFA and pure GSH and a combination of both is exhibited in Figure 4A. As observed, a strong fluorescent signal of CellROX was visible in the control cells due to PMA-induced ROS generation (Figure 4B,C). In the present study, an increase in total ROS was observed, indicating an increase in the oxidative stress in response to the treatment with EFA (Figure 4D) as reported in earlier studies also [9; 32; 33].  
Figure 4: Flow cytometry analysis depicting the measurement of reduction in mean fluorescence intensity (MFI) of PMA-induced oxidative stress in THP-1 monocyte/macrophage cells. (**A**) Dot plots representing the data of MFI reduction in cells treated with pure efa (EFA) with and without GSH in comparison with pure GSH (****: indicates \(p<0.0001\); ****: indicates \(p<0.001\)).Values are means \(\pm\) S.D. of three independent experiments. (**B–F**) represent dot plot of forward scatter to cellROX-derived red fluorescence showing the cellROX gate (gated events) and the below plots represent the dot plot depicting ROS-dependent cellROX fluorescence when cells treated with pure EFA with and without GSH. Untreated cells with no cellROX dye (**B**); untreated cells with cellROX dye (control—(**C**)).  
Forward scatter (FSC) vs. side scatter (SSC) dot plots were used to visualize the expression of fluorescence of the cells. Dot plots obtained from the flow cytometric analysis demonstrated that ROS-induced fluorescence was reduced by 1.2 fold in comparison with pure EFA, and was further reduced by 2.3 fold in the presence of GSH (Figure 4D,F).  
However, to further evaluate the ability of liposomal systems to reduce the ROS-dependent cellROX fluorescence, THP-1 cells were also incubated with the EFA liposomes (Figure 5A) and EFA + GSH liposomes (Figure 5B) for 24 h. The overlay image (Figure 5A,B) indicate that the combination of GSH with EFA was more active than the singly loaded EFA liposomes and the pure EFA as well. Hence, EFA-loaded liposomes effectively protected the oxidative damage by 1.8 fold relative to pure EFA. Data obtained from flow cytometric analysis reveal that EFA, when encapsulated into liposomes, could effectively quench the  
Figure 5: Flow cytometry analysis depicting the measurement of reduction in mean fluorescence intensity (MFI) of PMA-induced oxidative stress in of THP-1 monocyte/macrophage cells. (**A**,**B**) represent dot plot of forward scatter to cellROX-derived red fluorescence showing the cellROX gate (gated events) and the below plots represent the dot plot depicting ROS-dependent cellROX fluorescence when cells treated with pure EFA with and without GSH. Confocal fluorescent images acquired at \(\times\) 40 original magnification from cells treated with Liposomal EFA without GSH (**A**) and with GSH (**B**). Nucleus is stained with DAPI (blue); R123-loaded EFA liposomes shown as green fluorescence and oxidative stress is shown as red fluorescence. (**C**) represents the data of MFI reduction in cells treated with liposome loaded edavirenz (EFA) with and with GSH in comparison; (***** indicate \(p\) < 0.00001) significant difference of liposomal EFA and liposomal EFA + GSH. Values are means \(\pm\) S.D. of three independent experiments.  
intracellular accumulation of ROS, exhibiting a synergistic effect in combination with GSH (2.5 fold) as displayed in Figure 5C.